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Carcinogenesis Advance Access originally published online on January 27, 2007
Carcinogenesis 2007 28(6):1241-1246; doi:10.1093/carcin/bgm012
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Mutations in TP53 are a prognostic factor in colorectal hepatic metastases undergoing surgical resection

David G. Molleví, Teresa Serrano1, Mireia M. Ginestà, Joan Valls2, Jaume Torras3, Matilde Navarro4, Emilio Ramos3, Josep R. Germà4, Eduardo Jaurrieta3, Víctor Moreno2, Joan Figueras5, Gabriel Capellà and Alberto Villanueva*

Laboratory of Translational Research, Institut Català d'Oncologia-IDIBELL, L'Hospitalet de Llobregat, 08907 Barcelona, Spain
1 Department of Pathology, Hospital Universitari de Bellvitge-IDIBELL, L'Hospitalet de Llobregat, 08907 Barcelona, Spain
2 Department of Cancer Epidemiology, Institut Català d'Oncologia-IDIBELL, L'Hospitalet de Llobregat, 08907 Barcelona, Spain and Laboratori de Bioestadística i Epidemiologia, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain
3 Hepatobilliary Surgery Unit, Department of Surgery, Hospital Universitari de Bellvitge-IDIBELL, L'Hospitalet de Llobregat, Barcelona, Spain
4 Department of Medical Oncology, Institut Català d'Oncologia-IDIBELL, L'Hospitalet de Llobregat, 08907 Barcelona, Spain
5 Hepatobilliary Surgery Unit, Department of Surgery, Hospital Josep Trueta, 17007 Girona, Spain

* To whom correspondence should be addressed. Tel: +34 93 260 79 52; Fax: +34 93 260 74 66; Email: avillanueva{at}iconcologia.net


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this study was to analyze the prognostic value of TP53 mutations in a consecutive series of patients with hepatic metastases (HMs) from colorectal cancer undergoing surgical resection. Ninety-one patients with liver metastases from colorectal carcinoma were included. Mutational analysis of TP53, exons 4–10, was performed by single-strand conformation polymorphism and sequencing. P53 and P21 protein immunostaining was assessed. Multivariate Cox models were adjusted for gender, number of metastasis, resection margin, presence of TP53 mutations and chemotherapy treatment. Forty-six of 91 (50.05%) metastases showed mutations in TP53, observed mainly in exons 5–8, although 14.3% (n = 13) were located in exons 9 and 10. Forty percent (n = 22) were protein-truncating mutations. TP53 status associated with multiple (≥3) metastases (65.6%, P = 0.033), advanced primary tumor Dukes' stage (P = 0.011) and younger age (<57 years old, P = 0.03). Presence of mutation associated with poor prognosis in univariate (P = 0.017) and multivariate Cox model [hazard ratio (HR) = 1.80, 95% confidence interval (CI) = 1.07–3.06, P = 0.028]. Prognostic value was maintained in patients undergoing radical resection (R0 series, n = 79, P = 0.014). Mutation associated with a worse outcome in chemotherapy-treated patients (HR = 2.54, 95% CI = 1.12–5.75, P = 0.026). The combination of ≥3 metastases and TP53 mutation identified a subset of patients with very poor prognosis (P = 0.009). P53 and P21 protein immunostaining did not show correlation with survival. TP53 mutational status seems to be an important prognostic factor in patients undergoing surgical resection of colorectal cancer HMs.

Abbreviations: CI, confidence interval; CRC, colorectal carcinoma; DFS, disease-free survival; 5-FU, 5-fluorouracil; HM, hepatic metastasis; HR, hazard ratio; OS, overall survival; PCR, polymerase chain reaction; wt, wild type


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Colorectal carcinoma (CRC) is the second leading cause of cancer-related mortality in the Western world (1). The prognosis of CRC is based on the extent of colon wall invasion and the presence of lymph node and distal metastases (2). Hepatic or pulmonary metastases are important determinants of survival. Hepatic metastases (HMs) are diagnosed in 10–25% of patients at the time of primary tumor resection and associate with limited survival (3,4). Another 50–60% of patients will develop recurrent disease, most commonly in the liver, within the next 5 years.

For years, 5-fluorouracil (5-FU) has been the only drug approved to treatment of metastatic CRC, with a response rate of 20%. More recently, new drugs, such as irinotecan (CPT-11), a topoisomerase I inhibitor, and oxaliplatin, have shown, always in combination with 5-FU, significant improvements in response rates, disease-free survival (DFS) and overall survival (OS) (5,6). Despite advances in chemotherapy, surgical resection is the only therapeutic approach offering a chance of cure for patients with HMs. However, this therapeutic option is restricted to confined metastases that represent only 10–15% of cases (7). Complete surgical resection of metastases selected with standard clinical criteria results in 30–40% 5-year survival rates (7–9).

The advent of new techniques, such as pre-operative portal embolization (10), pre-operative detection with helical computed tomography (11) and intra-operative ultrasonography (12), has extended surgical indications. Thus, a better identification of candidate patients is now mandatory.

In the last two decades, the genes associated with initiation and progression of colorectal cancer have been well characterized (13). Nevertheless, the genetic basis of CRC distal dissemination remains largely unknown (14,15). Genome-wide screening procedures have identified recurrent chromosomal regions differentially gained or lost in HM with respect to primary CRC (16,17). Similarly, many genes de-regulated in HMs have been identified. Nevertheless, only a reduced number of these genes have been clearly associated with HM, and none of them have been shown to be consistently and specifically involved in HM (18).

TP53 tumor suppressor gene encodes a protein involved in cell cycle regulation, DNA replication and apoptosis (19), being activated during the adenoma–carcinoma transition. Approximately 40–50% of CRCs contain a mutated TP53 gene, usually associated with the allelic loss of the remaining wild-type (wt) allele. This percentage rises to 60% in HMs. In a recent pooled analyses (20), tumor site, type of mutation and adjuvant treatment were identified as important factors in determining the prognostic significance of TP53 mutations in primary colorectal cancer. The prognostic relevance of TP53 gene alterations after surgical resection of HMs remains controversial (21–24). However, these studies usually analyze a limited number of cases and provide no information about chemotherapy treatment.

The purpose of this study was to evaluate the clinical usefulness of TP53 mutations as prognostic marker after curative resection of HMs. P53 and P21 protein immunostaining were also assessed. Correlation between presence of gene mutation, immunostaining and clinico-pathological features, including chemotherapy administration, has been analyzed.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Patients
Between January 1996 and December 1998, a total of 104 consecutive patients harboring colorectal cancer liver metastases and undergoing surgical resection at Hospital Universitari de Bellvitge were initially considered. Thirteen cases were excluded for the analysis: eight cases were lost to follow-up and five died within 30 days after operation. Thus, a cohort of 91 patients was defined (30 female and 61 male), with a median age of 60 years (main characteristics are depicted in Table I). Patients were selected for resection using standard clinical criteria: (i) acceptable physical condition; (ii) tumors anatomically confined within the liver and (iii) absence of disseminated disease. Histological diagnosis was confirmed in all specimens. In 27 patients (29.7%), metastases were synchronous and the remaining 64 (70.3%) metachronous. A median of two metastases per patient (range 1–12) was detected. Bilobar HMs were present in 25 patients (27.5%). In 12 of 91 patients (13.2%), positive surgical margins were detected. Therefore, for prognostic assessment purposes, an R0 series of 79 patients was defined.


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  		Table I. Univariate analysis of clinico-pathological characteristics of patients included

 
Post-operative fluorouracil (5-FU)-based chemotherapy was administered after hepatic resection in 45 of 91 (49.5%) patients: 25 patients who had already received adjuvant 5-FU treatment and 20 who did not receive chemotherapy after primary tumor resection. No patient received neo-adjuvant treatment before metastasis resection. Systemic chemotherapy was initiated 4 weeks after resection, and six cycles were scheduled. 5-FU was administered daily for 5 days as an intravenous bolus of 425 mg/m2 body surface area, preceded each day by an infusion of leucovorin at a dose of 200 mg/m2. If a patient had previously received 5-FU, the systemic chemotherapy was administered as 5-FU in continuous infusion. The remaining 46 patients did not receive chemotherapy after liver resection: 33 had received adjuvant 5-FU previously and 13 declined. No significant differences were observed between patients receiving and not receiving chemotherapy regarding gender, number of metastases, primary Dukes' stage, pre-operative carcinoembryonic antigen (CEA), lobar affection and positive margins (data not shown). All patients gave written consent to participate and the Ethics Committee of the hospital cleared the study protocol.

Clinical follow-up after hepatic resection was prospectively recorded for all included patients, with a median follow-up of 78 months, range 67–102 months. Liver recurrence was detected by ultrasonography or computed tomography scan, which was performed every 6–12 months, or earlier, when symptoms developed. Follow-up protocol was maintained during the study period.

Endpoints were liver local recurrence for the DFS and death for OS. A median OS of 38 months [95% confidence interval (CI) = 33–43] was observed. At the end of follow-up period, 31 of 91 (34.1%) patients were still alive. Overall 2-, 3- and 5-year survivals are depicted in Figure 1. Median DFS was 15 months (95% CI = 11–19).


Figure 1
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  		Fig. 1. Kaplan–Meier plots for clinical features: OS and DFS after hepatic resection.

 
Detection of TP53 mutations
Formalin-fixed, paraffin-embedded tissue samples were retrieved from the files of the Pathology Department of the Hospital Universitari de Bellvitge. One metastasis per patient was selected for the study. When two or more HMs were available, selection was based on a higher content of tumor cells. Genomic DNA extraction was carried out using QIAamp Mini kit DNA, Qiagen (Qiagen, Valencia, CA) from five sections, 15 µm each, of paired liver metastases and normal hepatic parenchyma. The whole coding sequence of exons 4–10 of TP53 was amplified in seven independent polymerase chain reactions (PCRs) using flanking intronic-based primers. For each reaction, 5 µl of DNA was amplified, and nested PCR was carried out using 2 µl of previous PCR product as a template and internal intronic-based primers (all primers information is available upon request). For single-strand conformation polymorphism analysis, PCR products were diluted 1:16 in formamide:dye loading buffer, incubated for 3 min at 95°C, cooled on ice and loaded onto 6–8% polyacrylamide non-denaturing sequencing gels. Electrophoresis was carried out at room temperature under 7 W for 12–14h, and shifted bands excised from the gel, re-amplified and sequenced.

Immunohistochemistry
Slices (3 µm) of paraffin-embedded tissue were used. For antigen retrieval, the slides were boiled after deparaffinization in a pressure cooker for 2 min in citrated buffer (8.2 mM tri-sodium citrate and 1.98 mM citric acid, pH6), and endogen peroxidase was blocked with 3% H2O2 during 20 min. After blocking during 60 min with 1:5 dilution of goat serum, primary antibodies were incubated overnight at 4°C. Primary antibodies were monoclonal antibodies for P53 (clone PAB1801, Ab-6, Oncogene Science, Boston, MA), dilution 1:500 in phosphate-buffered saline, and P21 (clone 6B6, PharMingen, BD Biosciences, Erembodegem, Belgium), dilution 1:100 in phosphate-buffered saline. Reaction was visualized using EnVision anti-mouse antibody system, and developed using DAB-Plus kit (Dako, Copenhagen, Denmark). Slides were counter-stained with Harry's modified hematoxylin.

Blinded analysis of slides was performed by two independent observers (T.S. and D.G.M.). Five high-power fields (x400) were evaluated for P53 and P21 staining. The cut-off value for P53-positive immunohistochemical evaluation was 10% positive stained cells. For P21, cut-off value was set at 5% due to the lower P21 basal expression levels.

Statistical methods
Association between genetic aberrations and clinico-pathological variables was assessed by chi-square test. Survival was defined as the time from the date of liver resection until death, being censored for patients who were alive at the time of the last follow-up. Survival curves were estimated using the Kaplan–Meier method. Cox's proportional hazard models were fitted to estimate hazard ratios (HRs) and 95% CI and likelihood ratio tests were performed to assess statistical significance of the variables. Initially, univariate models were fitted, and in a second phase, multivariate models were used to analyze the conjoint effect of the variables, including interactions. P-values from all analyses were considered significant P ≤ 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Mutational analysis of TP53 in HMs
Fifty-five point mutations were identified in 46 of 91 liver metastases (50.5%) (Table II). In nine metastases, two mutations were simultaneously detected: in two cases both were located at the same exon (cases 6 and 13) whereas in seven cases mutations at different exons were detected (cases 7, 8, 10, 15, 17, 21 and 22) (Table II). Forty-six of 55 (83.6%) mutations were single base substitutions, including 35 transitions and 11 transversions, whereas the remaining nine mutations (16.4%) were single base insertions. Thirty-three of 55 mutations (60%) were missense, whereas 22 (40%) were protein-truncating mutations (13 non-sense and 9 frameshift). The majority of mutations were located at the DNA-binding domain (exons 5–8) but up to 13 of 55 (23.6%) were located at the tetramerization domain (exons 9 and 10) (Table II). Three mutational hot spot codons have been identified: 175 (n = 8), 213 (n = 6) and 342 (n = 4) (Table II).


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  		Table II. TP53 gene mutatisons in liver metastases of colorectal metastases

 
Overall, 26 of 91 (28.6%) metastases harbored TP53 mutations leading to amino acid changes and 20 (22.0%) contained protein-truncating mutations. Up to 14.3% (n = 13) of the metastases harbor tetramerization domain mutations (Table II).

TP53 mutations and clinico-pathological variables
TP53 mutations were more often detected in patients under 57 years (20 of 30, 66.7% versus ≥57 years old; 26 of 61, 42.6%, P = 0.030), and when three or more metastases were detected (21 of 32, 65.6%, P = 0.033). Also, TP53 mutations were more frequently detected in more advanced stages of the corresponding primary tumor (Dukes' C and D; 57.5 and 63%, respectively, P = 0.011). No association was observed between TP53 mutations and the remaining clinico-pathological variables studied, including age, primary tumor location, primary Duke's stage, primary tumor T stage, lymph node invasion, type of metastases, bilobar hepatic affection and CEA levels before hepatectomy (Table I).

Prognostic relevance of TP53 mutations
The presence of TP53 mutations in HMs was associated in univariate analysis with poorer prognosis (P = 0.017, Table III and Figure 2A). Accordingly, prognostic value was maintained in the R0 series (n = 79, HR = 2.07, 95% CI = 1.16–3.71, P = 0.014, Figure 2B). The same association was observed with DFS (P = 0.015). When mutations were grouped according to their functional impact, protein-truncating TP53 mutations associated with a better DFS (HR = 2.37, 95% CI = 1.14–4.96, P = 0.019) but this effect was no longer evident with OS (P = 0.13).


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  		Table III. Prognostic value of TP53 gene mutations and P53 and P21 protein expression analyzed by immunohistochemistry (univariate analysis)

 


Figure 2
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  		Fig. 2. OS Kaplan–Meier plots according TP53 mutational status in (A) complete series and (B) in R0 series only include those patients with negative surgical resection margins. Kaplan–Meier plots describing the association between TP53 status and number of metastases including whole series in forth categories (C). P-values correspond to likelihood ratio test and were considered significant if P ≤ 0.05.

 
In our series, the gender was a prognostic variable of better survival (median survival: 58 months in female versus 36 months in male, HR = 0.52, 95% CI = 0.29–0.94, P = 0.03, Table I). Similarly, patients with ≥3 metastases had a worse OS (28.5 months survival rate, HR = 1.86%, 95% CI = 1.11–3.12, P = 0.018). A modest trend towards better OS was observed in patients with unilobar liver metastases (P = 0.089). The presence of positive surgical margins and hepatic or pulmonary relapse were associated with a worse prognostic (Table I).

Prognostic value of TP53 mutations was assessed with a multivariate Cox's proportional hazard model (Table IV). After adjusting for significant clinico-pathological variables, TP53 mutations remained as a significant prognostic factor (HR = 1.80, 95% CI = 1.07–3.06, P = 0.028). Therefore, TP53 status is an independent prognostic factor. A relevant interaction between presence of TP53 mutations and post-operative 5-FU-based chemotherapy treatment was observed (P = 0.059). In our study, patients who did not receive post-operative chemotherapy had a worse prognosis independently of TP53 mutational status. Interestingly, mutation only associated with a worse outcome in chemotherapy-treated patients (5-FU-treated HR = 2.54, 95% CI = 1.12–5.75, P = 0.026 versus treated patients wtTP53). Alternative representation of interaction is shown in Table IV, where the relative HR for patients classified according chemotherapy treatment and TP53 mutational status has been estimated. Finally, the combination of mutated TP53 and ≥3 metastases identified a subset of patients with very poor prognosis (16 months; versus mutated TP53 and <3 metastases, wtTP53 and less than 3 metastases and wtTP53 and ≥3 metastases (38, 51.5 and 48 months, respectively, P = 0.009, Figure 2C).


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  		Table IV. Multivariate Cox model analysis describing the interaction among post-operative chemotherapy after hepatic resection and TP53 mutations

 
Immunostaining of P53 and P21 proteins
P53 over-expression was present in 56 metastases (61.5%). A poor correlation was detected between P53 immunostaining and the presence of mutations (kappa coefficient of agreement 0.12, P = 0.25): 31 of 56 (55.4%) positive P53 immunostaining metastases were mutated, whereas mutations were also detected in 15 of 35 (42.8%) negative P53 immunostaining cases. No significant differences in survival were observed between P53-positive (38.5 months) and P53-negative (35 months) liver metastases (Table III). P21 expression was lost in 59 cases (64.8%). No significant association with OS (P = 0.84) or DFS (P = 0.59) was observed when P21 immunostaining was considered. Also no association was detected between P21 protein levels and the mutational TP53 status (data not shown). Finally, no significant association between P53 or P21 immunostaining and other clinico-pathological features analyzed was observed (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In the present study, we have shown that TP53 gene mutation is an independent prognostic factor in patients undergoing radical surgery for CRC HMs. Interestingly, its prognostic value is restricted to those patients receiving 5-FU-based chemotherapy.

Surgical resection, when feasible, is the best available treatment for HM. Limitations of the standard clinical criteria in the selection of candidate patients make mandatory the search of novel prognostic markers. Because of the prominent role of TP53 gene alterations during colorectal carcinogenesis (25,26), mutation detection or protein expression have been proposed as potential prognostic markers. In primary colorectal cancer, the role of TP53 as a prognostic factor has been a matter of controversy. Recent studies analyzing a large number of patients have shown that, more than the simple presence or absence of TP53 mutations is the specific classes of mutations, the tumor site location and tumor stage are important factors in determining their prognostic significance (20,27,28).

In the present study, analyzing the most extensive series of HM reported so far, we have demonstrated that the presence of mutations in TP53 gene associates with tumor aggressiveness and is an independent prognostic factor. Previously, few studies have addressed the prognostic role of TP53 gene in HMs with controversial results. Sturm et al. (29), de Jong et al. (23) and Saw et al. (22) analyzing 41, 44 and 60 cases, respectively, concluded that TP53 mutational status did not add prognostic information. Yang et al.(30) described in a series of 37 patients a better survival for patients with mutated TP53.

TP53 mutations associate with established prognostic factors such as a high number of HM (21) and adds information to it and other factors such as gender (31) and positive surgical margins (9,32). The interaction observed between TP53 mutations and 5-FU chemotherapy can account for the prognostic usefulness reported. The role of TP53 mutations as predictors of 5-FU-based chemotherapy response in metastatic CRC is controversial (33) and little information is available about post-operative chemotherapy treatment and the prognostic role of TP53 gene after metastases resection. Throughout the study period, chemotherapy administration was indicated on a personalized manner, mainly based upon previous response to pre-operative chemotherapy. The interaction observed may also account for the contradictory results previously reported. The majority of studies (22,23,30) do not provide relevant information and Sturm et al. (29) did not assess the impact of personalized treatment. Our results suggest that the prognostic value observed for the presence of TP53 mutations may be partially related to a putative increased resistance to 5-FU.

In line with previous reports, up to 50% of metastases harbor TP53 mutations (21,24,34), but the spectrum and type of TP53 mutations are different from that of primary tumors (35). Mutations clustered in exons 5–8, but exons 9 and 10 mutations were over represented as previously reported by Miyaki et al. (24). Also, we have defined three TP53 hot spots: codons 175 (exon 5), 213 (exon 6) and 342 (exon 10), in agreement with Miyaki et al. (24) that reported codons 213 and 342 as specific hot spots in HMs that differs from primary CRC hot spot codons (175, 245, 248, 273 and 282). It is likely that the genetic area analyzed influences its prognostic value; studies that did not analyze exons 9 and/or 10 (29,32) failed to observe relevant clinico-pathological associations.

TP53 mutations in metastases not only differ in location but also in its functional impact. Protein-truncating mutations (non-sense and frameshift) account for 47.8% of all mutations whereas only a minority (7%) of primary tumors harbors this type of alterations (36). We have not been able to show any prognostic value of protein-truncating mutations that are evenly distributed along the TP53 gene.

It is difficult to understand the differential contribution of missense mutations located in exon 9 and 10 and protein-truncating mutations in HMs. While the majority of missense mutations disturb DNA-binding activity, protein-truncating mutations generate the loss of C-terminal region including tetramerization domain, apoptotic response domain and nuclear localization signals (37). It can be speculated that metastasis-specific mutations may confer selective growth advantages in the liver environment. Unfortunately, we have not been able to analyze and confirm (35) in matched primary tumors–HMs the differences observed in our series due to lack of available material. In an independent set of 27-matched primary tumor–HM, presence of TP53 mutations was detected in 33.3% (9 of 27) of primary tumors versus 51.8% (14 of 27) of HMs (data not shown). Three of the five acquired mutations were located at exons 9 (n = 2) and 10 (n = 1). Altogether these data suggest that the presence of TP53 mutations in primary tumors is not an accurate surrogate marker of TP53 status of corresponding metastases. Clearly, we consider that the differences observed between primary tumor versus HMs, with regard to incidence and molecular nature of aberrations, deserve additional studies using large and prospective series.

Immunohistochemical levels of P53 did not provide any prognostic information in line with Saw et al. (22) and in contrast with other reports (38,39). Noteworthy, differences in the percentage of positive cells cannot account for the discrepancies observed (38). No correlation has been observed between TP53 mutation and P53 protein immunostaining probably associated to the increased prevalence of protein-truncating mutations. Also P21 expression has no prognostic impact. No correlation between P53 and P21 status has been observed.

We have shown that TP53 mutational status of HMs contain prognostic information. However, its clinical translation is not straightforward. While routine fine needle aspirates of metastasis may not be feasible, mutation detection in plasma or serum DNA is a suitable alternative, especially when disseminated disease is present (40).

In summary, we have shown that mutational analysis of TP53 provides prognostic information in a large series of consecutive colorectal HMs undergoing surgical resection especially in those patients receiving 5-FU-based chemotherapy treatment and those with ≥3 metastases. The extent of TP53 mutation analysis including exons 9 and 10 is critical to obtain clinically relevant information. Our results suggest that a combined clinico-pathological and molecular assessment can be of help in identifying those patients unlikely to benefit from a potentially curative resection. These results should be further validated in prospective series where neo-adjuvant 5-FU-based chemotherapy is complemented with oxaliplatin or irinotecan.


   Acknowledgments
 
A.V. is an investigator from the Ramón y Cajal program. This work was supported by grants (SAF2002-02265 and AGL2004-07579-04) from Spanish Ministry of Science and Technology and (FIS03-0092 and FIS03-0114) from Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spanish Ministry of Health.

Conflict of Interest Statement: None declared.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 18, 2006; revised December 28, 2006; accepted January 16, 2007.


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