Radical radiotherapy and surgery achieve similar cure rates in muscle-invasive bladder cancer, but the choice of which treatment would be most beneficial cannot currently be predicted for individual patients. The primary aim of this study was to assess whether expression of any of a panel of DNA damage signaling proteins in tumor samples taken before irradiation could be used as a predictive marker of radiotherapy response, or rather was prognostic. Protein expression of MRE11, RAD50, NBS1, ATM, and H2AX was studied by immunohistochemistry in pretreatment tumor specimens from two cohorts of bladder cancer patients (validation cohort prospectively acquired) treated with radical radiotherapy and one cohort of cystectomy patients. In the radiotherapy test cohort (n = 86), low tumor MRE11 expression was associated with worse cancer-specific survival compared with high expression [43.1% versus 68.7% 3-year cause-specific survival (CSS), P = 0.012] by Kaplan-Meier analysis. This was confirmed in the radiotherapy validation cohort (n = 93; 43.0% versus 71.2%, P = 0.020). However, in the cystectomy cohort (n = 88), MRE11 expression was not associated with cancer-specific survival, commensurate with MRE11 being a predictive marker. High MRE11 expression in the combined radiotherapy cohort had a significantly better cancer-specific survival compared with the high-expression cystectomy cohort (69.9% versus 53.8% 3-year CSS, P = 0.021). In this validated immunohistochemistry study, MRE11 protein expression was shown and confirmed as a predictive factor associated with survival following bladder cancer radiotherapy, justifying its inclusion in subsequent trial designs. MRE11 expression may ultimately allow patient selection for radiotherapy or cystectomy, thus improving overall cure rates. Cancer Res; 70(18); 7017–26. ©2010 AACR.

Bladder cancer is the fifth most common cancer in the United Kingdom (1). Radical radiotherapy and surgical removal of the bladder (cystectomy) achieve similar cure rates in muscle-invasive disease (2), but they have never been compared in a randomized controlled trial. Recently in the United Kingdom, the SPARE selective bladder preservation trial feasibility study showed the significant barriers to recruitment encountered when attempting such a randomization (3). Treatment choices are thus largely governed by patient preferences and physician bias, as there are currently no means of predicting treatment response and subsequent survival (4). If predictive markers could be identified, patients might then be selected for the treatment most likely to benefit them personally, which would have the added advantage of maximizing overall cure rates.

DNA double-strand breaks (DSB) are the most lethal form of ionizing radiation–induced DNA damage. Failure to repair such breaks results in tumor cell death. Immediately following cellular exposure to ionizing radiation, the damage is detected by the MRE11-RAD50-NBS1 (MRN) complex, resulting in rapid recruitment of signaling and repair proteins and alteration of chromatin structure, including histone modifications, to permit protein access to the DNA (5). MRE11/RAD50 tethers broken DNA ends and NBS1 recruits ATM (6). On activation, ATM phosphorylates the histone H2AX, thus promoting DSB repair and amplifying DSB signaling (7), and it phosphorylates p53, NBS1, CHK2, and other proteins to activate cell cycle checkpoints (6). MRE11 is also involved in DNA end resection during DSB repair. If damage is overwhelming, however, the cell may die by apoptosis or senesce (7). Therefore, we hypothesized that low tumor expression of DNA DSB signaling proteins would be associated with better outcome following radical radiotherapy in bladder cancer due to decreased DNA repair. However, we would not expect it to be related to outcome following surgery, as the treatment efficacy of surgery is not mediated via DNA damage mechanisms. Data are sparse regarding the expression of these proteins in relation to radiotherapy outcomes. Arlehag and colleagues (8) found no association for ATM expression in colorectal cancer, but surprisingly, low expression of MRN complex proteins was associated with a worse radiotherapy response in breast cancer (9).

Approximately 50% of human cancers have TP53 mutations (10). More than 80% are in exons 5 to 8 (coding the p53 DNA binding domain); more than 75% of these are missense mutations. Both TP53 mutations and p53 protein expression by immunohistochemistry have been studied in tumors clinically, but no clear association has been shown between the two (11) and the literature on tumor p53 immunohistochemistry expression and radiotherapy outcomes is conflicting (1118).

In this study, we assessed protein expression by immunohistochemistry for ATM, MRE11, RAD50, NBS1, and H2AX in a cohort of bladder cancer patients treated with radical radiotherapy in a single institution from 1995 to 2000, and validated our results in a second cohort treated similarly from 2002 to 2005. We also studied TP53 mutations in exons 5 to 8. Having successfully validated the association between MRE11 expression and cause-specific survival (CSS) following radiotherapy, we used the cystectomy cohort from 1996 to 2005 from the same institution to establish whether the assay was prognostic for disease outcome or predictive of response to radiotherapy.

Ethical approval was obtained from Leeds (East) Local Research Ethical Committee (studies 02/060, 02/192, and 04/Q1206/62).

Study population

We studied two cohorts of patients, cohort A (1995–2000) and cohort B (2002–2005), treated with radical radiotherapy for transitional cell carcinoma of the bladder at the Leeds Cancer Centre, West Yorkshire, United Kingdom, and one cohort of patients treated with radical cystectomy at the Leeds Teaching Hospitals NHS Trust (1995–2005). Cohort B patients were prospectively recruited in clinic and gave informed consent for use of their tissue. Their outcome data were prospectively collected in clinic by use of a standard proforma. Details of radiotherapy treatments and cohort A patients have been described previously (19). Cohort A patients received 55 Gy in 20 fractions over 4 weeks using a computed tomography–planned three- or four-field two-dimensional cylinder technique, whereas for cohort B patients, a three-dimensional conformal technique was used and 11% of patients received additional treatments (Table 1). Details of the cystectomy technique have been described previously (2).

Table 1.

Demographics of test cohort A (1995–2000) and validation cohort B (2002–2005)

VariableTest set (1995–2000), no. of patients* (%)Validation set (2002–2005), no. of patients (%)Cystectomy set (1995–2005), no. of patients (%)P
Age (y) 
    Median (range) 75 (42–92) 77 (55–89) 68 (43–79) <0.001 
Gender 
    Male 65 (74.7) 72 (77.4) 66 (75.0) 0.90 
    Female 22 (25.3) 21 (22.6) 22 (25.0)  
Tumor stage 
    T1 3 (3.4) 3 (3.2) 0 (0) 0.34 
    T2 41 (47.1) 47 (50.5) 46 (52.3)  
    T3 36 (41.5) 31 (33.4) 28 (31.8)  
    T4 6 (6.9) 8 (8.6) 13 (14.8)  
    Tx 1 (1.1) 4 (4.3) 1 (1.1)  
Nodal class 
    N0 76 (87.4) 85 (91.4) 77 (87.5) 0.24 
    N+ 4 (4.6) 4 (4.3) 9 (10.2)  
    Nx 7 (8.0) 4 (4.3) 2 (2.3)  
Histologic grade§ 
    G3 73 (84.0) 79 (84.9) 80 (90.9) 0.45 
    <G3 13 (14.9) 10 (10.8) 8 (9.1)  
    Gx 1 (1.1) 4 (4.3) 0 (0)  
Hydronephrosis 
    No 69 (79.3) 62 (66.6) 48 (54.5) 0.0002 
    Yes 13 (14.9) 29 (31.2) 40 (45.5)  
    Unknown 5 (5.8) 2 (2.2) 0 (0)  
Neoadjuvant/concurrent chemotherapy 
    No 87 (100) 83 (89.2) 82 (93.2)  
    Yes 0 (0) 10 (10.8) 6 (6.8)  
Salvage cystectomy 
    No 79 (90.8) 86 (92.5) Not applicable  
    Yes 8 (9.2) 7 (7.5)   
Chemotherapy 
    No 81 (93.1) 88 (94.6) 84 (95.5)  
    Yes 6 (6.9) 5 (5.4) 4 (4.5)  
VariableTest set (1995–2000), no. of patients* (%)Validation set (2002–2005), no. of patients (%)Cystectomy set (1995–2005), no. of patients (%)P
Age (y) 
    Median (range) 75 (42–92) 77 (55–89) 68 (43–79) <0.001 
Gender 
    Male 65 (74.7) 72 (77.4) 66 (75.0) 0.90 
    Female 22 (25.3) 21 (22.6) 22 (25.0)  
Tumor stage 
    T1 3 (3.4) 3 (3.2) 0 (0) 0.34 
    T2 41 (47.1) 47 (50.5) 46 (52.3)  
    T3 36 (41.5) 31 (33.4) 28 (31.8)  
    T4 6 (6.9) 8 (8.6) 13 (14.8)  
    Tx 1 (1.1) 4 (4.3) 1 (1.1)  
Nodal class 
    N0 76 (87.4) 85 (91.4) 77 (87.5) 0.24 
    N+ 4 (4.6) 4 (4.3) 9 (10.2)  
    Nx 7 (8.0) 4 (4.3) 2 (2.3)  
Histologic grade§ 
    G3 73 (84.0) 79 (84.9) 80 (90.9) 0.45 
    <G3 13 (14.9) 10 (10.8) 8 (9.1)  
    Gx 1 (1.1) 4 (4.3) 0 (0)  
Hydronephrosis 
    No 69 (79.3) 62 (66.6) 48 (54.5) 0.0002 
    Yes 13 (14.9) 29 (31.2) 40 (45.5)  
    Unknown 5 (5.8) 2 (2.2) 0 (0)  
Neoadjuvant/concurrent chemotherapy 
    No 87 (100) 83 (89.2) 82 (93.2)  
    Yes 0 (0) 10 (10.8) 6 (6.8)  
Salvage cystectomy 
    No 79 (90.8) 86 (92.5) Not applicable  
    Yes 8 (9.2) 7 (7.5)   
Chemotherapy 
    No 81 (93.1) 88 (94.6) 84 (95.5)  
    Yes 6 (6.9) 5 (5.4) 4 (4.5)  

NOTE: P values are from Pearson's χ2 test unless otherwise stated.

*A comparison of the median and range of semiquantitative scores for the six proteins in the group of patients who had died of bladder cancer and those still alive at 3 y is shown in Supplementary Table S4.

Three patients in the validation set received neoadjuvant chemotherapy, two received concurrent carbogen and nicotinamide as part of a phase III clinical trial, and five patients received 100 mg/m2 of gemcitabine weekly ×4 concurrently with radiotherapy in a phase II clinical trial.

P value was from two-sided F test.

§All patients in cohort B had transitional cell carcinoma; four had some squamous elements and one had sarcomatoid differentiation.

Salvage chemotherapy for patients treated by radiotherapy and adjuvant chemotherapy for patients treated by surgery.

Formalin-fixed paraffin-embedded tumor samples taken at pretreatment transurethral resection of the bladder tumor (TURBT) were available in 91 cohort A, 93 cohort B, and 88 surgical cohort patients. For each patient, a H&E-stained section was reviewed by a consultant uropathologist and areas of invasive transitional cell carcinoma were outlined.

Immunohistochemistry of DNA damage signaling proteins and Ki67

Immunohistochemistry was undertaken using the standard streptavidin-biotin complex method. In brief, formalin-fixed paraffin-embedded sections (4 mm) were deparaffinized, rehydrated, and washed. Endogenous peroxidases were blocked using 3% hydrogen peroxide, followed by antigen retrieval in 10 mmol/L citrate buffer (pH 6.0) for 20 minutes. Slides were incubated with primary antibodies against MRE11 (1:150), RAD50 (1:100), NBS1 (1:2,000; ab214, ab89, and ab398, respectively, Abcam plc), Ki67 (1:400; Dako), and H2AX (1:700; Bethyl Labs) for 90 minutes at room temperature or with ATM antibody (1:50; Stratech) overnight at 4°C. Sections were incubated in biotinylated secondary antibody for 30 minutes, followed by streptavidin peroxidase (DakoCytomation) for a further 30 minutes. Bound antibodies were visualized using diaminobenzidine (DakoCytomation) and counterstained with hematoxylin.

Antibodies to MRE11, NBS1, RAD50, and ATM were titrated against the same formalin-fixed paraffin-embedded breast adenocarcinoma, the positive control for all subsequent experiments, including the cystectomy cohort samples, using a range of dilutions starting with the datasheet recommendation. The final dilution was chosen by two observers so that, on a scale of 1 to 3, the positive control scored 2. Normal urothelium was present in less than 15% of patients and could therefore not be used for internal reference purposes. Sections of normal tonsil were used for H2AX and Ki67 antibodies. The antibodies were omitted from the negative controls.

Digital images were captured from 6 to 10 random fields from within invasive tumor areas (×600 magnification) using the Olympus BX50 microscope and c-3030 camera, and 100 tumor cells were counted from each field as positively or negatively stained (by L.N. for cohort A, A.C. for cohort B, and M.T.W.T. for the cystectomy cohort). Five percent of sections from cohort A were scored by a second observer (A.C.) with comparable results. In addition, staining intensity (0–3; Fig. 1) was scored independently in a blinded manner by two observers (A.C. and A.E.K. for radiotherapy patients, and M.T.W.T. and A.E.K. for cystectomy patients), discordant scores (∼10%) were reviewed, and a consensus was reached. The median percentage of positive cells was multiplied by the modal intensity to give a semiquantitative score (SQS). Five percent of sections from cohort A were scored by a second observer (A.C.) with comparable results. Cohort A was used as a training set to establish cutoff algorithms, and the same cutoffs were used for the validation of cohort B and the surgical cohort (for MRE11 only).

Figure 1.

Kaplan-Meier CSS curves for (A) test, validation, and cystectomy cohorts; (B) MRE11 expression by quartile in test cohort A; (C) test cohort A comparing the groups above and below the 25th percentile (low indicates MRE11 expression below the 25th percentile, whereas high indicates MRE11 expression above this); (D) validation cohort B comparing the groups above and below the 25th percentile.

Figure 1.

Kaplan-Meier CSS curves for (A) test, validation, and cystectomy cohorts; (B) MRE11 expression by quartile in test cohort A; (C) test cohort A comparing the groups above and below the 25th percentile (low indicates MRE11 expression below the 25th percentile, whereas high indicates MRE11 expression above this); (D) validation cohort B comparing the groups above and below the 25th percentile.

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TP53 mutation detection and gene sequencing

Ten-micrometer-thick sections (one to three per patient) were stained with H&E, and areas containing at least 70% tumor cells were macrodissected for DNA extraction using the QIAamp DNA micro kit (Qiagen). Amplified DNA (primer sequences available on request) from TP53 exons 5 to 8 was screened for mutations by single-strand conformation polymorphism analysis. Briefly, PCR was carried out using FAM- or HEX-labeled primers. The products were diluted and subjected to capillary electrophoresis at 18°C and 30°C using a 3100 Genetic Analyzer (Applied Biosystems), in 5% GeneScan polymer (Applied Biosystems), 5% glycerol, 1× Tris-TAPS-EDTA buffer (Applied Biosystems). Data analysis was performed using GeneScan and Genotyper software (Applied Biosystems) and by visual inspection of electropherograms. Samples with possible mutations were sequenced using standard protocols and analyzed using ABI sequence analysis software by two independent observers.

Statistical analysis

Population demographics were compared among the three cohorts using Pearson χ2 tests and two-sided F tests. The outcome measure was survival time. In each cohort, Kaplan-Meier curves were plotted for CSS (deaths due to bladder cancer only, with other deaths censored) and the log-rank statistic was used to compare survival times across categories of MRE11 protein expression levels. Summary statistics of protein expression were calculated for the protein expression SQS and pairwise Spearman's rank correlations between markers. Cohort A scores were grouped into approximate quartiles. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated from Cox proportional hazard models adjusted for other model covariates, that is, age, hydronephrosis, grade, stage, high/low MRE11 expression, and TP53 mutation in the univariate model, and for the multivariate model, all these covariates except TP53 mutation (lowest quarter as the reference). Potentially interesting markers were then assessed in the same way for cohort B, and subsequently, MRE11 was assessed in the cystectomy cohort. Assuming high protein expression in 75% of samples and low expression in 25% of samples, with a 5% significance level, in a cohort of 88 patients, 60 CSS events would give an 87% power to detect a HR of 0.4, and 44 CSS events would give a 75% power to detect a HR of 0.4.

Expression of DSB signaling proteins

In cohort A, cohort B, and the cystectomy cohort, 87, 93, and 88 patient samples, respectively, had sufficient invasive tumor cells for analysis. Between cohorts A and B, the demographics of the study populations were similar (Table 1) despite the intervening 7 years, although the proportion with hydronephrosis [14.9% versus 31.2%; χ2(1) = 6.02, P = 0.018] was higher in cohort B. When all three cohorts were compared, the cystectomy cohort was significantly younger than cohorts A and B (median age, 68 versus 75 versus 77 years, respectively; P < 0.001) and a higher proportion had hydronephrosis [45.5% versus 14.9% versus 31.2%; χ2(2) = 17.27, P < 0.001]. This would be in keeping with the local practice of treating younger (hence fitter) patients and hydronephrosis patients with surgery. There was no significant difference in 3-year CSS among the three cohorts [61.8%, 65.1%, and 56.0%; χ2(2) = 1.52, P = 0.48; Fig. 1A]. The overall 3-year CSS of all three cohorts combined was 62.1%.

A range of nuclear expression was seen for all DNA damage signaling proteins in terms of the number of positive cells and staining intensity (relative to the positive controls; Table 2; Supplementary Fig. S1). In cohort A, dividing groups at the 25th, 50th, and 75th percentiles, there was no significant association between MRE11, NBS1, RAD50, ATM, H2AX, or Ki67 protein expression and CSS (Fig. 1B, MRE11; Table 2). However, inspection of the curves for cohort A showed visual differences between patients with values of MRE11 expression less than the 25th percentile and patients with values higher than the 25th percentile. Improved 3-year CSS was demonstrable in cohort A for patients whose tumors had high MRE11 expression (Fig. 1C; 68.7% if >25th versus 43.1% if <25th percentile, SQS cutoff point of 130 at 25th percentile; HR, 0.42; 95% CI, 0.21–0.84; P = 0.012). This was confirmed in cohort B with 3-year CSS of 71.2% if >25th and 43.0% if <25th percentile, with a SQS cutoff point of 76 at 25th percentile (Fig. 1D; HR, 0.43; 95% CI, 0.21–0.87; P = 0.020); for the combined A + B cohorts, 3-year CSS was 69.9% and 43%, respectively (Fig. 2A; HR, 0.43; 95% CI, 0.26–0.71; P < 0.001).

Table 2.

Test cohort A (1995–2000)

ProteinPercentage positive nuclei: median (range)Intensity: mode (range)Semiquantitative score: median (range)Died of bladder cancer at 3 y: number; median score (range)Alive at 3 y: number; median score (range)QuartileNo. of cases3-y CSSP
MRE11 91.2 (17.1–98.6) 2 (0–3) 182 (28–296) 31; 167 (48–294) 45; 186 (28–296) ≤25th 21 43.1 0.069 
25–50th 23 65.5 
50–75th 21 71.4 
≥75th 21 71.4 
NBS1 62.6 (6.0–93.2) 2 (0–3) 125 (6–280) 30; 122 (6–239) 45; 110 (11–280) ≤25th 21 57.1 0.76 
25–50th 22 68.2 
50–75th 22 67.2 
≥75th 21 61.8 
RAD50 95.3 (24.3–99.9) 2 (0–3) 193 (24–299) 31; 192 (84–297) 45; 191 (24–299) ≤25th 21 66.3 0.94 
25–50th 23 60.9 
50–75th 22 61.9 
≥75th 21 62.0 
ATM 85.2 (24.0–97.3) 2 (0–3) 174 (6–292) 30; 172 (48–285) 45; 170 (48–292) ≤25th 22 72.7 0.83 
25–50th 22 54.5 
50–75th 21 70.8 
≥75th 21 56.7 
H2AX 7.4 (1–60.0) 1 (0–3) 10 (1–149) 29; 16 (1–120) 45; 8 (1–149) ≤25th 20 79.2 0.49 
25–50th 21 65.5 
50–75th 23 63.6 
≥75th 21 50.8 
Ki67 27.8 (2.0–74.0)   30; 30 (2–74) 45; 26 (5–52) ≤25th 21 63.3 0.56 
25–50th 23 60.1 
50–75th 21 74.8 
≥75th 21 51.9 
ProteinPercentage positive nuclei: median (range)Intensity: mode (range)Semiquantitative score: median (range)Died of bladder cancer at 3 y: number; median score (range)Alive at 3 y: number; median score (range)QuartileNo. of cases3-y CSSP
MRE11 91.2 (17.1–98.6) 2 (0–3) 182 (28–296) 31; 167 (48–294) 45; 186 (28–296) ≤25th 21 43.1 0.069 
25–50th 23 65.5 
50–75th 21 71.4 
≥75th 21 71.4 
NBS1 62.6 (6.0–93.2) 2 (0–3) 125 (6–280) 30; 122 (6–239) 45; 110 (11–280) ≤25th 21 57.1 0.76 
25–50th 22 68.2 
50–75th 22 67.2 
≥75th 21 61.8 
RAD50 95.3 (24.3–99.9) 2 (0–3) 193 (24–299) 31; 192 (84–297) 45; 191 (24–299) ≤25th 21 66.3 0.94 
25–50th 23 60.9 
50–75th 22 61.9 
≥75th 21 62.0 
ATM 85.2 (24.0–97.3) 2 (0–3) 174 (6–292) 30; 172 (48–285) 45; 170 (48–292) ≤25th 22 72.7 0.83 
25–50th 22 54.5 
50–75th 21 70.8 
≥75th 21 56.7 
H2AX 7.4 (1–60.0) 1 (0–3) 10 (1–149) 29; 16 (1–120) 45; 8 (1–149) ≤25th 20 79.2 0.49 
25–50th 21 65.5 
50–75th 23 63.6 
≥75th 21 50.8 
Ki67 27.8 (2.0–74.0)   30; 30 (2–74) 45; 26 (5–52) ≤25th 21 63.3 0.56 
25–50th 23 60.1 
50–75th 21 74.8 
≥75th 21 51.9 

NOTE: The table shows the DNA damage signaling protein median percentage score, intensity, and SQS with ranges; median expression scores with ranges in patients who died of bladder cancer or were still alive at 3 y (Ki67 was scored as a percentage of positive nuclei alone); and associations of protein expression by quartile with CSS.

Figure 2.

MRE11 as a prognostic/predictive marker. Kaplan-Meier CSS curves for (A) combined cohort A and cohort B comparing the groups above and below the 25th percentile; (B) 1995–2005 cystectomy cohort comparing the groups above and below the 25th percentile; (C) MRE11 as a predictive factor: high MRE11 (>25%) in radiotherapy (RT) versus cystectomy patients; (D) MRE11 as a predictive factor: low MRE11 (≤25%) in radiotherapy versus cystectomy patients.

Figure 2.

MRE11 as a prognostic/predictive marker. Kaplan-Meier CSS curves for (A) combined cohort A and cohort B comparing the groups above and below the 25th percentile; (B) 1995–2005 cystectomy cohort comparing the groups above and below the 25th percentile; (C) MRE11 as a predictive factor: high MRE11 (>25%) in radiotherapy (RT) versus cystectomy patients; (D) MRE11 as a predictive factor: low MRE11 (≤25%) in radiotherapy versus cystectomy patients.

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When normal urothelium was present, normal/tumor differences in MRE11 expression levels were found in 8 of 9 (89%) of cases, with lower tumor expression in seven cases and higher tumor expression in one. By SQS, tumor MRE11 expression was significantly correlated with NBS1 expression (r = 0.22, P = 0.003), RAD50 and ATM were significantly correlated (r = 0.20, P = 0.008), and ATM and NBS1 were negatively correlated with Ki67 expression (r = −0.19, P = 0.010 and r = −0.23, P = 0.002 respectively). None of the other proteins correlated with Ki67 expression. There was no association between protein expression and stage or grade of tumor (data not shown).

In the cystectomy cohort, MRE11 expression did not significantly influence 3-year CSS (Fig. 2B; 53.8% for >25th percentile versus 62.2% for <25th percentile, SQS cutoff point of 58 at 25th percentile; HR, 1.30; 95% CI, 0.65–2.64; P = 0.46). MRE11 expression did not influence 3-year CSS when the cystectomy cohort was combined with cohort A or cohort B to have a balanced number of radiotherapy and cystectomy patients (n = 88 + 86 and n = 88 + 93, respectively; Supplementary Fig. S2). For individuals with high MRE11 expressing tumors, radiotherapy patients (cohort A + cohort B) had better 3-year CSS compared with cystectomy patients (Fig. 2C; 69.9% versus 53.8%; HR, 0.60; 95% CI, 0.39–0.93; P = 0.021). In individuals with low MRE11 expressing tumors, there was a nonsignificant poorer outcome in radiotherapy cases compared with cystectomy cases, but case numbers were small (44 radiotherapy, 22 cystectomy; Fig. 2D; 42.8% versus 62.2%; HR, 1.78; 95% CI, 0.84–3.76; P = 0.13). These results would be supportive of MRE11 being a predictive marker for radiotherapy response rather than a prognostic marker in bladder cancer.

TP53 mutation and radiotherapy outcome

Of the 160 bladder tumor samples tested for TP53 mutations, 66 had at least one TP53 mutation. Eighty-three mutations were found in total, with nine samples having more than one mutation; six were silent, three frameshift, one splice site, one intronic, and 72 missense. Dysfunctional mutations were defined as those with evidence of an in vitro functional effect (20). There were mutation clusters around bases 13203, 14060, and 14508 (Fig. 3A), with codons 175, 245, and 280 in exons 5, 7, and 8, respectively, being most commonly mutated (Fig. 3B). There was no significant difference in CSS between those patients having tumors with a predicted dysfunctional TP53 mutation and the remainder (data not shown).

Figure 3.

A, frequency and position of base mutations in TP53. B, frequency of mutations by codon position in exons 5 to 8 of p53.

Figure 3.

A, frequency and position of base mutations in TP53. B, frequency of mutations by codon position in exons 5 to 8 of p53.

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Multivariate analysis of predictors for CSS

Using a multivariate Cox proportional hazards analysis (Table 3), hydronephrosis was a significant prognostic factor in cohort A (P = 0.001), cohorts A and B combined (P = 0.003), and the cystectomy cohort (P < 0.001). In cohort A, MRE11 protein expression was a borderline significant independent predictor for CSS after treatment with radiotherapy (P = 0.076). However, this was confirmed as an independent predictor of CSS in cohort B (P = 0.010), with combined analysis of the two cohorts increasing the statistical significance of the MRE11 result (P < 0.001). In contrast, MRE11 was not found to be a significant independent predictor of CSS in the cystectomy cohort (P = 0.29).

Table 3.

Multivariate cause-specific analyses using Cox proportional hazards regression analysis to determine HRs and P values in the 1995–2000 and 2002–2005 radiotherapy data sets (cohorts A and B) and the 1995–2005 cystectomy data set

Category (87 cohort A, 93 cohort B, 88 cystectomy)Cohort ACohort BCombined A + BCystectomy
Univariate modelMultivariate modelUnivariate modelMultivariate modelUnivariate modelMultivariate modelUnivariate modelMultivariate model
HR (95% CI)HR (95% CI)HR (95% CI)HR (95% CI)HR (95% CI)HR (95% CI)HR (95% CI)HR (95% CI)
Age (y) 1.00 (0.96–1.03) 0.98 (0.95–1.02) 1.02 (0.98–1.07) 1.03 (0.98–1.08) 1.01 (0.98–1.04) 1.00 (0.98–1.03) 1.03 (0.99–1.08) 1.03 (0.99–1.07) 
(P = 0.96) (P = 0.38) (P = 0.36) (P = 0.27) (P = 0.63) (P = 0.77) (P = 0.12) (P = 0.77) 
Hydronephrosis 
    No (69, 62, 48)     
    Yes (13, 29, 40) 3.72 (1.75–7.87) 4.21 (1.74–10.15) 1.84 (0.91–3.69) 1.71 (0.78–3.78) 2.37 (1.43–3.94) 2.29 (1.33–3.94) 3.20 (1.73–5.90) 3.37 (1.79–6.36) 
(P < 0.001) (P = 0.0014) (P = 0.09) (P = 0.18) (P < 0.001) (P = 0.003) (P < 0.001) (P < 0.001) 
Grade 
    <G3 (13, 10, 8)     
    G3 (73, 79, 80) 1.00 (0.41–2.41) 1.26 (0.51–3.11) 1.20 (0.37–3.94) 1.15 (0.35–3.85) 1.05 (0.52–2.13) 1.10 (0.55–2.24) 2.79 (0.68–11.50) 2.68 (0.63–11.40) 
(P = 1.00) (P = 0.62) (P = 0.76) (P = 0.82) (P = 0.88) (P = 0.78) (P = 0.16) (P = 0.18) 
Tumor stage 
    1 + 2 (44, 50, 46)     
    3 + 4 (42, 39, 41) 1.32 (0.68–2.58) 1.26 (0.62–2.52) 2.08 (1.05–4.12) 1.96 (0.94–4.08) 1.66 (1.03–2.68) 1.60 (0.96–2.65) 1.96 (1.07–3.56) 1.63 (0.89–3.00) 
(P = 0.42) (P = 0.52) (P = 0.037) (P = 0.073) (P = 0.038) (P = 0.069) (P = 0.028) (P = 0.11) 
MRE11 
    Low (21, 23, 22)     
    High (65, 70, 66) 0.46 (0.22–0.97) 0.50 (0.23–1.08) 0.46 (0.22–0.96) 0.36 (0.17–0.78) 0.47 (0.28–0.78) 0.39 (0.23–0.66) 1.45 (0.70–3.02) 1.50 (0.70–3.20) 
(P = 0.042) (P = 0.076) (P = 0.037) (P = 0.010) (P = 0.004) (P < 0.001) (P = 0.32) (P = 0.29) 
Functional TP53 mutation 
    0 (57, 55)      
    1 (30, 18) 1.36 (0.70–2.63)  0.66 (0.27–1.63)  1.04 (0.62–1.74)    
  (P = 0.37)  (P = 0.37)  (P = 0.89)   
Category (87 cohort A, 93 cohort B, 88 cystectomy)Cohort ACohort BCombined A + BCystectomy
Univariate modelMultivariate modelUnivariate modelMultivariate modelUnivariate modelMultivariate modelUnivariate modelMultivariate model
HR (95% CI)HR (95% CI)HR (95% CI)HR (95% CI)HR (95% CI)HR (95% CI)HR (95% CI)HR (95% CI)
Age (y) 1.00 (0.96–1.03) 0.98 (0.95–1.02) 1.02 (0.98–1.07) 1.03 (0.98–1.08) 1.01 (0.98–1.04) 1.00 (0.98–1.03) 1.03 (0.99–1.08) 1.03 (0.99–1.07) 
(P = 0.96) (P = 0.38) (P = 0.36) (P = 0.27) (P = 0.63) (P = 0.77) (P = 0.12) (P = 0.77) 
Hydronephrosis 
    No (69, 62, 48)     
    Yes (13, 29, 40) 3.72 (1.75–7.87) 4.21 (1.74–10.15) 1.84 (0.91–3.69) 1.71 (0.78–3.78) 2.37 (1.43–3.94) 2.29 (1.33–3.94) 3.20 (1.73–5.90) 3.37 (1.79–6.36) 
(P < 0.001) (P = 0.0014) (P = 0.09) (P = 0.18) (P < 0.001) (P = 0.003) (P < 0.001) (P < 0.001) 
Grade 
    <G3 (13, 10, 8)     
    G3 (73, 79, 80) 1.00 (0.41–2.41) 1.26 (0.51–3.11) 1.20 (0.37–3.94) 1.15 (0.35–3.85) 1.05 (0.52–2.13) 1.10 (0.55–2.24) 2.79 (0.68–11.50) 2.68 (0.63–11.40) 
(P = 1.00) (P = 0.62) (P = 0.76) (P = 0.82) (P = 0.88) (P = 0.78) (P = 0.16) (P = 0.18) 
Tumor stage 
    1 + 2 (44, 50, 46)     
    3 + 4 (42, 39, 41) 1.32 (0.68–2.58) 1.26 (0.62–2.52) 2.08 (1.05–4.12) 1.96 (0.94–4.08) 1.66 (1.03–2.68) 1.60 (0.96–2.65) 1.96 (1.07–3.56) 1.63 (0.89–3.00) 
(P = 0.42) (P = 0.52) (P = 0.037) (P = 0.073) (P = 0.038) (P = 0.069) (P = 0.028) (P = 0.11) 
MRE11 
    Low (21, 23, 22)     
    High (65, 70, 66) 0.46 (0.22–0.97) 0.50 (0.23–1.08) 0.46 (0.22–0.96) 0.36 (0.17–0.78) 0.47 (0.28–0.78) 0.39 (0.23–0.66) 1.45 (0.70–3.02) 1.50 (0.70–3.20) 
(P = 0.042) (P = 0.076) (P = 0.037) (P = 0.010) (P = 0.004) (P < 0.001) (P = 0.32) (P = 0.29) 
Functional TP53 mutation 
    0 (57, 55)      
    1 (30, 18) 1.36 (0.70–2.63)  0.66 (0.27–1.63)  1.04 (0.62–1.74)    
  (P = 0.37)  (P = 0.37)  (P = 0.89)   

NOTE: Numbers in boldface represent significance at the 5% level.

To our knowledge, this is the first study to have investigated the DSB signaling proteins ATM, MRE11, RAD50, NBS1, and H2AX in bladder cancer radiotherapy patients. We expected that patients with low expression of these proteins would have improved outcomes following radiotherapy due to reduced DNA DSB repair. Whereas we found no correlation for ATM, RAD50, NBS1 and H2AX, we found the opposite effect for MRE11. In two independent test and validation cohorts (the second prospectively acquired) with similar 3-year CSS rates (61.8% and 65.1%), comparable to those of other centers (13, 15, 17, 2124), our data show that low tumor MRE11 protein expression level is an independent factor associated with worse CSS following radical radiotherapy for bladder cancer. Although not validated, similar results have been reported in breast cancer (9), with high expression of MRN complex proteins associated with improved prognosis and better outcome following adjuvant radiotherapy. Additionally, Rhee and colleagues (25) showed that overexpression of NBS1 using an adenoviral vector resulted in radiosensitization in a head and neck squamous cell carcinoma cell line.

As MRE11 expression could be a general prognostic marker in bladder cancer rather than predictive of radiotherapy treatment response per se, we also studied a cohort of cystectomy patients treated at the same institution within the same era and found that, for these patients, MRE11 expression was not associated with CSS, making its role as a prognostic marker unlikely. However, the role of MRE11 expression as a predictive marker was strengthened when we compared the cystectomy and radiotherapy cohorts; in high MRE11 expressing tumors, outcomes were better after radiotherapy than after cystectomy, with a 16% absolute improvement in 3-year CSS. Results for patients with tumors of low MRE11 expression were not significant due to small numbers, but in this group, patients who had cystectomy seemed to do better than those who had radiotherapy. As is routinely the case for human epidermal growth factor receptor 2 (HER2) testing in patients with breast cancer (26), MRE11 expression as assessed by immunohistochemistry in diagnostic specimens may guide the patient's choice to either have a cystectomy or radiotherapy.

We used the 25th percentile of each cohort as a cutoff point because stratification by quartiles was more appropriate in the absence of a linear relationship between MRE11 and CSS (using the SAS version 9.1 PHREG procedure). The significance of the 25th percentile cutoff point was maintained despite a difference in its numerical value (130 versus 76). However, use of the 130 cutoff point in cohort B still gave a significant result (P = 0.006; data not shown). Variations in measures of marker expression may be due to differences in preanalytic (e.g., length and type of fixation), analytic (e.g., test reagents, methods), and postanalytic (e.g., interpretation, calibration of automation) factors (2628). All these factors must be standardized within each laboratory for the test to be useful clinically, and test material, with known levels of marker expression, must be incorporated into each assay run, as for HER2 testing (26). A similar approach will be required for MRE11 expression in muscle-invasive bladder cancer using samples from phase III clinical studies with known outcome data to establish a standardized protocol for test performance and interpretation, followed by a prospective clinical trial, where patients' treatments are selected on the basis of marker expression (29).

We believe that we are the first to report the association of MRE11 expression and radiotherapy outcome in bladder cancer, but interestingly, Soderlund and colleagues (9) found a similar result in breast cancer, although in that study the result was not validated. This implies that this finding might have larger relevance to the cancer community, and thus this marker should also be tested in other important common tumor sites such as the prostate, colorectum, and lung.

Our limited data suggest that the low expression of MRE11 that we observed in tumors is in fact reduced expression relative to that seen in normal urothelium. Reduced MRE11 protein expression due to MRE11 mutations, epigenetic silencing by promoter hypermethylation, loss of heterozygosity at 11q21, alterations in transcription or translation, or posttranslational modifications could result in MRN complex instability. Although we initially hypothesized that patients would have a better outcome due to reduced DNA repair, surprisingly we found the opposite result. Failure of induction of the DNA damage signaling cascade following DNA damage could in fact lead to radioresistance as, through less efficient activation of the downstream apoptotic cell death pathway and/or due to lack of checkpoint arrest, the cells would continue to proliferate. Giannini and colleagues (30) found cells containing an MRE11 frameshift mutation to have an impaired S-phase checkpoint, and Zhang and colleagues (31) found that siRNA-mediated knockdown of NBS1 expression in B-lymphoblasts resulted in impaired checkpoint activation, reduced apoptosis, and radioresistance.

In our study, the presence in tumor of a dysfunctional tumor TP53 mutation had no significant effect on patient survival following radiotherapy. We observed the mutations coding for codons 175, 245, 248, and 280 (32, 33), which are relatively common in bladder cancer. Although MRE11 and p53 are both involved in cell cycle control, we found no association between MRE11 protein expression and TP53 mutation.

The literature on the role of p53 in radiosensitivity is conflicting: In vitro, TP53 mutations are found to be associated with either radiosensitivity (23) or radioresistance (34), and in clinical series, TP53 mutations are associated with both improved and worse outcomes following ionizing radiation (3537). Also, no clear association has been shown between TP53 mutations and p53 protein expression by immunohistochemistry (11). In a large systematic review of p53 expression and TP53 mutations and outcomes in colorectal cancer, Munro and colleagues (36) concluded that the heterogeneous results and publication bias meant that no clear consensus could be established.

However, we have validated, in two independent cohorts, MRE11 expression by immunohistochemistry as a potential predictive marker for outcome following radical radiotherapy for muscle-invasive bladder cancer. In our cystectomy series from the same era, MRE11 was not associated with outcome, implying that MRE11 expression is not a prognostic factor in bladder cancer. Ultimately, if these results are validated in clinical trials, patients may be selected for either radiotherapy or surgery on the basis of MRE11 expression by immunohistochemistry in their pretreatment TURBT specimen, thus increasing overall cure rates for this disease.

A.E. Kiltie, as an invited speaker, had part of her registration and accommodation reimbursed at the BAUS Section of Oncology Annual Meeting, November 2009.

We thank the Northern and Yorkshire Cancer Registry and Information Service for providing us with some cause-of-death data, Filomena Esteves for advice on immunohistochemical staining, Madeleine McCarthy and Sanjeev Kotwal for help with data retrieval from patients' notes, and Andy Sharpe for DNA extractions. We also thank Prof. Penny Jeggo and Drs. Janet Hall and Anderson Ryan for helpful discussions.

Grant Support: Yorkshire Cancer Research grant L319 and Cancer Research UK grants C19217/A6082 and C15140/A11505.

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.

1
Cancer incidence—UK statistics [Internet]. London: Cancer Research UK; [updated 2009 Aug 27;cited 2010 Mar 24]. Available from: http://info.cancerresearchuk.org/cancerstats/incidence/
.
2
Kotwal
S
,
Choudhury
A
,
Johnston
C
,
Paul
AB
,
Whelan
P
,
Kiltie
AE
. 
Similar treatment outcomes for radical cystectomy and radical radiotherapy in invasive bladder cancer treated at a United Kingdom specialist treatment center
.
Int J Radiat Oncol Biol Phys
2008
;
70
:
456
63
.
3
Moynihan
C
,
Hall
E
,
Lewis
R
,
Birtle
A
,
Mead
GM
,
Huddart
R
. 
SPARE: a qualitative study investigating randomization barriers in a Selective Bladder Preservation trial (SBP)
.
J Clin Oncol
2009
;
27
:
15s
.
4
Colquhoun
AJ
,
Jones
GD
,
Moneef
MA
, et al
. 
Improving and predicting radiosensitivity in muscle invasive bladder cancer
.
J Urol
2003
;
169
:
1983
92
.
5
Iijima
K
,
Ohara
M
,
Seki
R
,
Tauchi
H
. 
Dancing on damaged chromatin: functions of ATM and the RAD50/MRE11/NBS1 complex in cellular responses to DNA damage
.
J Radiat Res (Tokyo)
2008
;
49
:
451
64
.
6
Lavin
MF
. 
ATM and the Mre11 complex combine to recognize and signal DNA double-strand breaks
.
Oncogene
2007
;
26
:
7749
58
.
7
Jackson
SP
,
Bartek
J
. 
The DNA-damage response in human biology and disease
.
Nature
2009
;
461
:
1071
8
.
8
Arlehag
L
,
Adell
G
,
Knutsen
A
,
Thorstenson
S
,
Sun
XF
. 
ATM expression in rectal cancers with or without preoperative radiotherapy
.
Oncol Rep
2005
;
14
:
313
7
.
9
Soderlund
K
,
Stal
O
,
Skoog
L
,
Rutqvist
LE
,
Nordenskjold
B
,
Askmalm
MS
. 
Intact Mre11/Rad50/Nbs1 complex predicts good response to radiotherapy in early breast cancer
.
Int J Radiat Oncol Biol Phys
2007
;
68
:
50
8
.
10
Hollstein
M
,
Shomer
B
,
Greenblatt
M
, et al
. 
Somatic point mutations in the p53 gene of human tumors and cell lines: updated compilation
.
Nucleic Acids Res
1996
;
24
:
141
6
.
11
Hall
PA
,
McCluggage
WG
. 
Assessing p53 in clinical contexts: unlearned lessons and new perspectives
.
J Pathol
2006
;
208
:
1
6
.
12
Garcia del Muro
X
,
Condom
E
,
Vigues
F
, et al
. 
p53 and p21 Expression levels predict organ preservation and survival in invasive bladder carcinoma treated with a combined-modality approach
.
Cancer
2004
;
100
:
1859
67
.
13
Jahnson
S
,
Risberg
B
,
Karlsson
MG
,
Westman
G
,
Bergstrom
R
,
Pedersen
J
. 
p53 and Rb immunostaining in locally advanced bladder cancer: relation to prognostic variables and predictive value for the local response to radical radiotherapy
.
Eur Urol
1995
;
28
:
135
42
.
14
Moonen
L
,
Ong
F
,
Gallee
M
, et al
. 
Apoptosis, proliferation and p53, cyclin D1, and retinoblastoma gene expression in relation to radiation response in transitional cell carcinoma of the bladder
.
Int J Radiat Oncol Biol Phys
2001
;
49
:
1305
10
.
15
Osen
I
,
Fossa
SD
,
Majak
B
,
Rotterud
R
,
Berner
A
. 
Prognostic factors in muscle-invasive bladder cancer treated with radiotherapy: an immunohistochemical study
.
Br J Urol
1998
;
81
:
862
9
.
16
Qureshi
KN
,
Griffiths
TR
,
Robinson
MC
, et al
. 
Combined p21WAF1/CIP1 and p53 overexpression predict improved survival in muscle-invasive bladder cancer treated by radical radiotherapy
.
Int J Radiat Oncol Biol Phys
2001
;
51
:
1234
40
.
17
Rotterud
R
,
Berner
A
,
Holm
R
,
Skovlund
E
,
Fossa
SD
. 
p53, p21 and mdm2 expression vs the response to radiotherapy in transitional cell carcinoma of the bladder
.
BJU Int
2001
;
88
:
202
8
.
18
Wu
CS
,
Pollack
A
,
Czerniak
B
, et al
. 
Prognostic value of p53 in muscle-invasive bladder cancer treated with preoperative radiotherapy
.
Urology
1996
;
47
:
305
10
.
19
Sak
SC
,
Harnden
P
,
Johnston
CF
,
Paul
AB
,
Kiltie
AE
. 
APE1 and XRCC1 protein expression levels predict cancer-specific survival following radical radiotherapy in bladder cancer
.
Clin Cancer Res
2005
;
11
:
6205
11
.
20
Petitjean
A
,
Mathe
E
,
Kato
S
, et al
. 
Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database
.
Hum Mutat
2007
;
28
:
622
9
.
21
Borgaonkar
S
,
Jain
A
,
Bollina
P
, et al
. 
Radical radiotherapy and salvage cystectomy as the primary management of transitional cell carcinoma of the bladder. Results following the introduction of a CT planning technique
.
Clin Oncol (R Coll Radiol)
2002
;
14
:
141
7
.
22
Chung
PW
,
Bristow
RG
,
Milosevic
MF
, et al
. 
Long-term outcome of radiation-based conservation therapy for invasive bladder cancer
.
Urol Oncol
2007
;
25
:
303
9
.
23
Ribeiro
JC
,
Barnetson
AR
,
Fisher
RJ
,
Mameghan
H
,
Russell
PJ
. 
Relationship between radiation response and p53 status in human bladder cancer cells
.
Int J Radiat Biol
1997
;
72
:
11
20
.
24
Rodel
C
,
Grabenbauer
GG
,
Rodel
F
, et al
. 
Apoptosis, p53, bcl-2, and Ki-67 in invasive bladder carcinoma: possible predictors for response to radiochemotherapy and successful bladder preservation
.
Int J Radiat Oncol Biol Phys
2000
;
46
:
1213
21
.
25
Rhee
JG
,
Li
D
,
Suntharalingam
M
,
Guo
C
,
O'Malley
BW
 Jr.
,
Carney
JP
. 
Radiosensitization of head/neck squamous cell carcinoma by adenovirus-mediated expression of the Nbs1 protein
.
Int J Radiat Oncol Biol Phys
2007
;
67
:
273
8
.
26
Wolff
AC
,
Hammond
ME
,
Schwartz
JN
, et al
. 
American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer
.
Arch Pathol Lab Med
2007
;
131
:
18
43
.
27
Bussolati
G
,
Leonardo
E
. 
Technical pitfalls potentially affecting diagnoses in immunohistochemistry
.
J Clin Pathol
2008
;
61
:
1184
92
.
28
Gown
AM
. 
Current issues in ER and HER2 testing by IHC in breast cancer
.
Mod Pathol
2008
;
21
Suppl 2
:
S8
15
.
29
Cordon-Cardo
C
. 
p53 and RB: simple interesting correlates or tumor markers of critical predictive nature?
J Clin Oncol
2004
;
22
:
975
7
.
30
Giannini
G
,
Ristori
E
,
Cerignoli
F
, et al
. 
Human MRE11 is inactivated in mismatch repair-deficient cancers
.
EMBO Rep
2002
;
3
:
248
54
.
31
Zhang
Y
,
Lim
CU
,
Williams
ES
, et al
. 
NBS1 knockdown by small interfering RNA increases ionizing radiation mutagenesis and telomere association in human cells
.
Cancer Res
2005
;
65
:
5544
53
.
32
Oren
M
. 
Decision making by p53: life, death and cancer
.
Cell Death Differ
2003
;
10
:
431
42
.
33
Xu
X
,
Stower
MJ
,
Reid
IN
,
Garner
RC
,
Burns
PA
. 
A hot spot for p53 mutation in transitional cell carcinoma of the bladder: clues to the etiology of bladder cancer
.
Cancer Epidemiol Biomarkers Prev
1997
;
6
:
611
6
.
34
Hinata
N
,
Shirakawa
T
,
Zhang
Z
, et al
. 
Radiation induces p53-dependent cell apoptosis in bladder cancer cells with wild-type- p53 but not in p53-mutated bladder cancer cells
.
Urol Res
2003
;
31
:
387
96
.
35
Eriksen
JG
,
Alsner
J
,
Steiniche
T
,
Overgaard
J
. 
The possible role of TP53 mutation status in the treatment of squamous cell carcinomas of the head and neck (HNSCC) with radiotherapy with different overall treatment times
.
Radiother Oncol
2005
;
76
:
135
42
.
36
Munro
AJ
,
Lain
S
,
Lane
DP
. 
P53 abnormalities and outcomes in colorectal cancer: a systematic review
.
Br J Cancer
2005
;
92
:
434
44
.
37
Saffari
B
,
Bernstein
L
,
Hong
DC
, et al
. 
Association of p53 mutations and a codon 72 single nucleotide polymorphism with lower overall survival and responsiveness to adjuvant radiotherapy in endometrioid endometrial carcinomas
.
Int J Gynecol Cancer
2005
;
15
:
952
63
.

Supplementary data