Numerous pangenomic studies identified protein-coding genes and signaling pathways involved in bladder carcinogenesis. However, noncoding somatic alterations remain unexplored. A recent study revealed a mutational hotspot in intron 6 of GPR126 gene in 2.7% of a large breast cancer series. As GPR126 is highly expressed in bladder tissues, we investigated here the prevalence and the prognostic significance of these mutations in bladder cancer. We analyzed a cohort of 103 bladder cancers including 44 nonmuscle-invasive bladder cancers (NMIBC) and 59 muscle-invasive bladder cancers (MIBC). GPR126 mutations were analyzed by high-resolution melting and Sanger sequencing, and GPR126 expression levels were assessed using real-time quantitative RT-PCR. In NMIBC, somatic GPR126 noncoding mutations occurred in 47.7% of samples and were negatively associated with GPR126 mRNA levels. GPR126 mutations had higher frequencies in nonsmoker patients and were associated with a prior history of NMIBC. GPR126 overexpression was detected in 70.5% of samples. GPR126 mutation and overexpression status were not associated with outcome. In MIBC, somatic GPR126 mutations occurred in 44.1% of samples. Mutations were more frequent in females. GPR126 overexpression was detected in 27.1% of the sample. A trend toward significance was observed between GPR126 overexpression and better outcome. We identified the second most frequent mutational hotspot after TERT promoter (∼70%) in bladder cancer, with a mutation rate of approximately 50%.

Implications:

The GPR126 intronic mutational hotspot could be a promising clinical biomarker candidate to monitor tumor burden using circulating tumor DNA in bladder cancer.

Bladder cancer is the sixth most common cause of cancer mortality that causes nearly 150,000 deaths per year worldwide. Its incidence has increased markedly in recent decades (1, 2). The risk factors are different, including tobacco use, Schistosoma infection, chemical exposure, diet and lifestyle trends, atmospheric pollution, and genetic susceptibilities (1, 2). Urothelial carcinoma is the predominant histologic type, accounting for 90% of all cases (2). At initial diagnosis, bladder cancer could be characterized by two different forms with specific clinical and pathologic phenotypes: Nonmuscle-invasive bladder cancers (NMIBC) represent about two thirds of newly diagnosed cases. These have a 60% recurrence rate (3) and, in 10% to 20% of cases, evolve to muscle-invasive tumors (4). Muscle-invasive bladder cancers (MIBC) occurring in one third of cases frequently cause distant metastases. Survival greatly differs between early and advanced bladder cancer (5). Moreover, current prognostic factors, namely tumor–node–metastasis (TNM) stage and pathologic grade (6), are insufficient to predict outcome at the individual level. New effective molecular markers that may also serve as therapeutic targets are urgently needed.

The different clinical and pathologic bladder cancer phenotypes are driven by specific molecular alterations. Several pangenomic studies have focused on delineating genomic changes and gene expression in the various stages of bladder cancer development and progression. Recently, molecular classifications have emerged from high-throughput analyses (7–11). Specific molecular pathways and potential new target genes not previously described in bladder carcinogenesis have been identified (12–15). Alterations in coding regions are now being described in bladder cancer as in various other tumor types (16, 17). However, exome represents only 1% to 2% of the human genome. Noncoding DNA is still largely unexplored while representing the large majority of the genome. Thus, few whole-genome sequencing analyses have been recently performed in order to identify alterations in noncoding sequences (18–21). Hotspots or regions emerged as highly mutated along all the genome: promoter sequences like TERT, long noncoding RNA (long ncRNA) such as NEAT1 or MALAT1, and untranslated regions (UTR) such as NOTCH1. Consequences of these mutations on the expression of corresponding mRNA or protein translation remain (except for TERT promoter mutations) unclear. Characterization of the involvement of these alterations in carcinogenesis has not yet been completely explored.

GPR126 is one of the most remarkable targets, because it is one of the major genes showing frequent somatic noncoding mutations identified with a frequency of 2.7% in a recent whole-genome sequencing study of 560 breast cancers (22). This gene, also named ADGRG6, is located on 6q24.2. It encodes for the G Protein-Coupled Receptor 126, which is involved in adult height. Somatic mutations are two hotspots of single nucleotide in intron 6 of GPR126 gene (NM_198569.2): substitutions that affect guanine at genomic position chr6:142706206 and cytosine at genomic position chr6:142706209 from Homo sapiens genome assembly GRCh37 (hg19) (or c.1222+1226 and c1222+1229 from NM_198569.2). Interestingly, these two mutated bases, within a 4 bp core motif, are flanked on either side by a stretch of 11 bp of palindromic sequence forming inverted repeats (Fig. 1). This gene has been identified as one of the most mutated in noncoding region in breast cancers, but data do not exist for other types of cancer.

Figure 1.

Representation of the two hotspots of mutation in the intron 6 of GPR126. Flanking regions from both sides of the hotspots are palindromic.

Figure 1.

Representation of the two hotspots of mutation in the intron 6 of GPR126. Flanking regions from both sides of the hotspots are palindromic.

Close modal

Interestingly, among 52 normal tissues, bladder tissue is one of the three tissues (with the liver tissue and the placenta) that most express the human GPR126 gene in the database available from GTEx Consortium (23). This gene is also highly expressed in bladder and liver cancers, as compared with the other cancer types from the data sets of cBioPortal (24), suggesting physiologic and pathological roles in these tissues. However, this pathologic role in cancer cells is still unknown.

Thus in the present study, we analyzed these intronic mutations of this gene in a large series of 103 bladder cancers, including 44 NMIBC and 59 MIBC. The impact of mutations on the expression of GPR126 has been evaluated, together with the putative prognostic value of these mutations and expressions in this cancer type.

Patients and samples

The series consisted of 103 patients who had undergone transurethral bladder resection or a radical cystectomy in our hospital—Cochin hospital, Paris, France—between January 2002 and January 2007. All patients signed a written-informed consent. This study received approval from an Institutional Review Board and was conducted according to the principles outlined in the Declaration of Helsinki.

Immediately after surgery, tumor samples from each patient were frozen in liquid nitrogen and stored at −80°C (for DNA and RNA extraction). Each tumor was reviewed by two pathologists (M. Sibony and D. Damotte) blinded to the clinical outcomes. Tumors were restaged according to the 2009 TNM classification of bladder tumors (25) and were graded according to the WHO 2016 tumor-grading scheme. Standard follow-up visits were done according to current guidelines. Data were obtained from the patients' medical records. Complete clinical, histologic, and survival information were available for the series.

Specimens of normal bladder tissue from 18 patients undergoing surgery unrelated to bladder tumors (transurethral resection of prostate, prostatic adenectomy) were used as sources of normal bladder tissues.

DNA mutation analysis

The assessment of the two GPR126 noncoding mutational hotspots (as well as the PIK3CA protein-coding mutational hotspots) was performed by a screening using high-resolution melting (HRM) followed by Sanger sequencing of the samples with an altered profile in HRM, in order to validate the HRM data and determine the nomenclature of mutations characterized. Analysis of FGFR3 most frequent point mutations was performed by PCR-SNaPshot method as described previously (26).

The nucleotide sequences of the primers used were as follows: GPR126: F: 5′-CCAGTGCATATTTCACATGGACTCT-3′ and R: 5′-CAGTAGAGGATGTGTCAATCCTGGA-3′ (PCR product of 134 bp). For PIK3CA HRM: F: 5′-CAGCTCAAAGCAATTTCTACACGA-3′ R: 5′-TCCATTTTAGCACTTACCTGTGACTC-3′ (exon 9; 94 bp) and F: 5′-AGCAAGAGGCTTTGGAGTATTTCAT-3′ R: 5′-GTGGAAGATCCAATCCATTTTTGTT-3′ (exon 20; 86 bp), and for PIK3CA sequencing: F: 5′-TGTATTTGCTTTTTCTGTAAATCAT-3′, R: 5′-CATTTTAGCACTTACCTGTGACTC-3′ (exon 9; 229 bp), and F: 5′-AGCATGCCAATCTCTTCATAAATCTT-3′, R: 5′-CAGTGTGGAATCCAGAGTGAGCTT-3′ (exon 20; 274 bp). For FGFR3 multiplex PCR: F: 5′-AGTGGCGGTGGTGGTGAGGGAG-3′, R: 5′-GCACCGCCGTCTGGTTGG-3′ (exon 7; 115 bp), F: 5′-CAACGCCCATGTCTTTGCAG-3′, R: 5′-AGGCGGCAGAGCGTCACAG-3′ (exon 10; 138 bp), F: 5′-GACCGAGGACAACGTGATG-3′, R: 5′-GTGTGGGAAGGCGGTGTTG-3′ (exon 15, 160 bp) and for FGFR3 SNaPshot: 5′-T46CGTCATCTGCCCCCACAGAG-3′ (R248C), 5′-T36TCTGCCCCCACAGAGCGCT-3′ and 5′-T28TCTGCCCCCACAGAGCGCT-3′ (S249C), 5′-T29GGTGGAGGCTGACGAGGCG-3′ (G372C), 5′-T43ACGAGGCGGGCAGTGTGT-3′ (Y375C), 5′-T34CCTGTTCATCCTGGTGGTGG-3′ (A393E), 5′-T50GCACAACCTCGACTACTACAAG-3′ (K652E/Q), 5′-T20CACAACCTCGACTACTACAAGA-3′ (K652M/T).

Real-time quantitative RT-PCR

The theoretical basis of real-time quantitative RT-PCR, RNA extraction, cDNA synthesis, and PCR amplification conditions previously described in detail (27) is developed in Supplementary Text S1.

One endogenous RNA control gene was selected, namely TBP (NM_003194), which encodes the TATA box-binding protein. Each sample was normalized based on its TBP content. Results, expressed as N-fold differences in GPR126 gene expression relative to the TBP gene, and termed “NGPR126,” were determined as NGPR126 = 2ΔCtsample, where the ΔCt value of the sample was determined by subtracting the average Ct value of GPR126 gene from the average Ct value of the TBP gene. The NGPR126 values of the samples were subsequently normalized such that the median of the NGPR126 values for the 18 normal bladder tissues was 1. The NGPR126 values of three or more were considered as marked GPR126 gene overexpression in bladder tumor RNA samples.

The nucleotide sequences of the TBP and GPR126 primers used were as follows: GPR126-F: 5′-CCATTGGAAATGGAAGCCCAGTC-3′, GPR126-R: 5′-AGTTGGCACATCCCCACACTGAGT-3′ for GPR126 gene (product of 88 bp) and TBP-F: 5′-TGCACAGGAGCCAAGAGTGAA-3′ and TBP-R: 5′- CACATCACAGCTCCCCACCA-3′ for TBP gene (product of 132 bp).

Statistical analysis

The clinicopathologic features of NMIBC and MIBC were tested for their association with tumor recurrence and survival using χ2 test for qualitative variables, χ2 test with Yates' correction, or Fisher test if appropriate.

The distribution of mRNA levels was analyzed using median values and ranges. Relationships between mutation profiles and mRNA levels of GPR126 gene expression and clinical histologic and biological parameters were tested using the nonparametric tests, namely the χ2 test (relation between two qualitative parameters) and the Mann–Whitney U test (relation between one qualitative parameter and one quantitative parameter).

For MIBC, overall survival (OS) was calculated from the date of surgery until death or the last follow-up. Recurrence-free survival (RFS) was defined as the time elapsed from the date of surgery until the first local relapse or first metastasis. For NMIBC, progression-free survival (PFS) was defined as the time elapsed from the date of surgery until progression to muscle-invasive disease. Patients were censored if they had not experienced the end-point of interest at the time of the last follow-up. Survival curves were derived from Kaplan–Meier estimates. The log-rank test was used to compare survival distributions between subgroups.

Differences were judged significant at a confidence level of >95% (P < 0.05).

Patients

There were 17 women and 86 men, with a median age of 67.6 years (range, 40–91). Pathologic staging showed NMIBC in 44 patients (20 low-grade Ta, 10 high-grade Ta, and 14 high-grade T1) and high-grade MIBC in 59 patients. For NMIBC, the mean follow-up was 31 months (range, 1–158 months). Among the 44 cases of NMIBC, 22 (50%) had one or more recurrences of NMIBC during the follow-up. Progression to a muscle-invasive tumor was observed in 8 patients (18.2%). For MIBC, the mean follow-up was 29 months (range, 1–152 months). During the follow-up period, 31 patients (52.5%) died of bladder cancer, and 3 (5.1%) died from unrelated causes. Clinical, histologic, and survival characteristics of the series NMIBC and MIBC are presented in Tables 1 and 2, respectively.

Table 1.

Clinical, pathologic, and survival characteristics of the 44 NMIBC

Whole populationNo recurrenceRecurrenceMuscle-invasive progression
Number of patients (%)Number (%)Number (%)P valueaNumber (%)P valueb
Total population 44 (100.0) 14 (31.8) 22 (50.0)  8 (18.2)  
Age (years) 
 ≥60 34 (77.3) 10 (29.4) 16 (47.1) 0.77 8 (23.5) 0.17 
 <60 10 (22.7) 4 (40.0) 6 (60.0)  0 (0.0)  
Sex 
 Male 41 (93.2) 13 (31.7) 20 (48.8) >0.99 8 (19.5) >0.99 
 Female 3 (6.8) 1 (33.3) 2 (66.7)  0 (0.0)  
Smoking status 
 Nonsmoker 19 (43.2) 4 (21.1) 12 (63.2) 0.13 3 (15.8) 0.97 
 Smoker 25 (56.8) 10 (40.0) 10 (40.0)  5 (20.0)  
History of NMIBC 
 No 24 (54.5) 11 (45.8) 10 (41.7) 0.049 3 (12.5) 0.50 
 Yes 20 (45.5) 3 (15.0) 12 (60.0)  5 (25.0)  
Associated pTis 
 No 42 (95.4) 14 (33.3) 22 (52.4) NAc 6 (14.3) 0.030 
 Yes 2 (4.6) 0 (0.0) 0 (0.0)  2 (100)  
Grade 
 Low grade 20 (45.5) 9 (45.0) 10 (50.0) 0.27 1 (5.0) 0.094 
 High grade 24 (54.5) 5 (20.8) 12 (50.0)  7 (29.2)  
Tumor stage 
 Ta 30 (68.2) 10 (33.3) 18 (60.0) 0.75 2 (6.7) 0.008 
 T1 14 (31.8) 4 (28.6) 4 (28.6)  6 (42.9)  
FGFR3 status 
 Mutated 22 (50.0) 5 (22.7) 16 (72.7) 0.028 1 (4.5) 0.051 
 Not mutated 22 (50.0) 9 (40.9) 6 (27.3)  7 (31.8)  
PIK3CA status 
 Mutated 7 (15.9) 4 (57.1) 2 (28.6) 0.18 1 (14.3) >0.99 
 Not mutated 37 (84.1) 10 (27.0) 20 (54.1)  7 (18.9)  
Whole populationNo recurrenceRecurrenceMuscle-invasive progression
Number of patients (%)Number (%)Number (%)P valueaNumber (%)P valueb
Total population 44 (100.0) 14 (31.8) 22 (50.0)  8 (18.2)  
Age (years) 
 ≥60 34 (77.3) 10 (29.4) 16 (47.1) 0.77 8 (23.5) 0.17 
 <60 10 (22.7) 4 (40.0) 6 (60.0)  0 (0.0)  
Sex 
 Male 41 (93.2) 13 (31.7) 20 (48.8) >0.99 8 (19.5) >0.99 
 Female 3 (6.8) 1 (33.3) 2 (66.7)  0 (0.0)  
Smoking status 
 Nonsmoker 19 (43.2) 4 (21.1) 12 (63.2) 0.13 3 (15.8) 0.97 
 Smoker 25 (56.8) 10 (40.0) 10 (40.0)  5 (20.0)  
History of NMIBC 
 No 24 (54.5) 11 (45.8) 10 (41.7) 0.049 3 (12.5) 0.50 
 Yes 20 (45.5) 3 (15.0) 12 (60.0)  5 (25.0)  
Associated pTis 
 No 42 (95.4) 14 (33.3) 22 (52.4) NAc 6 (14.3) 0.030 
 Yes 2 (4.6) 0 (0.0) 0 (0.0)  2 (100)  
Grade 
 Low grade 20 (45.5) 9 (45.0) 10 (50.0) 0.27 1 (5.0) 0.094 
 High grade 24 (54.5) 5 (20.8) 12 (50.0)  7 (29.2)  
Tumor stage 
 Ta 30 (68.2) 10 (33.3) 18 (60.0) 0.75 2 (6.7) 0.008 
 T1 14 (31.8) 4 (28.6) 4 (28.6)  6 (42.9)  
FGFR3 status 
 Mutated 22 (50.0) 5 (22.7) 16 (72.7) 0.028 1 (4.5) 0.051 
 Not mutated 22 (50.0) 9 (40.9) 6 (27.3)  7 (31.8)  
PIK3CA status 
 Mutated 7 (15.9) 4 (57.1) 2 (28.6) 0.18 1 (14.3) >0.99 
 Not mutated 37 (84.1) 10 (27.0) 20 (54.1)  7 (18.9)  

NOTE: P < 0.05 are in bold.

aχ2 test, χ2 test with Yates' correction, or Fisher test if appropriate (recurrence vs. no recurrence).

bχ2 test, Yates's χ2 test, or Fisher test if appropriate (muscle-invasive progression vs. others).

cNot applicable.

Table 2.

Clinical, pathologic, and survival characteristics of the 59 MIBC

Whole populationRFSOS
Number of patients (%)Number of events (%)aP valuebNumber of events (%)cP valueb
Total population 59 (100.0) 39 (66.1)  34 (57.6)  
Age (years) 
 ≥60 43 (72.8) 33 (76.7) 0.0046 30 (69.8) 0.002 
 <60 16 (27.2) 6 (37.5)  4 (25.0)  
Sex 
 Male 45 (76.3) 27 (60.0) 0.15 26 (57.8) 0.97 
 Female 14 (23.7) 12 (85.7)  8 (57.1)  
Smoking statusd 
 Nonsmoker 9 (17.0) 7 (77.8) 0.50 7 (77.8) 0.25 
 Smoker 44 (83.0) 26 (59.1)  22 (50.0)  
History of NMIBC 
 No 35 (59.3) 21 (60.0) 0.23 20 (57.1) 0.93 
 Yes 24 (40.7) 18 (75.0)  14 (58.3)  
Associated pTis 
 No 52 (88.1) 36 (69.2) 0.21 32 (61.5) 0.12 
 Yes 7 (11.9) 3 (42.9)  2 (28.6)  
Tumor stage 
 T2 21 (35.6) 13 (61.9) 0.61 8 (38.1) 0.024 
 ≥T3 38 (64.4) 26 (68.4)  26 (68.4)  
Lymph node statuse 
 N− 37 (64.9) 20 (54.1) 0.019 17 (45.9) 0.004 
 N+ 20 (35.1) 17 (85.0)  17 (85.0)  
FGFR3 status 
 Mutated 6 (10.2) 4 (66.7) >0.99 4 (66.7) >0.99 
 Not mutated 53 (89.9) 35 (66.0)  30 (56.6)  
PIK3CA status 
 Mutated 6 (10.2) 2 (33.3) 0.17 2 (33.3) 0.39 
 Not mutated 53 (89.9) 37 (69.8)  32 (60.4)  
Whole populationRFSOS
Number of patients (%)Number of events (%)aP valuebNumber of events (%)cP valueb
Total population 59 (100.0) 39 (66.1)  34 (57.6)  
Age (years) 
 ≥60 43 (72.8) 33 (76.7) 0.0046 30 (69.8) 0.002 
 <60 16 (27.2) 6 (37.5)  4 (25.0)  
Sex 
 Male 45 (76.3) 27 (60.0) 0.15 26 (57.8) 0.97 
 Female 14 (23.7) 12 (85.7)  8 (57.1)  
Smoking statusd 
 Nonsmoker 9 (17.0) 7 (77.8) 0.50 7 (77.8) 0.25 
 Smoker 44 (83.0) 26 (59.1)  22 (50.0)  
History of NMIBC 
 No 35 (59.3) 21 (60.0) 0.23 20 (57.1) 0.93 
 Yes 24 (40.7) 18 (75.0)  14 (58.3)  
Associated pTis 
 No 52 (88.1) 36 (69.2) 0.21 32 (61.5) 0.12 
 Yes 7 (11.9) 3 (42.9)  2 (28.6)  
Tumor stage 
 T2 21 (35.6) 13 (61.9) 0.61 8 (38.1) 0.024 
 ≥T3 38 (64.4) 26 (68.4)  26 (68.4)  
Lymph node statuse 
 N− 37 (64.9) 20 (54.1) 0.019 17 (45.9) 0.004 
 N+ 20 (35.1) 17 (85.0)  17 (85.0)  
FGFR3 status 
 Mutated 6 (10.2) 4 (66.7) >0.99 4 (66.7) >0.99 
 Not mutated 53 (89.9) 35 (66.0)  30 (56.6)  
PIK3CA status 
 Mutated 6 (10.2) 2 (33.3) 0.17 2 (33.3) 0.39 
 Not mutated 53 (89.9) 37 (69.8)  32 (60.4)  

NOTE: P < 0.05 are in bold.

aFirst recurrence (local or metastatic).

bχ2 test, χ2 test with Yates' correction, or Fisher test if appropriate.

cDeath.

dData available for 53 patients.

eData available for 57 patients.

DNA noncoding mutations of GPR126

In the global cohort, 45.6% (47/103) of the tumor samples had somatic mutations in intron 6 of GPR126. These GPR126 variants were absent in the gnomAD database gathering genomic data of 15,496 whole-genome sequences from unrelated individuals (assessed in April 2017; http://gnomad.broadinstitute.org/) confirming the somatic feature of these mutations.

No difference was observed between the two groups of tumor: 21 (47.7%) of the 44 NMIBC tumor samples and 26 (44.1%) of the 59 MIBC were GPR126 mutated (Table 3A). The intron 6 of GPR126 exhibited recurrent mutations at two genomic positions: position c.1222+1226 (G>A) and position c.1222+1229 (C>T; Fig. 1). In the NMIBC group, we found 12 (c.1222+1226 G>A), 5 (c.1222+1229 C>T), and 3 with the two substitutions. One tumor sample was altered in HRM but could not be identified with Sanger sequencing due to the lower sensitivity of this method. In MIBC group, we found 16 (c.1222+1226 G>A), 5 (1222+1229 C>T), and 5 with the two substitutions There was no difference between the distribution of GPR126 mutations in the two groups (P = 0.88).

Table 3A.

Mutations and overexpression frequency of GPR126 in bladder tumors

MutatedNot mutatedOverexpressionNo overexpression
GPR126 profileN (%)N (%)PaN (%)N (%)Pa
All tumors 47 (45.6) 56 (54.4)  47 (45.6) 56 (54.4)  
NMIBC 21 (47.7) 23 (52.3) 0.71 31 (70.5) 13 (29.5) 0.000013 
MIBC 26 (44.1) 33 (55.9)  16 (27.1) 43 (72.9)  
MutatedNot mutatedOverexpressionNo overexpression
GPR126 profileN (%)N (%)PaN (%)N (%)Pa
All tumors 47 (45.6) 56 (54.4)  47 (45.6) 56 (54.4)  
NMIBC 21 (47.7) 23 (52.3) 0.71 31 (70.5) 13 (29.5) 0.000013 
MIBC 26 (44.1) 33 (55.9)  16 (27.1) 43 (72.9)  

NOTE: P < 0.05 are in bold.

aχ2 test.

In the NMIBC group, GPR126 mutations were significantly overrepresented in the nonsmoker group (P = 0.017) and in patients with a prior history of NMIBC (P = 0.0069; Table 4). These mutations co-occurred with FGFR3 mutations (P = 0.035), but not with PIK3CA mutations. RFS and PFS were not influenced by the GPR126 mutation status (data not showed).

Surprisingly, in the MIBC group, GPR126 mutations affected significantly more females than males (P = 0.0029; Table 5). No other association was observed between GPR126 mutation status and other clinical pathologic and biological parameters (Table 5), as well as RFS and OS (data not showed).

In the total population, pooling both MIBC and NMIBC, no additional correlation (Supplementary Table S1) nor survival difference was observed (data not shown).

mRNA expression of GPR126

In the global cohort, 45.6% (47/103) bladder tumors showed GPR126 overexpression (≥3-fold above median for normal bladder samples). GPR126 overexpression was more frequent in NMIBC group (70.5%; 31/44) than in MIBC group (27.1%; 16/59; P = 0.000013; Table 3A).

In the NMIBC group, GPR126 mRNA levels were higher in the GPR126 nonmutated group than in the GPR126-mutated group, with median RNA values of 4.75 and 3.09, respectively (P = 0.021; Table 3B,Table 4,Table 5). GPR126 overexpression status was associated neither with clinical pathologic and biological parameters (Supplementary Table S2), nor with RFS and PFS (data not showed).

Table 3B.

Relationships between GPR126 mutations and mRNA levels in bladder tumors

GPR126-normalized expression vs. normal samples
NMedianPa
NMIBC total 44 4.02 (1.16–16.9)  
NMIBC wt 23 4.75 (1.16–16.9) 0.021 
NMIBC mutated 21 3.09 (1.32–9.75)  
MIBC total 59 1.17 (0.01–21.2)  
MIBC wt 33 1.22 (0.07–21.1) 0.92 
MIBC mutated 26 1.09 (0.01–9.26)  
GPR126-normalized expression vs. normal samples
NMedianPa
NMIBC total 44 4.02 (1.16–16.9)  
NMIBC wt 23 4.75 (1.16–16.9) 0.021 
NMIBC mutated 21 3.09 (1.32–9.75)  
MIBC total 59 1.17 (0.01–21.2)  
MIBC wt 33 1.22 (0.07–21.1) 0.92 
MIBC mutated 26 1.09 (0.01–9.26)  

NOTE: P < 0.05 are in bold.

aMann–Whitney test.

Table 4.

Relationship between GPR126 mutation status and standard clinical, pathologic, and biological parameters in NMIBC

GPR126 mutatedGPR126 not mutated
Total population (%)N (%)N (%)Pa
Total population 44 (100.0) 21 (47.7) 23 (52.3)  
Age (years) 
 ≥60 34 (77.3) 19 (55.9) 15 (44.1) 0.10 
 <60 10 (22.7) 2 (20.0) 8 (80.0)  
Sex 
 Male 41 (93.2) 18 (43.9) 23 (56.1) 0.10 
 Female 3 (6.8) 3 (100.0) 0 (0.0)  
Smoking status 
 Nonsmoker 19 (43.2) 13 (68.4) 6 (31.6) 0.017 
 Smoker 25 (56.8) 8 (32.0) 17 (68.0)  
History of NMIBC 
 No 24 (54.5) 7 (29.2) 17 (70.8) 0.0069 
 Yes 20 (45.5) 14 (70.0) 6 (30.0)  
Associated pTis 
 No 42 (95.4) 20 (47.6) 22 (52.4) >0.99 
 Yes 2 (4.6) 1 (50.0) 1 (50.0)  
Grade 
 Low grade 20 (45.5) 8 (40.0) 12 (60.0) 0.35 
 High grade 24 (54.5) 13 (54.2) 11 (45.8)  
Tumor stage 
 Ta 30 (68.2) 14 (46.7) 16 (53.3) 0.84 
 T1 14 (31.8) 7 (50.0) 7 (50.0)  
FGFR3 status 
 Mutated 22 (50.0) 14 (63.6) 8 (36.4) 0.035 
 Not mutated 22 (50.0) 7 (31.8) 15 (68.2)  
PIK3CA status 
 Mutated 7 (15.9) 5 (71.4) 2 (28.6) 0.34 
 Not mutated 37 (84.1) 16 (43.2) 21 (56.8)  
GPR126 mutatedGPR126 not mutated
Total population (%)N (%)N (%)Pa
Total population 44 (100.0) 21 (47.7) 23 (52.3)  
Age (years) 
 ≥60 34 (77.3) 19 (55.9) 15 (44.1) 0.10 
 <60 10 (22.7) 2 (20.0) 8 (80.0)  
Sex 
 Male 41 (93.2) 18 (43.9) 23 (56.1) 0.10 
 Female 3 (6.8) 3 (100.0) 0 (0.0)  
Smoking status 
 Nonsmoker 19 (43.2) 13 (68.4) 6 (31.6) 0.017 
 Smoker 25 (56.8) 8 (32.0) 17 (68.0)  
History of NMIBC 
 No 24 (54.5) 7 (29.2) 17 (70.8) 0.0069 
 Yes 20 (45.5) 14 (70.0) 6 (30.0)  
Associated pTis 
 No 42 (95.4) 20 (47.6) 22 (52.4) >0.99 
 Yes 2 (4.6) 1 (50.0) 1 (50.0)  
Grade 
 Low grade 20 (45.5) 8 (40.0) 12 (60.0) 0.35 
 High grade 24 (54.5) 13 (54.2) 11 (45.8)  
Tumor stage 
 Ta 30 (68.2) 14 (46.7) 16 (53.3) 0.84 
 T1 14 (31.8) 7 (50.0) 7 (50.0)  
FGFR3 status 
 Mutated 22 (50.0) 14 (63.6) 8 (36.4) 0.035 
 Not mutated 22 (50.0) 7 (31.8) 15 (68.2)  
PIK3CA status 
 Mutated 7 (15.9) 5 (71.4) 2 (28.6) 0.34 
 Not mutated 37 (84.1) 16 (43.2) 21 (56.8)  

NOTE: P < 0.05 are in bold.

aχ2 test, χ2 test with Yates' correction, or Fisher test if appropriate.

Table 5.

Relationship between GPR126 mutation status and standard clinical, pathologic, and biological parameters in MIBC

GPR126 mutatedGPR126 not mutated
Total population (%)N (%)N (%)Pa
Total population 59 (100.0) 26 (44.1) 33 (55.9)  
Age (years) 
 ≥60 43 (72.9) 19 (44.2) 24 (55.8) 0.98 
 <60 16 (27.1) 7 (43.8) 9 (56.3)  
Sex 
 Male 45 (76.3) 15 (33.3) 30 (66.7) 0.0029 
 Female 14 (23.7) 11 (78.6) 3 (21.4)  
Smoking statusb 
 Nonsmoker 9 (17.0) 4 (44.4) 5 (55.6) 0.85 
 Smoker 44 (83.0) 21 (47.7) 23 (52.3)  
History of NMIBC 
 No 35 (59.3) 13 (37.1) 22 (62.9) 0.20 
 Yes 24 (40.7) 13 (54.2) 11 (45.8)  
Associated pTis 
 No 52 (88.1) 22 (42.3) 30 (57.7) 0.74 
 Yes 7 (11.9) 4 (57.1) 3 (42.9)  
Tumor stage 
 T2 21 (35.6) 9 (42.9) 12 (57.1) 0.89 
 ≥T3 38 (64.4) 17 (44.7) 21 (55.3)  
Lymph node statusc 
 N− 37 (64.9) 16 (43.2) 21 (56.8) 0.81 
 N+ 20 (35.1) 8 (40.0) 12 (60.0)  
FGFR3 status 
 Mutated 6 (10.2) 3 (50.0) 3 (50.0) >0.99 
 Not mutated 53 (89.9) 23 (43.4) 30 (56.6)  
PIK3CA status 
 Mutated 6 (10.2) 3 (50.0) 3 (50.0) >0.99 
 Not mutated 53 (89.9) 23 (43.4) 30 (56.6)  
GPR126 mutatedGPR126 not mutated
Total population (%)N (%)N (%)Pa
Total population 59 (100.0) 26 (44.1) 33 (55.9)  
Age (years) 
 ≥60 43 (72.9) 19 (44.2) 24 (55.8) 0.98 
 <60 16 (27.1) 7 (43.8) 9 (56.3)  
Sex 
 Male 45 (76.3) 15 (33.3) 30 (66.7) 0.0029 
 Female 14 (23.7) 11 (78.6) 3 (21.4)  
Smoking statusb 
 Nonsmoker 9 (17.0) 4 (44.4) 5 (55.6) 0.85 
 Smoker 44 (83.0) 21 (47.7) 23 (52.3)  
History of NMIBC 
 No 35 (59.3) 13 (37.1) 22 (62.9) 0.20 
 Yes 24 (40.7) 13 (54.2) 11 (45.8)  
Associated pTis 
 No 52 (88.1) 22 (42.3) 30 (57.7) 0.74 
 Yes 7 (11.9) 4 (57.1) 3 (42.9)  
Tumor stage 
 T2 21 (35.6) 9 (42.9) 12 (57.1) 0.89 
 ≥T3 38 (64.4) 17 (44.7) 21 (55.3)  
Lymph node statusc 
 N− 37 (64.9) 16 (43.2) 21 (56.8) 0.81 
 N+ 20 (35.1) 8 (40.0) 12 (60.0)  
FGFR3 status 
 Mutated 6 (10.2) 3 (50.0) 3 (50.0) >0.99 
 Not mutated 53 (89.9) 23 (43.4) 30 (56.6)  
PIK3CA status 
 Mutated 6 (10.2) 3 (50.0) 3 (50.0) >0.99 
 Not mutated 53 (89.9) 23 (43.4) 30 (56.6)  

NOTE: P < 0.05 are in bold.

aχ2 test, χ2 test with Yates' correction, or Fisher test if appropriate.

bData available for 53 patients.

cData available for 57 patients.

In the MIBC group, no difference was detected between the GPR126 mRNA level medians of the GPR126 nonmutated group and the GPR126-mutated group (Table 3B). GPR126 overexpression status was not associated with clinical pathologic and biological parameters (Supplementary Table S3), except occurring at a lower frequency in patients with positive lymph node (P = 0.04). RFS and OS of the 16 patients with GPR126-overexpressing tumors as compared with 43 patients with normal GPR126 expression showed a trend toward a difference for a better outcome: 10-year RFS 34.2% versus 9.2%; 10-year OS 50.5% versus 28.0% (Supplementary Fig. S1).

In the total population, pooling both MIBC and NMIBC, no additional correlation (Supplementary Table S4) nor survival difference was observed (data not shown).

Recently, whole-genome sequencing studies focused on noncoding regions (18, 20). Although these studies remain rare due to the cost and complexity of whole-genome analysis, they identified somatic alteration hotspots in various noncoding regions, including promoter, long ncRNA, UTR, and intronic sequences. These recurrent alterations can now be investigated by targeted techniques in various cancers. Our study focused on a noncoding mutational hotspot in the intron 6 of GPR126 gene, recently identified in a large series of 560 breast cancers with an observed frequency of 2.7% (22). We confirmed this frequency of GPR126 noncoding mutations in an independent series of breast tumor DNAs (4.9%; 17/350; unpublished data). No data concerning these GPR126 mutations were available for other types of cancers.

Little is known about the physiologic roles of GPR126. It is part of the adhesion G protein–coupled receptors family with 33 members. Gpr126 was identified to be essential for Schwann cells to initiate myelination through induction of key transcriptional factors (28). Deletion of Gpr126 in mice leads to mid-gestation embryonic lethality due to internal hemorrhaging and failures in cardiovascular development (29). Recently, Gpr126 was identified as a regulator of developmental and pathologic angiogenesis through modulation of VEGFR2 receptor signaling (30).

Our results show that GPR126 somatic mutations, within 2 hotspots separated by 3 bp, occur with a very high prevalence of 45.6% (47/103 samples) in our bladder cancer series which makes this mutational hotspot as the most frequent in our genome after TERT promoter hotspot (∼70%; ref. 31), with a rate similar to inactivating TP53 mutations (∼50%; ref. 31) in this cancer type. This GPR126 mutational prevalence of 45.6% in bladder cancer is very superior to its observed rate (3%–5%) in breast cancer (22). These GPR126 mutations were absent from 15,496 genomes publically available on the genome Aggregation Database (gnomAD; http://gnomad.broadinstitute.org/) confirming the somatic feature of these mutations.

Our data also showed a significant overexpression of GPR126 in the NMIBC group. In this specific subgroup of patients, tumors with the intronic GPR126 mutations showed a lower GPR126 mRNA level as compared with the GPR126 wild-type tumors, suggesting a possible repression of the overexpression effect. Further studies (in vitro analyses) are necessary to elucidate the genetic (or epigenetic) mechanism responsible for the putative dysregulation of GPR126 mRNA expression by these somatic GPR126 mutations.

The association with survival was tested depending on mutation and overexpression of GPR126 status. No significant effect was observed on PFS and RFS for the NMIBC group, and on OS and RFS for the MIBC group. However, a trend toward significance was observed in the MIBC patients: the group overexpressing GPR126 showed a better RFS (34.2% vs. 9.2% after 10 years) and a better OS (50.5% vs. 28.0% after 10 years). These observations remain to be confirmed on larger series of MIBC patients.

The main application of such a prevalent somatic mutation (∼50%) could be its use as a clinical biomarker in bladder cancer. For example, circulating tumor DNA (ctDNA) emerged as a promising biomarker in oncology, when the somatic alterations can be found in the plasma among the circulating cell-free DNA (32, 33). It could have diagnostic, prognostic, theranostic, and disease monitoring applications. For instance, in bladder cancers, in a study monitoring 12 patients for up to 20 years, ctDNA has been shown to be detectable both in urine and blood at early stage of the disease, even in patients with noninvasive disease (34). Moreover, high level of ctDNA in urine correlated with progressive and invasive diseases, compared with low levels in patients with recurrent and noninvasive diseases. Significantly higher levels of ctDNA were detected before disease progression occurred, suggesting ctDNA as an interesting tool for disease surveillance both in urine and blood. Urine appears as an interesting sample for bladder cancers, as urinary ctDNA originates from cell-free DNA in circulation passing through glomerular filtration, and also from cells shedding from genitourinary tract (35).

In the case of GPR126, this single mutational hotspot is very attractive for the development of an easy quantitative polymerase chain reaction (qPCR) or droplet digital PCR (ddPCR) tool which could be used for nearly half patients with bladder cancer. In particular, a direct application could be the monitoring of a treatment effect or the early detection of a relapse. Although being far less studied than exonic mutations, noncoding mutations have already been identified in plasma samples. For example, TERT promoter mutation hotspot has been detected in ctDNA in 46% (12/26) of patients in a series of metastatic bladder cancers. Intronic mutations of about 40 genes were detected in ctDNA in the same series (36).

The GPR126 mutations (unlike the FGFR3 mutations) occur indistinctly in NMIBC and MIBC (47.7% and 44.1%, respectively), and thus could be relevant as a diagnostic marker, with GPR126 mutations detected in ctDNA or in the urine being an additional argument for the diagnosis of malignancy. However, its potential to distinguish between malignant and benign tumors remains to be explored.

In conclusion, we report here the identification of the second most prevalent hotspot of somatic mutation in bladder cancer after TERT mutations, in intronic sequence of GPR126 gene. Direct clinical application could be as a biomarker of tumor burden in ctDNA.

No potential conflicts of interest were disclosed.

Conception and design: C. Le Goux, I. Bieche

Development of methodology: N. Sirab, D. Damotte, I. Bieche

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Garinet, G. Pignot, S. Vacher, C. Le Goux, N.B. Delongchamps, M. Sibony, Y. Allory, I. Bieche

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Garinet, S. Vacher, A. Schnitzler, W. Chemlali, D. Damotte, I. Bieche

Writing, review, and/or revision of the manuscript: S. Garinet, G. Pignot, S. Vacher, N. Sirab, Y. Allory, I. Bieche

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Sibony, I. Bieche

Study supervision: M. Zerbib, I. Bieche

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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