Clinical Value of Mitochondrial Mutations in Colorectal Cancer
http://www.100md.com
《临床肿瘤学》
the Institut National de Sante et de la Recherche Medicale (INSERM) U490, Laboratoire de Toxicologie Moleculaire
Ple de Cancerologie, Hpital Europeen Georges Pompidou, Paris
Centre Hospitalier Universitaire (CHU) Dijon, Service d'Anatomo-Pathologie
INSERM Equipe Mixte INSERM (EMI) 0106, CHU, Registre Bourguignon des Tumeurs
Centre de Pathologie, Rue Nicolas Bornier
Centre Georges Franois Leclerc, Service d'Anatomo-Pathologie, Dijon, France
ABSTRACT
PURPOSE: Prognostic factors that could select high-risk recurrence colorectal cancer patients and predict chemosensitivity are needed. Since mutations of mitochondrial DNA (mtDNA) have been described in different types of cancers and since they may play a role in response to anticancer agents, we investigated in a population-based series of colorectal cancer patients the clinical value of mtDNA mutations.
PATIENTS AND METHODS: The displacement loop (D-loop) region of mtDNA was sequenced on a series of 365 patients recorded in the Digestive Cancer Registry of Cte-d'Or (France) between 1998 and 2000. Clinicopathologic characteristics were correlated to the presence of a D-loop mutation. Survival rates were compared with the log-rank test. A multivariate survival analysis was performed.
RESULTS: D-loop mutations were found in 38.3% of the tumors. The 3-year survival rate was 53.5% in patients with D-loop mutation versus 62.1% in patients without (P = .05). After adjustment for age, stage, and microsatellite instability status, the relative risk of death in patients with D-loop mutation was 1.40 (95% CI, 1.02 to 1.93; P = .034) as compared with those without. In stage III colon cancers, adjuvant chemotherapy was beneficial only for patients without D-loop mutation (3-year survival, 78.3% v 45.4%, P < .02). In those with D-loop mutation who received adjuvant chemotherapy, the relative risk of death was 4.30 (95% CI, 1.23 to 15.00; P < .02).
CONCLUSION: The D-loop region is a hotspot for somatic mutations in colorectal tumors. Moreover, presence of tumor D-loop mutation appears to be a factor of poor prognosis in colorectal patients and a factor of resistance to fluorouracil-based adjuvant chemotherapy in stage III colon cancers.
INTRODUCTION
Colorectal cancer is one of the most common human malignancies, with more than 300,000 cases both in the United States and in the European Union each year.1 Prognosis remains poor despite progress in its management.2 Surgery is the primary treatment for colorectal cancer, but over the last two decades, adjuvant and palliative treatments have been developed with the aim of improving survival. There is a need to gain additional clues to explain chemosensitivity. Although much knowledge has been collected concerning alterations in cancer cell nuclear DNA (nDNA), less attention has been paid to mutations within mitochondrial DNA (mtDNA) despite extensive evidence of mitochondrial involvement in apoptosis. mtDNA is a 16,569 base-pair (bp), double-stranded, circular DNA composed of genes and of a noncoding region, the displacement loop (D-loop), located between nucleotides 16,024 and 576, which contains essential transcription and replication elements. It has been reported that mtDNA was more susceptible to mutations than nDNA3,4 and frequently mutated in different types of cancers,5-16 including 10% to 70% of colorectal carcinomas.5,7,17-20 These findings suggest a potential role of mitochondrial genome in tumor carcinogenesis. Several hotspots of mtDNA mutations have been described. In particular a polymorphic polycytosine CnTC6 sequence (named D310 sequence) of the D-loop was found as a major target for mtDNA mutations in cancers.5 Since most studies have not sequenced the entire mitochondrial genome and have focused their analysis only on a few coding regions or exclusively on the D-loop,5,7,17,18,20 available data about mtDNA mutations in colorectal cancers are few and inconsistent. The aims of this work were to find in colorectal cancer a mutational hotspot by sequencing the complete mitochondrial genome in 11 patients, to assess the relations between this mutational hotspot and the clinicopathologic characteristics of colorectal cancer, and to determine the chemosensitivity impact of these mutations in a population-based series of 365 patients.
PATIENTS AND METHODS
Patients
As a first step, tumors and matched healthy colon tissues (taken from surgical margins) were collected at Hpital Laennec (Paris, France) from 11 patients (three males and eight females; median age, 62 years) who underwent colorectal cancer resection in the department of surgery between 1997 and 1999. These cases were selected according to the tumor phenotype determined by the genotyping of five microsatellites (BAT-26, BAT-25, D2S123, D5S346 and D17S250). The unstable phenotype (MSI-H) was assigned following the criteria defined by the international consensus conference on microsatellite instability.21 Five MSI-H and six microsatellite stable (MSS) tumors were retained for futher analysis.
As a second step, tumor and healthy colon tissues were obtained from 365 patients (198 males and 167 females; mean age, 71.7 years; standard deviation, 12.2) who underwent colorectal cancer resection in the Cte d'Or area (Burgundy, France) between January 1998 and December 2000. Samples were obtained from a population-based frozen-tissue bank. This includes all patients with resected colorectal cancer who live in the administrative area of Cte-d'Or. Due to the small size of samples, carcinomas resected by polypectomy were not included in this study (n = 100). Data were merged with that of a digestive cancer registry, which routinely collects data on patient characteristics, stage at diagnosis, treatment, and survival. One major advantage of this type of analysis is the absence of bias concerning the selection of patients, as compared with hospital or clinical trials series. Tumor site was classified according to the International Classification of Diseases into colon cancers (C18, 250 cases) and rectal cancers (C19 to C20, 115 cases). Cancer extension at the time of diagnosis was classified according to the TNM classification: 31 (8.5%) were stage I, 157 (42.7%) were stage II, 93 (25.8%) were stage III, and 84 (23%) were stage IV. Forty-one tumors (11.2%) were MSI-H, 322 were MSS, and two cases were inconclusive. Data on adjuvant and palliative chemotherapy was recorded. The French Consensus Conference on colorectal cancer recommends adjuvant chemotherapy in stage III colon cancer and palliative chemotherapy in advanced colorectal cancers—stage IV and in nonresected cases.22 Adjuvant chemotherapy was fluorouracil (FU) -based. Only two patients received FU/oxaliplatin based chemotherapy in adjuvant setting.
Tissue Sample Preparation and DNA Extraction
Tumor and nontumor matching tissues were obtained immediately after surgical resection, frozen in liquid nitrogen, and stored at –80°C until DNA extraction. Before DNA extraction, the presence of tumor cells in the tumor fragment was assessed by hematoxylin-eosine-safran coloration, and fragments with more than 70% of tumor cells were retained for DNA extraction. The samples were treated with proteinase K, and total cellular DNA was extracted using QIAmp DNA Mini Kit (Qiagen, Courtaboeuf, France) according to the manufacturer's recommendations.
Polymerase Chain Reaction Amplification
To amplify the entire mtDNA of the first 11 patients, 8 pairs of primers described previously19 were used. Large polymerase chain reaction (PCR) products (1 to 3 kb each) were generated to avoid amplification of nuclear pseudogenes. For amplification, 100 ng of mtDNA were used in a final 50 μL master mix containing 1x final concentration of 10x cloned Pfu DNA polymerase reaction buffer (Stratagene, Amsterdam, Holland), 250 μmol/L of each deoxy nucleotide triphosphate (dNTP), 0.5 μmol/L of each primer and 1 μL (2.5 UI) of Pfu DNA polymerase (Stratagene). Total DNA was subjected to step-down PCR protocol on a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA) at 95°C for 2 minutes for a total of 35 cycles; 95°C for 30 seconds; 50°C to 68°C for 30 seconds (according to each pair of primers); 72°C for 1 to 3 minutes (according to each amplified fragment length); and a final extension at 72°C for 10 minutes (detailed protocol available on request).
For D-loop amplification of the remaining patients, the primers used were F47: 5'-CGCACGGACTACAACCACGAC-3' (forward) and R15: 5'-CTGTGGGGGGTGTCTTTGGG (reverse). Fifty to 100 ng of mtDNA were used in a final 20 μL master mix containing 1x final concentration of 10x PCR buffer (Qiagen), 250 μmol/L of each dNTP, 1.5 mmol/L MgCl2, 0.5 μmol/L of each primer, and 0.1 μL of HotStartTaq DNA Polymerase (Qiagen). Total DNA was subjected to the following step-down PCR protocol: 95°C for 15 minutes, 35 cycles; 95°C for 30 seconds; 64°C for 30 seconds; 72°C for 2 minutes; and a final extension at 72°C for 10 minutes. PCR products were then purified using G-50 Sephadex superfine (Amersham Biosciences, Orsay, France) on Multiscreen support (Millipore, Bedford, MA).
Direct Sequencing of mtDNA
Purified PCR products were sequenced using a Big Dye Terminator cycle sequencing kit (Applied Biosystems) on an ABI Prism 3700 DNA Analyser automated sequencer (Applied Biosystems). Seventy-three primer pairs described previously19 were used to sequence the entire mtDNA of the first 11 patients and one primer, DS2: 5'-ACCTACGTTCAATATTACAGGCG-3', was used to sequence the D310 homopolymeric C-tract. For sequencing, 2 μL of purified PCR product were used in a final 20 μL mix containing 1 μL of Big Dye Terminator V 3.1, 0.875x final concentration of 5x Big Dye Terminator sequencing buffer (Applied Biosystems) and 0.5 μmol/L of each primer. DNA was then submitted to the following cycle conditions: 94°C for 4 minutes; 25 cycles, 96°C for 30 seconds, 50°C for 15 seconds, and 60°C for 4 minutes. The results of DNA sequence analysis were compared with the published reference mtDNA sequence (GenBank, access number J01415) using Autoassembler software (Applied Biosystems). Sequence variations found in both healthy and tumor tissues mtDNA were scored as germ-line polymorphisms. Each was then checked against the Mitomap database (http://www.mitomap.org). Those not recorded in the database were categorized as novel mtDNA polymorphisms. Any mtDNA sequences that differed between tumor and matched healthy tissue mtDNA were scored as somatic mutations. All somatic mutations found were further validated by a new independent amplification and sequencing.
Statistical Analysis
Associations among categoric data were analyzed using 2 tests for heterogeneity. The crude survival rates were calculated using the Kaplan-Meier method. Survival curves were compared using the log-rank test. Patients who died within 30 days after resection were excluded from the survival analysis. The life status was known for the 365 patients in January 2004. A special survey was conducted in order to collect this data. Information about life status was ascertained from dearth certificates, registrar of the place of birth and place of residence, or from practitioners.
The median follow-up was 39 months. Multivariate survival analysis was performed using a Cox proportional hazards model. Analysis was carried out using the STATA software package (Statistical Software for Professionals, College Station, Texas).
Ethics Committee
This study was evaluated by the Consultative Committee Protecting Persons in Biomedical Research (Dijon, France), according to French law.
RESULTS
Analysis of the Entire mtDNA in 11 Patients
The entire mtDNA sequencing of the first 11 patients has allowed identification of a total of 10 somatic mutations in seven patients (table 1). Eight mutations were found in the D-loop region and two were found in the coding region of mtDNA, within MTCO1 and MTCO3, genes which led to the following amino acid changes: I87T and G234S respectively. Among the D-loop mutations, six were located in the D310 repeat (insertion or deletion of one to three base pairs). The other two were located in position 16,390 (G16390A) and 300 (300delA). Two different D-loop mutations were associated in one patient (A11B11) and two D-loop mutations were associated with a coding region mutation in another patient (A7B7). In total, six (54%) of 11 patients with a colorectal cancer exhibited mutations in the D310 sequence, which can be considered as a hotspot of mtDNA mutations in colorectal carcinoma patients. The frequency of the mtDNA mutations was 66% in MSS and 40% in MSI-H tumors (P = not significant).
Furthermore, 133 polymorphisms were identified in these 11 patients, of which 98 were previously recorded and 35 were considered as new polymorphisms.
Analysis of a 400-bp Fragment of the D-Loop (nucleotide position, 190-590) Containing the D1310 Repeat
A somatic D-loop mutation was identified in 142 of the 365 tumors. The D310 mononucleotide repeat was found mutated in 132 of them (36%). The alterations detected in the D310 repeat were insertions or deletions of one to several base pairs. Seventy-two (50.7%) of the total mutated patients had homoplasmic mutations (Fig 1A) and sixty-six (46.5%) had heteroplasmic mutations (Fig 1B). Four of them (2.8%) had homoplasmic and heteroplasmic mutations at the same time. Eleven patients had mutations outside the D310, associated for three of them with a mutation of the D310 repeat. Thus, only eight of the 140 patients had a D-loop mutation outside the D310 sequence.
The D310 sequence is polymorphic in the human population. The number of cytosines in the 7-bp stretch varied from six to 13. The most frequent sequences for the D310 region are C7TC6, C8TC6, and C9TC6, which were present respectively in 178 of 365 (49%), 113 of 365 (31%) and 15 of 365 (4%) of the nonmalignant tumor tissues in our series. The prevalence of D310 alterations increased significantly with the number of cytosines in the sequence. Indeed, the C7TC6 sequence was altered in 16 patients (9%) of the 178, the C8TC6 was altered in 56 patients (49.5%) of the 113, and the C9TC6 sequence was altered in 11 patients (73%) of the 15, respectively (P < 10–16).
MtDNA Mutations, Clinicopathologic Data, Survival and Response to Chemotherapy
Clinicopathologic data. After completion of the detailed sequence analysis in all specimens, clinicopathologic data were correlated with the molecular analysis. No significant association between sex, age, tumor site, stage at diagnosis, or MSI status and the presence of mtDNA mutation were found (table 2).
Overall, 121 patients with colorectal cancer received chemotherapy. Among them, 82 received adjuvant chemotherapy: patient had stage I disease, 19 patients had stage II, 54 patients had stage III (42 colon and 12 rectal cancers) and 8 had stage IV. Thirty-nine stage IV cancers received palliative chemotherapy. The use of chemotherapy was significantly associated with age of patients. For stage III colon cancer, the proportion of patients receiving chemotherapy was 100.0% for patients younger than 65 years, 71.4% for the 65 to 74 years group, and 47.1% thereafter, respectively (P < .001). The corresponding percentages for stage IV colorectal cancers were 60.7%, 75.0%, and 32.1%, respectively (P < .004).
Survival of Colorectal Cancer Patients
In univariate analysis, after exclusion of 21 patients who died within 30 days after surgical resection, the 3-year overall survival of colorectal cancer patients with tumor mutation in the D-loop sequence was lower than that of patients with no tumor mutation in the D-loop sequence: 53.5% versus 62.1%, respectively (P = .05; Fig 2). In an overall multivariate analysis, D-loop sequence status was an independent predictor of survival, with a relative risk of death after adjustment for age, tumor stage, tumor site, and MSI status of 1.41 (95% CI, 1.02 to 1.95; P = .034) in patients with a D-loop mutation when compared with those without D-loop mutation (Table 3). When rectal cancer cases were excluded from the analysis, 3-year overall survival of colon cancer patients with tumor mutation in the D-loop was 54.5% compared with 64.7% for patients without mutation (P = .0387).
A subgroup analysis was performed examining stage II, stage III and stage IV colorectal patients. No significant difference was observed in 3-year overall survival of patients presenting a D-loop mutation as compared with those without D-loop mutation. However, the prognosis tended to be worse among patients with D-loop mutation: 72.9% (95% CI, 59.0% to 82.8%) compared with 76.7% (95% CI, 65.8% to 84.5%) for stage II disease; 50.1% (95% CI, 30.4% to 66.9%) compared with 65.8% (95% CI, 52.0% to 76.5%) for stage III disease; and 13% (95% CI, 3.9% to 27.7%) compared with 11.8% (95% CI, 4.1% to 23.9%) for stage IV.
Mutation of the D-Loop and Response to Chemotherapy
Because only 19 of the 156 stage II colorectal cancer patients received adjuvant chemotherapy, no further subgroup analysis was performed for them.
Considering the subgroup of stage III colon cancer patients for whom adjuvant chemotherapy has been demonstrated efficient in terms of survival,22 the 3-year survival rate was 47.4% for patients presenting a D-loop mutation as compared with 70.0% (P < .02) for those without D-loop mutation (Fig 3A). Adjuvant chemotherapy was beneficial only for patients without tumor D-loop mutation: the 3-year survival rate for patients with D-loop mutation was 45.4% compared with 78.3% for patients without mutation (P < .02; Fig 3B). Among patients not receiving chemotherapy, the 3-year survival rate for cases with tumor D-loop mutation was 41.0% compared with 42.9% for cases without mutation (P = not significant; Fig 3C). After adjustment for age and MSI status, both chemotherapy and the absence of D-loop mutation were significantly and independently related to a longer survival (Table 4). The relative risk of death for stage III colon cancer patients with tumor D-loop mutation was 2.51 (95% CI, 1.04 to 6.02; P = .04) as compared with those without tumor mutation. When considering the group of stage III colon cancers treated with adjuvant chemotherapy, the relative risk of death for patients with tumor D-loop mutation was 4.30 (95% CI, 1.23 to 15.00; P < .02).
Among patients with advanced colorectal cancers receiving chemotherapy, the 1-year survival rate was slightly worse for those with D-loop mutation as compared to those without mutation (63.2% [95% CI, 37.9 to 80.4] v 71.4% [95% CI, 50.9 to 84.6]; P = not significant). The survival rates became similar at the 3-year limit, 12.5% (95% CI, 2.1 to 32.8) and 13.8% (95% CI, 3.1 to 32.2), respectively.
DISCUSSION
The sequencing of the entire mitochondrial genome from healthy and tumor mtDNA of 11 colorectal cancer patients allowed demonstration that the D-loop, especially the D310 mononucleotide repeat within the D-loop, was a hotspot for somatic mutations in colorectal tumors because 54% of them were found mutated in this region. This result was confirmed by the study of a population-based series of 365 colorectal cancers for which the D310 sequence was found mutated in 36% (95% CI, 32% to 42%) of the cases. Most of these mutations were insertions or deletions in the polymorphic C7/8/9/10TC6 tract of the D310 sequence. These results are similar to those obtained in previous studies including few patients, which found a D310 mutation in 28% to 44% of colorectal cancers.5,17,18 However, and surprisingly, the only study in which a sequencing of the entire mitochondrial genome was done from 10 human colorectal cell lines did not find any mutation in the noncoding region of the D-loop.19 Despite these contradictory results, we considered our findings sufficient to justify restriction of further sequencing to the D-loop region since most of the mtDNA mutations identified in various human cancers were found in this region, especially in the D310 microsatellite sequence.5,15 For example, among 27 lung tumors for which one third of the mitochondrial genome was sequenced, nine tumors were found to have somatic mutation5: eight tumors showed a D-loop mutation, of which seven mutations were insertions or deletions in the mononucleotide repeat sequence D310 between nucleotides 303 and 316-318. These results made the authors consider that the D310 sequence was a mutational hotspot. They analyzed it, therefore, on 220 other various primary tumors that were found to be mutated in 22% of the cases. Interestingly in our series, the number of cytosines in the D310 repeat in nonmalignant tissues strongly influenced the prevalence of mutations in the D310 sequence. Similar results have been found in lung cancers.23 The majority of D310 mutations were observed when the number of cytosines was more than seven (ie, eight or nine). These findings suggest that as for nDNA the length of base repetition could influence the rate of replicative errors. Therefore, this variation in the number of cytosines in the D310 repeat could explain discrepancies among previously published results.5,7,17-20 No association was observed between the presence of a microsatellite instablity phenotype and the presence of mutation in the D310 sequence or within the D-loop sequence. In one series,17 a correlation was found between the presence of D310 mutations and the presence of mutations in a repeated coding sequence within MTND1 and MTND5 genes, suggesting a molecular mechanism similar to that observed in tumors with a microsatellite instability phenotype for which there is evidence of nuclear genetic instability through the presence of frameshift mutations in coding repeated gene sequences.24 In the present study, sequencing of the entire mitochondrial genome in 11 cases showed mutations within MTCO1 and MTCO3 genes in a nonrepeated sequence. These two mutated tumors were associated with an nDNA microsatellite-stable tumor phenotype and were associated with a D310 sequence alteration in only one of the two cases. These results do not support a general defect in DNA repair for the occurrence of the D310 mutations in colorectal cancer.
The role of mtDNA mutations in tumor development and progression has not yet been resolved. The D-loop is a noncoding sequence of the mitochondrial genome that is implicated in mtDNA replication and transcription. Within this sequence region, the D310 sequence is located in the conserved sequence block II, which is an essential element for mtDNA replication25 because it contains the H-strand replication origin. Mutations in the D310 sequence may therefore affect the rate of mtDNA replication. Some authors recently found a decrease in the copy number of mtDNA in 70% of 24 hepatocellular carcinoma mutated in the D-loop, which is consistent with this hypothesis.26 Futhermore, in colorectal cancer cells, alterations in the tumor mitochondrial gene expression have been found. High levels of MTND2 mRNA have been detected in primary tumors of 15 patients when compared with matched healthy tissues.27 Moreover, mtDNA analysis revealed an increase of MTRNR2, MTND4, MTND4L, MTCYB, MTCO2, MTATP6 and MTATP8 gene expression in HT-29 adenocarcinoma cell line.28 On the contrary, a progressive decrease was shown in the mean level of the MTCO3 gene expression through colon adenomas to carcinomas as compared with healthy mucosae.29 However mtDNA was not sequenced in these studies. As the noncoding D-loop contains the major mtDNA transcription promoters and as frequent D-loop mutations have been found in our series of colorectal cancers, we hypothesized that such mutations may alter mtDNA transcription. According to this hypothesis, somatic mutation in the D-loop may lead to respiratory chain alteration, which is responsible for high reactive oxygen species levels release and could contribute to nuclear genome damage and cancer initiation and promotion.30-32 Moreover, respiratory chain alteration may cause a dysfunction in mitochondrion-induced apoptosis.33,34 These results are consistent with the hypothesis of Warburg,35 for whom an alteration of the respiratory machinery resulting in compensatory increase in glycolytic ATP production was considered to be a key event in the carcinogenesis. Malignant cells satisfy their energy needs by producing a large proportion of their ATP through glycolytic ATP mechanisms rather than through oxydative phosphorylation. Some authors have also recently found a bioenergetic signature in several human carcinomas, including colorectal cancers, characterized by a low proteomic-based bioenergetic cellular index.36,37 Study of mitochondrial gene expression by a quantitative real time polymerase chain reaction method and of mitochondrial respiratory chain functionality by measurement of oxygen consumption in several mutated and nonmutated colorectal tumor cell lines should be assessed to confirm these hypotheses.
Our results suggest for the first time that somatic mutations in the D-loop are not only a potential tumor marker, but an independent prognostic factor in colorectal cancer. In a multivariate analysis, the relative risk (RR) of death in patients with D-loop mutation was significantly higher (RR, 1.41; 95% CI, 1.02 to 1.95; P = .034) than that of patients without D-loop mutation, independently of other prognostic factors including tumor stage, which is the most important one in colorectal cancer. Interestingly, subgroup analyses showed the limited benefit of adjuvant FU-based chemotherapy in stage III colon cancer patients when their tumor mtDNA was mutated in the D-loop. These results, which should be confirmed in a large prospective randomized study considering the small number of patients taken into account for the analysis and its retrospective nature, suggest that mutation in the D-loop region induced resistance to FU-based chemotherapy in colon cancer. Although no experimental data supports the role of D-loop mutation in the resistance to this drug, in vitro experiments show some evidence that several chemotherapy drugs modulate or interfere with mithochondrial function and require a functional respiratory chain to be efficient,38-41 leading to the hypothesis that mtDNA determines the cellular response to some cancer therapeutic agents.39,40,42 Singh et al40 have determined the in vitro survival of the HSL2 cell line (Rho+ cells) and its derivative cell line lacking mtDNA (Rho0 cells) after exposure to different anticancer agents, and found that Rho0 cells were resistant to adriamycin-induced cell death, whereas the Rho+ cells were sensitive to adriamycin-induced cell death. They showed that exposure to adriamycin leads to mtDNA mutations, providing evidence of their role in cellular response to this chemotherapy. However, in this study the resistance to adriamycin was not due to changes in the apoptotic cell death. This result is consistent with the fact that cytochrome c release and caspase activation were maintained after exposure to staurosporine, a widely used apoptosis inducer, in Rho0 cells.41 Two others experimental studies have shown that the mitochondrial respiratory chain was essential to obtain a response to CD437 and BMD188, two pro-apoptotic anticancer agents.39,42 Because FU interferes with the apoptotic process,43 we speculate that the potential respiratory chain alteration induced by D-loop mutations could be in part a cause of the resistance found to FU. Sensitivity/resistance of colorectal cancer cell lines to this specific drug clearly need to be investigated in presence and absence of D-loop mutation to confirm this hypothesis.
Survival rates calculated from incident population—based cases should be distinguished from data established from a series of hospital patients or from patients included in clinical trials. Due to differences in the patient selection process, which affects these latter two types of studies, differences can be notable. This present study has the advantage of being representative of the real survival in a well-defined French population. Identification of new prognostic factors that would allow selection of high-recurrence-risk stage II and stage III colon cancer patients for whom intensification of adjuvant chemotherapy would be favorable is needed. Such an intensification of chemotherapy with new efficient drugs like irinotecan or oxaliplatin should be assessed in patients with D-loop mutation since our results have suggested in these patients a resistance to adjuvant chemotherapy based on FU alone.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
Acknowledgment
We thank Xavier Jeunemaitre, MD, PhD, for providing sequencing facilities at the European Georges Pompidou Hospital, Anne Berger, MD, PhD, for providing tumor tissues, Anne-Marie Houllier and Philippe Coudol for their technical assistance.
NOTES
Supported by L'Association de Recherche Contre le Cancer (ARC No. 3329); La Societe Nationale Franaise de Gastroenterologie; La Region Ile de France, La Fondation de France, Paris, France.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Cai J, Wallace DC, Zhivotovsky B, et al: Separation of cytochrome c-dependent caspase activation from thiol-disulfide redox change in cells lacking mitochondrial DNA. Free Radic Biol Med 29:334-342, 2000
Hail N Jr, Youssef EM, Lotan R: Evidence supporting a role for mitochondrial respiration in apoptosis induction by the synthetic retinoid CD437. Cancer Res 61:6698-6702, 2001
Sakaguchi Y, Stephens LC, Makino M, et al: Apoptosis in normal tissues induced by 5-fluorouracil: Comparison between bolus injection and prolonged infusion. Anticancer Res 14:1489-1492, 1994(Astrid Lièvre, Caroline C)
Ple de Cancerologie, Hpital Europeen Georges Pompidou, Paris
Centre Hospitalier Universitaire (CHU) Dijon, Service d'Anatomo-Pathologie
INSERM Equipe Mixte INSERM (EMI) 0106, CHU, Registre Bourguignon des Tumeurs
Centre de Pathologie, Rue Nicolas Bornier
Centre Georges Franois Leclerc, Service d'Anatomo-Pathologie, Dijon, France
ABSTRACT
PURPOSE: Prognostic factors that could select high-risk recurrence colorectal cancer patients and predict chemosensitivity are needed. Since mutations of mitochondrial DNA (mtDNA) have been described in different types of cancers and since they may play a role in response to anticancer agents, we investigated in a population-based series of colorectal cancer patients the clinical value of mtDNA mutations.
PATIENTS AND METHODS: The displacement loop (D-loop) region of mtDNA was sequenced on a series of 365 patients recorded in the Digestive Cancer Registry of Cte-d'Or (France) between 1998 and 2000. Clinicopathologic characteristics were correlated to the presence of a D-loop mutation. Survival rates were compared with the log-rank test. A multivariate survival analysis was performed.
RESULTS: D-loop mutations were found in 38.3% of the tumors. The 3-year survival rate was 53.5% in patients with D-loop mutation versus 62.1% in patients without (P = .05). After adjustment for age, stage, and microsatellite instability status, the relative risk of death in patients with D-loop mutation was 1.40 (95% CI, 1.02 to 1.93; P = .034) as compared with those without. In stage III colon cancers, adjuvant chemotherapy was beneficial only for patients without D-loop mutation (3-year survival, 78.3% v 45.4%, P < .02). In those with D-loop mutation who received adjuvant chemotherapy, the relative risk of death was 4.30 (95% CI, 1.23 to 15.00; P < .02).
CONCLUSION: The D-loop region is a hotspot for somatic mutations in colorectal tumors. Moreover, presence of tumor D-loop mutation appears to be a factor of poor prognosis in colorectal patients and a factor of resistance to fluorouracil-based adjuvant chemotherapy in stage III colon cancers.
INTRODUCTION
Colorectal cancer is one of the most common human malignancies, with more than 300,000 cases both in the United States and in the European Union each year.1 Prognosis remains poor despite progress in its management.2 Surgery is the primary treatment for colorectal cancer, but over the last two decades, adjuvant and palliative treatments have been developed with the aim of improving survival. There is a need to gain additional clues to explain chemosensitivity. Although much knowledge has been collected concerning alterations in cancer cell nuclear DNA (nDNA), less attention has been paid to mutations within mitochondrial DNA (mtDNA) despite extensive evidence of mitochondrial involvement in apoptosis. mtDNA is a 16,569 base-pair (bp), double-stranded, circular DNA composed of genes and of a noncoding region, the displacement loop (D-loop), located between nucleotides 16,024 and 576, which contains essential transcription and replication elements. It has been reported that mtDNA was more susceptible to mutations than nDNA3,4 and frequently mutated in different types of cancers,5-16 including 10% to 70% of colorectal carcinomas.5,7,17-20 These findings suggest a potential role of mitochondrial genome in tumor carcinogenesis. Several hotspots of mtDNA mutations have been described. In particular a polymorphic polycytosine CnTC6 sequence (named D310 sequence) of the D-loop was found as a major target for mtDNA mutations in cancers.5 Since most studies have not sequenced the entire mitochondrial genome and have focused their analysis only on a few coding regions or exclusively on the D-loop,5,7,17,18,20 available data about mtDNA mutations in colorectal cancers are few and inconsistent. The aims of this work were to find in colorectal cancer a mutational hotspot by sequencing the complete mitochondrial genome in 11 patients, to assess the relations between this mutational hotspot and the clinicopathologic characteristics of colorectal cancer, and to determine the chemosensitivity impact of these mutations in a population-based series of 365 patients.
PATIENTS AND METHODS
Patients
As a first step, tumors and matched healthy colon tissues (taken from surgical margins) were collected at Hpital Laennec (Paris, France) from 11 patients (three males and eight females; median age, 62 years) who underwent colorectal cancer resection in the department of surgery between 1997 and 1999. These cases were selected according to the tumor phenotype determined by the genotyping of five microsatellites (BAT-26, BAT-25, D2S123, D5S346 and D17S250). The unstable phenotype (MSI-H) was assigned following the criteria defined by the international consensus conference on microsatellite instability.21 Five MSI-H and six microsatellite stable (MSS) tumors were retained for futher analysis.
As a second step, tumor and healthy colon tissues were obtained from 365 patients (198 males and 167 females; mean age, 71.7 years; standard deviation, 12.2) who underwent colorectal cancer resection in the Cte d'Or area (Burgundy, France) between January 1998 and December 2000. Samples were obtained from a population-based frozen-tissue bank. This includes all patients with resected colorectal cancer who live in the administrative area of Cte-d'Or. Due to the small size of samples, carcinomas resected by polypectomy were not included in this study (n = 100). Data were merged with that of a digestive cancer registry, which routinely collects data on patient characteristics, stage at diagnosis, treatment, and survival. One major advantage of this type of analysis is the absence of bias concerning the selection of patients, as compared with hospital or clinical trials series. Tumor site was classified according to the International Classification of Diseases into colon cancers (C18, 250 cases) and rectal cancers (C19 to C20, 115 cases). Cancer extension at the time of diagnosis was classified according to the TNM classification: 31 (8.5%) were stage I, 157 (42.7%) were stage II, 93 (25.8%) were stage III, and 84 (23%) were stage IV. Forty-one tumors (11.2%) were MSI-H, 322 were MSS, and two cases were inconclusive. Data on adjuvant and palliative chemotherapy was recorded. The French Consensus Conference on colorectal cancer recommends adjuvant chemotherapy in stage III colon cancer and palliative chemotherapy in advanced colorectal cancers—stage IV and in nonresected cases.22 Adjuvant chemotherapy was fluorouracil (FU) -based. Only two patients received FU/oxaliplatin based chemotherapy in adjuvant setting.
Tissue Sample Preparation and DNA Extraction
Tumor and nontumor matching tissues were obtained immediately after surgical resection, frozen in liquid nitrogen, and stored at –80°C until DNA extraction. Before DNA extraction, the presence of tumor cells in the tumor fragment was assessed by hematoxylin-eosine-safran coloration, and fragments with more than 70% of tumor cells were retained for DNA extraction. The samples were treated with proteinase K, and total cellular DNA was extracted using QIAmp DNA Mini Kit (Qiagen, Courtaboeuf, France) according to the manufacturer's recommendations.
Polymerase Chain Reaction Amplification
To amplify the entire mtDNA of the first 11 patients, 8 pairs of primers described previously19 were used. Large polymerase chain reaction (PCR) products (1 to 3 kb each) were generated to avoid amplification of nuclear pseudogenes. For amplification, 100 ng of mtDNA were used in a final 50 μL master mix containing 1x final concentration of 10x cloned Pfu DNA polymerase reaction buffer (Stratagene, Amsterdam, Holland), 250 μmol/L of each deoxy nucleotide triphosphate (dNTP), 0.5 μmol/L of each primer and 1 μL (2.5 UI) of Pfu DNA polymerase (Stratagene). Total DNA was subjected to step-down PCR protocol on a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA) at 95°C for 2 minutes for a total of 35 cycles; 95°C for 30 seconds; 50°C to 68°C for 30 seconds (according to each pair of primers); 72°C for 1 to 3 minutes (according to each amplified fragment length); and a final extension at 72°C for 10 minutes (detailed protocol available on request).
For D-loop amplification of the remaining patients, the primers used were F47: 5'-CGCACGGACTACAACCACGAC-3' (forward) and R15: 5'-CTGTGGGGGGTGTCTTTGGG (reverse). Fifty to 100 ng of mtDNA were used in a final 20 μL master mix containing 1x final concentration of 10x PCR buffer (Qiagen), 250 μmol/L of each dNTP, 1.5 mmol/L MgCl2, 0.5 μmol/L of each primer, and 0.1 μL of HotStartTaq DNA Polymerase (Qiagen). Total DNA was subjected to the following step-down PCR protocol: 95°C for 15 minutes, 35 cycles; 95°C for 30 seconds; 64°C for 30 seconds; 72°C for 2 minutes; and a final extension at 72°C for 10 minutes. PCR products were then purified using G-50 Sephadex superfine (Amersham Biosciences, Orsay, France) on Multiscreen support (Millipore, Bedford, MA).
Direct Sequencing of mtDNA
Purified PCR products were sequenced using a Big Dye Terminator cycle sequencing kit (Applied Biosystems) on an ABI Prism 3700 DNA Analyser automated sequencer (Applied Biosystems). Seventy-three primer pairs described previously19 were used to sequence the entire mtDNA of the first 11 patients and one primer, DS2: 5'-ACCTACGTTCAATATTACAGGCG-3', was used to sequence the D310 homopolymeric C-tract. For sequencing, 2 μL of purified PCR product were used in a final 20 μL mix containing 1 μL of Big Dye Terminator V 3.1, 0.875x final concentration of 5x Big Dye Terminator sequencing buffer (Applied Biosystems) and 0.5 μmol/L of each primer. DNA was then submitted to the following cycle conditions: 94°C for 4 minutes; 25 cycles, 96°C for 30 seconds, 50°C for 15 seconds, and 60°C for 4 minutes. The results of DNA sequence analysis were compared with the published reference mtDNA sequence (GenBank, access number J01415) using Autoassembler software (Applied Biosystems). Sequence variations found in both healthy and tumor tissues mtDNA were scored as germ-line polymorphisms. Each was then checked against the Mitomap database (http://www.mitomap.org). Those not recorded in the database were categorized as novel mtDNA polymorphisms. Any mtDNA sequences that differed between tumor and matched healthy tissue mtDNA were scored as somatic mutations. All somatic mutations found were further validated by a new independent amplification and sequencing.
Statistical Analysis
Associations among categoric data were analyzed using 2 tests for heterogeneity. The crude survival rates were calculated using the Kaplan-Meier method. Survival curves were compared using the log-rank test. Patients who died within 30 days after resection were excluded from the survival analysis. The life status was known for the 365 patients in January 2004. A special survey was conducted in order to collect this data. Information about life status was ascertained from dearth certificates, registrar of the place of birth and place of residence, or from practitioners.
The median follow-up was 39 months. Multivariate survival analysis was performed using a Cox proportional hazards model. Analysis was carried out using the STATA software package (Statistical Software for Professionals, College Station, Texas).
Ethics Committee
This study was evaluated by the Consultative Committee Protecting Persons in Biomedical Research (Dijon, France), according to French law.
RESULTS
Analysis of the Entire mtDNA in 11 Patients
The entire mtDNA sequencing of the first 11 patients has allowed identification of a total of 10 somatic mutations in seven patients (table 1). Eight mutations were found in the D-loop region and two were found in the coding region of mtDNA, within MTCO1 and MTCO3, genes which led to the following amino acid changes: I87T and G234S respectively. Among the D-loop mutations, six were located in the D310 repeat (insertion or deletion of one to three base pairs). The other two were located in position 16,390 (G16390A) and 300 (300delA). Two different D-loop mutations were associated in one patient (A11B11) and two D-loop mutations were associated with a coding region mutation in another patient (A7B7). In total, six (54%) of 11 patients with a colorectal cancer exhibited mutations in the D310 sequence, which can be considered as a hotspot of mtDNA mutations in colorectal carcinoma patients. The frequency of the mtDNA mutations was 66% in MSS and 40% in MSI-H tumors (P = not significant).
Furthermore, 133 polymorphisms were identified in these 11 patients, of which 98 were previously recorded and 35 were considered as new polymorphisms.
Analysis of a 400-bp Fragment of the D-Loop (nucleotide position, 190-590) Containing the D1310 Repeat
A somatic D-loop mutation was identified in 142 of the 365 tumors. The D310 mononucleotide repeat was found mutated in 132 of them (36%). The alterations detected in the D310 repeat were insertions or deletions of one to several base pairs. Seventy-two (50.7%) of the total mutated patients had homoplasmic mutations (Fig 1A) and sixty-six (46.5%) had heteroplasmic mutations (Fig 1B). Four of them (2.8%) had homoplasmic and heteroplasmic mutations at the same time. Eleven patients had mutations outside the D310, associated for three of them with a mutation of the D310 repeat. Thus, only eight of the 140 patients had a D-loop mutation outside the D310 sequence.
The D310 sequence is polymorphic in the human population. The number of cytosines in the 7-bp stretch varied from six to 13. The most frequent sequences for the D310 region are C7TC6, C8TC6, and C9TC6, which were present respectively in 178 of 365 (49%), 113 of 365 (31%) and 15 of 365 (4%) of the nonmalignant tumor tissues in our series. The prevalence of D310 alterations increased significantly with the number of cytosines in the sequence. Indeed, the C7TC6 sequence was altered in 16 patients (9%) of the 178, the C8TC6 was altered in 56 patients (49.5%) of the 113, and the C9TC6 sequence was altered in 11 patients (73%) of the 15, respectively (P < 10–16).
MtDNA Mutations, Clinicopathologic Data, Survival and Response to Chemotherapy
Clinicopathologic data. After completion of the detailed sequence analysis in all specimens, clinicopathologic data were correlated with the molecular analysis. No significant association between sex, age, tumor site, stage at diagnosis, or MSI status and the presence of mtDNA mutation were found (table 2).
Overall, 121 patients with colorectal cancer received chemotherapy. Among them, 82 received adjuvant chemotherapy: patient had stage I disease, 19 patients had stage II, 54 patients had stage III (42 colon and 12 rectal cancers) and 8 had stage IV. Thirty-nine stage IV cancers received palliative chemotherapy. The use of chemotherapy was significantly associated with age of patients. For stage III colon cancer, the proportion of patients receiving chemotherapy was 100.0% for patients younger than 65 years, 71.4% for the 65 to 74 years group, and 47.1% thereafter, respectively (P < .001). The corresponding percentages for stage IV colorectal cancers were 60.7%, 75.0%, and 32.1%, respectively (P < .004).
Survival of Colorectal Cancer Patients
In univariate analysis, after exclusion of 21 patients who died within 30 days after surgical resection, the 3-year overall survival of colorectal cancer patients with tumor mutation in the D-loop sequence was lower than that of patients with no tumor mutation in the D-loop sequence: 53.5% versus 62.1%, respectively (P = .05; Fig 2). In an overall multivariate analysis, D-loop sequence status was an independent predictor of survival, with a relative risk of death after adjustment for age, tumor stage, tumor site, and MSI status of 1.41 (95% CI, 1.02 to 1.95; P = .034) in patients with a D-loop mutation when compared with those without D-loop mutation (Table 3). When rectal cancer cases were excluded from the analysis, 3-year overall survival of colon cancer patients with tumor mutation in the D-loop was 54.5% compared with 64.7% for patients without mutation (P = .0387).
A subgroup analysis was performed examining stage II, stage III and stage IV colorectal patients. No significant difference was observed in 3-year overall survival of patients presenting a D-loop mutation as compared with those without D-loop mutation. However, the prognosis tended to be worse among patients with D-loop mutation: 72.9% (95% CI, 59.0% to 82.8%) compared with 76.7% (95% CI, 65.8% to 84.5%) for stage II disease; 50.1% (95% CI, 30.4% to 66.9%) compared with 65.8% (95% CI, 52.0% to 76.5%) for stage III disease; and 13% (95% CI, 3.9% to 27.7%) compared with 11.8% (95% CI, 4.1% to 23.9%) for stage IV.
Mutation of the D-Loop and Response to Chemotherapy
Because only 19 of the 156 stage II colorectal cancer patients received adjuvant chemotherapy, no further subgroup analysis was performed for them.
Considering the subgroup of stage III colon cancer patients for whom adjuvant chemotherapy has been demonstrated efficient in terms of survival,22 the 3-year survival rate was 47.4% for patients presenting a D-loop mutation as compared with 70.0% (P < .02) for those without D-loop mutation (Fig 3A). Adjuvant chemotherapy was beneficial only for patients without tumor D-loop mutation: the 3-year survival rate for patients with D-loop mutation was 45.4% compared with 78.3% for patients without mutation (P < .02; Fig 3B). Among patients not receiving chemotherapy, the 3-year survival rate for cases with tumor D-loop mutation was 41.0% compared with 42.9% for cases without mutation (P = not significant; Fig 3C). After adjustment for age and MSI status, both chemotherapy and the absence of D-loop mutation were significantly and independently related to a longer survival (Table 4). The relative risk of death for stage III colon cancer patients with tumor D-loop mutation was 2.51 (95% CI, 1.04 to 6.02; P = .04) as compared with those without tumor mutation. When considering the group of stage III colon cancers treated with adjuvant chemotherapy, the relative risk of death for patients with tumor D-loop mutation was 4.30 (95% CI, 1.23 to 15.00; P < .02).
Among patients with advanced colorectal cancers receiving chemotherapy, the 1-year survival rate was slightly worse for those with D-loop mutation as compared to those without mutation (63.2% [95% CI, 37.9 to 80.4] v 71.4% [95% CI, 50.9 to 84.6]; P = not significant). The survival rates became similar at the 3-year limit, 12.5% (95% CI, 2.1 to 32.8) and 13.8% (95% CI, 3.1 to 32.2), respectively.
DISCUSSION
The sequencing of the entire mitochondrial genome from healthy and tumor mtDNA of 11 colorectal cancer patients allowed demonstration that the D-loop, especially the D310 mononucleotide repeat within the D-loop, was a hotspot for somatic mutations in colorectal tumors because 54% of them were found mutated in this region. This result was confirmed by the study of a population-based series of 365 colorectal cancers for which the D310 sequence was found mutated in 36% (95% CI, 32% to 42%) of the cases. Most of these mutations were insertions or deletions in the polymorphic C7/8/9/10TC6 tract of the D310 sequence. These results are similar to those obtained in previous studies including few patients, which found a D310 mutation in 28% to 44% of colorectal cancers.5,17,18 However, and surprisingly, the only study in which a sequencing of the entire mitochondrial genome was done from 10 human colorectal cell lines did not find any mutation in the noncoding region of the D-loop.19 Despite these contradictory results, we considered our findings sufficient to justify restriction of further sequencing to the D-loop region since most of the mtDNA mutations identified in various human cancers were found in this region, especially in the D310 microsatellite sequence.5,15 For example, among 27 lung tumors for which one third of the mitochondrial genome was sequenced, nine tumors were found to have somatic mutation5: eight tumors showed a D-loop mutation, of which seven mutations were insertions or deletions in the mononucleotide repeat sequence D310 between nucleotides 303 and 316-318. These results made the authors consider that the D310 sequence was a mutational hotspot. They analyzed it, therefore, on 220 other various primary tumors that were found to be mutated in 22% of the cases. Interestingly in our series, the number of cytosines in the D310 repeat in nonmalignant tissues strongly influenced the prevalence of mutations in the D310 sequence. Similar results have been found in lung cancers.23 The majority of D310 mutations were observed when the number of cytosines was more than seven (ie, eight or nine). These findings suggest that as for nDNA the length of base repetition could influence the rate of replicative errors. Therefore, this variation in the number of cytosines in the D310 repeat could explain discrepancies among previously published results.5,7,17-20 No association was observed between the presence of a microsatellite instablity phenotype and the presence of mutation in the D310 sequence or within the D-loop sequence. In one series,17 a correlation was found between the presence of D310 mutations and the presence of mutations in a repeated coding sequence within MTND1 and MTND5 genes, suggesting a molecular mechanism similar to that observed in tumors with a microsatellite instability phenotype for which there is evidence of nuclear genetic instability through the presence of frameshift mutations in coding repeated gene sequences.24 In the present study, sequencing of the entire mitochondrial genome in 11 cases showed mutations within MTCO1 and MTCO3 genes in a nonrepeated sequence. These two mutated tumors were associated with an nDNA microsatellite-stable tumor phenotype and were associated with a D310 sequence alteration in only one of the two cases. These results do not support a general defect in DNA repair for the occurrence of the D310 mutations in colorectal cancer.
The role of mtDNA mutations in tumor development and progression has not yet been resolved. The D-loop is a noncoding sequence of the mitochondrial genome that is implicated in mtDNA replication and transcription. Within this sequence region, the D310 sequence is located in the conserved sequence block II, which is an essential element for mtDNA replication25 because it contains the H-strand replication origin. Mutations in the D310 sequence may therefore affect the rate of mtDNA replication. Some authors recently found a decrease in the copy number of mtDNA in 70% of 24 hepatocellular carcinoma mutated in the D-loop, which is consistent with this hypothesis.26 Futhermore, in colorectal cancer cells, alterations in the tumor mitochondrial gene expression have been found. High levels of MTND2 mRNA have been detected in primary tumors of 15 patients when compared with matched healthy tissues.27 Moreover, mtDNA analysis revealed an increase of MTRNR2, MTND4, MTND4L, MTCYB, MTCO2, MTATP6 and MTATP8 gene expression in HT-29 adenocarcinoma cell line.28 On the contrary, a progressive decrease was shown in the mean level of the MTCO3 gene expression through colon adenomas to carcinomas as compared with healthy mucosae.29 However mtDNA was not sequenced in these studies. As the noncoding D-loop contains the major mtDNA transcription promoters and as frequent D-loop mutations have been found in our series of colorectal cancers, we hypothesized that such mutations may alter mtDNA transcription. According to this hypothesis, somatic mutation in the D-loop may lead to respiratory chain alteration, which is responsible for high reactive oxygen species levels release and could contribute to nuclear genome damage and cancer initiation and promotion.30-32 Moreover, respiratory chain alteration may cause a dysfunction in mitochondrion-induced apoptosis.33,34 These results are consistent with the hypothesis of Warburg,35 for whom an alteration of the respiratory machinery resulting in compensatory increase in glycolytic ATP production was considered to be a key event in the carcinogenesis. Malignant cells satisfy their energy needs by producing a large proportion of their ATP through glycolytic ATP mechanisms rather than through oxydative phosphorylation. Some authors have also recently found a bioenergetic signature in several human carcinomas, including colorectal cancers, characterized by a low proteomic-based bioenergetic cellular index.36,37 Study of mitochondrial gene expression by a quantitative real time polymerase chain reaction method and of mitochondrial respiratory chain functionality by measurement of oxygen consumption in several mutated and nonmutated colorectal tumor cell lines should be assessed to confirm these hypotheses.
Our results suggest for the first time that somatic mutations in the D-loop are not only a potential tumor marker, but an independent prognostic factor in colorectal cancer. In a multivariate analysis, the relative risk (RR) of death in patients with D-loop mutation was significantly higher (RR, 1.41; 95% CI, 1.02 to 1.95; P = .034) than that of patients without D-loop mutation, independently of other prognostic factors including tumor stage, which is the most important one in colorectal cancer. Interestingly, subgroup analyses showed the limited benefit of adjuvant FU-based chemotherapy in stage III colon cancer patients when their tumor mtDNA was mutated in the D-loop. These results, which should be confirmed in a large prospective randomized study considering the small number of patients taken into account for the analysis and its retrospective nature, suggest that mutation in the D-loop region induced resistance to FU-based chemotherapy in colon cancer. Although no experimental data supports the role of D-loop mutation in the resistance to this drug, in vitro experiments show some evidence that several chemotherapy drugs modulate or interfere with mithochondrial function and require a functional respiratory chain to be efficient,38-41 leading to the hypothesis that mtDNA determines the cellular response to some cancer therapeutic agents.39,40,42 Singh et al40 have determined the in vitro survival of the HSL2 cell line (Rho+ cells) and its derivative cell line lacking mtDNA (Rho0 cells) after exposure to different anticancer agents, and found that Rho0 cells were resistant to adriamycin-induced cell death, whereas the Rho+ cells were sensitive to adriamycin-induced cell death. They showed that exposure to adriamycin leads to mtDNA mutations, providing evidence of their role in cellular response to this chemotherapy. However, in this study the resistance to adriamycin was not due to changes in the apoptotic cell death. This result is consistent with the fact that cytochrome c release and caspase activation were maintained after exposure to staurosporine, a widely used apoptosis inducer, in Rho0 cells.41 Two others experimental studies have shown that the mitochondrial respiratory chain was essential to obtain a response to CD437 and BMD188, two pro-apoptotic anticancer agents.39,42 Because FU interferes with the apoptotic process,43 we speculate that the potential respiratory chain alteration induced by D-loop mutations could be in part a cause of the resistance found to FU. Sensitivity/resistance of colorectal cancer cell lines to this specific drug clearly need to be investigated in presence and absence of D-loop mutation to confirm this hypothesis.
Survival rates calculated from incident population—based cases should be distinguished from data established from a series of hospital patients or from patients included in clinical trials. Due to differences in the patient selection process, which affects these latter two types of studies, differences can be notable. This present study has the advantage of being representative of the real survival in a well-defined French population. Identification of new prognostic factors that would allow selection of high-recurrence-risk stage II and stage III colon cancer patients for whom intensification of adjuvant chemotherapy would be favorable is needed. Such an intensification of chemotherapy with new efficient drugs like irinotecan or oxaliplatin should be assessed in patients with D-loop mutation since our results have suggested in these patients a resistance to adjuvant chemotherapy based on FU alone.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
Acknowledgment
We thank Xavier Jeunemaitre, MD, PhD, for providing sequencing facilities at the European Georges Pompidou Hospital, Anne Berger, MD, PhD, for providing tumor tissues, Anne-Marie Houllier and Philippe Coudol for their technical assistance.
NOTES
Supported by L'Association de Recherche Contre le Cancer (ARC No. 3329); La Societe Nationale Franaise de Gastroenterologie; La Region Ile de France, La Fondation de France, Paris, France.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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