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Abstract

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

We previously reported that mitochondrial transcription factor A (mtTFA) preferentially recognizes cisplatin-damaged DNA via physical interaction with p53 and is upregulated by treatment with cisplatin and fluorouracil (5-FU). The aim of the present study was to evaluate whether expression of mtTFA predicts the clinical outcome in patients with metastatic colorectal cancer treated with modified 5-fluorouracil, leucovorin and oxaliplatin 6 (mFOLFOX6). Fifty-nine patients with metastatic lesions from colorectal cancer treated with mFOLFOX6 were included in this study. The subjects consisted of 25 women and 34 men with a median age of 62 years. The patients were treated with oxaliplatin (85 mg/m2) plus leucovorin (200 mg/m2) as a 2-h infusion on day 1, followed by 5-FU (400 mg/m2) bolus and 46-h continuous infusion of 2400 mg/m2. The expressions of mtTFA and p53 of resected primary tumors were examined by immunohistochemical analysis. Of the 59 patients, 33 (complete response 1, partial response 32) achieved a confirmed response to therapy. The positive cytoplasmic staining rate for mtTFA was 44.1% and that for p53 was 59.3%, respectively. Strong expression of mtTFA was detected in eight of 33 complete response/partial response (24.2%) and in 18 of 26 SD/PD (69.2%), indicating that mtTFA expression was significantly correlated with response to chemotherapy (P < 0.01). Median overall survival was significantly longer in patients without mtTFA expression (P = 0.0493). Multivariate analysis revealed that mtTFA expression significantly affected overall survival (hazard ratio 2.10, P = 0.036). Immunohistochemical study of mtTFA may be useful for predicting the clinical outcome of metastatic colorectal cancer patients treated with FOLFOX. (Cancer Sci 2011; 102: 578–582)

Somatic cells have an average of 100–500 mitochondria with 1–15 mitochondrial DNA (mtDNA) molecules per mitochondrion.(1) Mitochondrial DNA is a circular molecule that is only 16 569 bp in length(2) and encodes 13 respiratory-chain subunits. Mitochondrial biogenesis requires gene products from both the nucleus and the mitochondrion.

Platination of mitochondrial DNA has also been reported.(3–6) Some in vitro experiments and in vivo studies have revealed higher incorporation of cisplatin into mitochondrial DNA than into genomic DNA.(3,5,6) However, a higher degree of mitochondrial DNA platination may partly result from the following features of this type of DNA. Mitochondria contain double-stranded circular DNA, which has no histones, and as a result lacks nucleosomal organization.(7) Therefore, mitochondrial DNA is more easily accessible for DNA-damaging agents than genomic DNA. Moreover, mitochondria do not efficiently repair DNA because they do not perform nucleotide excision repair.(8) The impact of mitochondrial DNA platination on cell viability is not well elucidated. Mitochondria are known to be involved in apoptotic processes.(9,10) Thus, the binding of platinum-based anticancer drugs to mitochondrial DNA may play a role in the induction of cytotoxic effects.

Oxaliplatin is a third-generation platinum (Pt) drug that has been approved for treatment of colorectal cancer.(11) Its main mode of action is through formation of Pt adducts on genomic DNA. The Pt-DNA adducts have been shown to stall replication and transcription and recruit DNA damage recognition proteins, which leads to apoptosis.(12,13) Moreover, oxaliplatin can induce mitochondrial apoptosis even in the absence of nuclear DNA, indicating that oxaliplatin sensitivity may be involved in mitochondria.(14)

Mitochondrial transcription factor A (mtTFA) is a member of the high mobility group (HMG)-box protein family(15) that stimulates transcription of mitochondrial genes by binding to the mitochondrial D-loop region. Nuclear HMG-box proteins, such as HMG1/HMG2, are ubiquitous in higher eukaryotic cells and bind preferentially to cisplatin-damaged DNA.(16–19) Mitochondrial transcription factor A is essential not only for mitochondrial gene expression but also for mtDNA maintenance and repair.(20) In addition, increased apoptosis has been observed in mtTFA knockout animals, suggesting that mtTFA is involved in apoptosis.(21) mtTFA binds to four-way DNA junctions(22) and recognizes cisplatin-damaged DNA.(23) We previously reported that mtTFA preferentially recognizes cisplatin-damaged DNA via physical interaction with p53 and is upregulated by the treatment with cisplatin and fluorouracil (5-FU).(10) Moreover, binding of purified mtTFA protein (100 ng) to 32P-labeled double-stranded oligonucleotides (22-mers) with or without oxaliplatin treatment was analyzed. We confirmed that mtTFA preferentially recognizes oxaliplatin-damaged DNA as well as cisplatin-damaged DNA by electrophoretic mobility shift assay (Kimitoshi Kohno, unpublished data, 2010). Therefore, it is thought that expression of mtTFA and oxaliplatin are closely related. Compared with radio-sensitive cell lines, radio-resistant cell lines had higher mtTFA levels. Downregulation of mtTFA using an RNA interference technique enhanced the susceptibility of radio-resistant cells to gamma rays.(24)

On the basis of these findings, we tested the hypothesis whether mtTFA would predict the clinical outcome of patients with advanced colorectal cancer treated with 5-fluorouracil, leucovorin and oxaliplatin (FOLFOX).

Patients and Methods

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Patients.  From January 2006 to April 2008, we retrospectively analyzed 59 patients (34 men, 25 women) who had metastatic lesions (Stage IV) from colorectal cancer treated with mFOLFOX6 as a first-line chemotherapy at the Osaka Rosai Hospital. The studied specimens were obtained from 59 patients who underwent surgery for primary colorectal carcinoma between 1997 and 2007 in the Department of Surgery at the Osaka Rosai Hospital. All of the patients with synchronous metastasis enrolled underwent non-curative operation. Table 1 shows the histological classifications and other clinicopathological factors. This study was approved by the Institutional Review Board at the Osaka Rosai Hospital and all individual patients gave informed consent for biological study.

Table 1.   Patient and clinicopathological data
  1. A, ascending colon; C, cecum; D, descending colon; mFOLFOX6, modified 5-fluorouracil, leucovorin and oxaliplatin 6; muc, mucinous adenocarcinoma; por, poorly differentiated adenocarcinoma; P, proctos; R, rectum; S, sigmoid colon; T, transverse colon; tub1, well differentiated adenocarcinoma; tub2, moderately differentiated adenocarcinoma.

mFOLFOX6Median (range) 8 (4–22) 
Age (years)Median (range)62 (29–84) 
GenderMale3457.60%
Female2542.40%
Histologytub11322.00%
tub23762.70%
por 610.20%
muc 35.10%
LocationC 23.40%
A 46.80%
T 46.80%
D 46.80%
S1932.20%
R2644.10%
P 00%

Anti-mtTFA antibody.  Polyclonal antibodies against human mtTFA were generated by multiple immunizations of a New Zealand white rabbit using synthetic peptides as described previously.(10) The sequence of synthetic peptide for mtTFA was KRTIKKQRKYGAEEC (K plus amino acids 233–246). A dilution of 1:2000 in phosphate-buffered saline (PBS) containing 2% bovine serum albumin was used to immunostain the paraffin-embedded sections.

Immunohistochemistry.  Tumor samples resected from patients with colorectal carcinoma were fixed in formalin and embedded in paraffin. Immunohistochemistry was performed using a streptavidin–biotin–peroxidase complex method.(25) Tissue sections (2-μm thick) were pretreated twice for 5 min per treatment with citrate buffer (0.01 mol/L; pH 6.0) at 100°C in a microwave oven. Endogenous peroxidase activity was blocked by preincubation of the slides in 3% H2O2 in absolute methanol for 5 min. Each slide was preincubated in normal goat serum for 10 min and then incubated with polyclonal mtTFA antibody for 60 min. The antibody-treated slides were then washed thoroughly, incubated in goat anti-rabbit immunoglobulin for 10 min, washed and incubated with streptavidin-biotinylated horseradish peroxidase complex for 5 min. Finally, diaminobenzidine was used as a chromogen and the sections were lightly counterstained with hematoxylin. Substitution of PBS for the primary antibody was used as the negative control.

Histopathology.  The sections were examined by two independent observers who were blinded to the clinical status of the patients. Staining was evaluated in each section by counting the frequency of labeled cells in five high-power fields containing 100 tumor cells each. This count was classified using the following criteria: positive was defined as moderate to strong cytoplasmic staining in more than 50% of the tumor cells.

Statistical analysis.  Statistical analysis was performed using the chi-square and Mann–Whitney U tests. The Kaplan–Meier method was applied for survival analysis(26) and the log-rank test was used to calculate statistical significance. Univariate and multivariate analyses were performed using a Cox proportional hazards model.(27) The differences were considered statistically significant if the P value was <0.05. All analyses were performed using the StatView statistical package (version 5.0, SAS Institute, Inc., Cary, NC, USA).

Results

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Clinicopathological features and mtTFA expression.  The patient population consisted of 34 (57.6%) men and 25 (42.4%) women. The mean age at the time of tumor resection was 62 years (range, 29–84 years). For investigation of the associations between mtTFA expression and clinicopathological features, the tumors were divided into two groups: tumors with strong mtTFA expression and tumors with weak mtTFA expression. Mitochondrial transcription factor A showed only cytoplasmic expression (Fig. 1). Table 1 summarizes the clinicopathological data of all 59 tumors.

image

Figure 1.  Immunohistochemical mitochondrial transcription factor A (mtTFA) expression in colorectal adenocarcinoma. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue specimens. Immunostaining of colorectal adenocarcinoma considered to have weak (A) or strong (B) expression of mtTFA (original magnification, ×400).

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We examined how closely mtTFA expression was related to various clinicopathological characteristics (Table 2). The expression of mtTFA was not associated with age, gender, tumor differentiation, location, adverse events, liver metastasis, lymph node metastasis or p53 expression (Table 2).

Table 2.   Clinicopathological variables and mtTFA expression
VariablemtTFA(+) (n = 26)mtTFA(−) (n = 33)P value
  1. A, ascending colon; C, cecum; D, descending colon; meta, metastasis; muc, mucinous adenocarcinoma; mtTFA, mitochondrial transcription factor A; por, poorly differentiated adenocarcinoma; P, proctos; R, rectum; S, sigmoid colon; T, transverse colon; tub1, well differentiated adenocarcinoma; tub2, moderately differentiated adenocarcinoma.

Age (years)
 ≥6412 (41.4%)17 (58.6%)0.957
 <6414 (46.7%)16 (53.3%)
Gender
 Male15 (44.1%)19 (55.9%)0.992
 Female11 (44.0%)14 (56.0%)
Histology
 tub1/tub224 (48.0%)26 (52.0%)0.152
 por/muc2 (22.2%)7 (77.8%)
Location
 C/A/T4 (40.0%)6 (60.0%)0.776
 D/S/R22 (44.9%)27 (55.1%)
Adverse events
 <Grade317 (41.5%)24 (58.5%)0.543
 ≥Grade39 (50.0%)9 (50.0%)
Lymph node
 Meta(+)17 (42.5%)23 (57.5%)0.725
 Meta(−)9 (47.4%)10 (52.4%)
Liver
 Meta(+)20 (48.8%)21 (51.2%)0.271
 Meta(−)6 (33.3%)12 (66.7%)
p53 expression
 (+)16 (45.7%)19 (54.3%)0.758
 (−)10 (41.7%)14 (58.3%)
Metastasis
 Synchronous14 (41.2%)20 (58.8%)0.609
 Metachronous12 (48.0%)13 (52.0%)

mtTFA expression and response of patients treated with FOLFOX.  Expression of mtTFA was assessed in a cohort of 59 patients (26 strong mtTFA expression and 33 weak mtTFA expression) who had received FOLFOX therapy. The response rates were 30.8% in strong mtTFA expression and 75.8% in weak mtTFA expression (P = 0.008, Fig. 2A). The response rates were 57.1% in strong p53 expression and 54.2% in weak p53 expression (P = 0.82, Fig. 2B).

image

Figure 2.  Relationship between mitochondrial transcription factor A (mtTFA) or p53 expression and response to 5-fluorouracil, leucovorin and oxaliplatin. Response rates were 30.8% in tumors with strong mtTFA expression and 75.8% in tumors with weak mtTFA expression (A, P = 0.008). Response rates were 57.1% in tumors with strong p53 expression and 54.2% in tumors with weak p53 expression (B, P = 0.82).

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mtTFA expression and prognosis.  We assessed how mtTFA expression affected clinical parameters such as progression-free survival (PFS) and overall survival (OS). Survival analysis was performed for the 59 patients (stage IV) in terms of several individual variables. Patients whose tumors had strong mtTFA expression had lower OS (Fig. 3A, P = 0.0493) and PFS (Fig. 3B, P = 0.046) than patients whose tumors had weak mtTFA expression. Expression of mtTFA had a strong association with survival among the variables tested (Cox proportional hazard model; P = 0.036). The relative hazard ratio for OS in patients whose tumors had weak mtTFA expression was 2.10 relative to patients whose tumors had strong mtTFA expression (95% confidence interval, 0.08–0.92, Table 3). These results indicate that mtTFA is an independent significant prognostic factor.

image

Figure 3.  Kaplan–Meier curves for overall (A) and progression-free (B) survival in patients with colorectal cancer. Positive mitochondrial transcription factor A (mtTFA) expression was associated with poor overall survival.

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Table 3.   Multivariate analyses of prognostic variables for overall survival by Cox proportional hazards model
VariableHR (95% CI)P value
  1. A, ascending colon; C, cecum, CI, confidence interval; D, descending colon, F, female; HR, hazard ratio; M, male; mFOLFOX6, modified 5-fluorouracil, leucovorin and oxaliplatin 6; mtTFA, mitochondrial transcription factor A; P, proctos; R, rectum; S, sigmoid colon; T, transverse colon.

Age 1.71 (0.99–1.13)0.088
GenderM/F0.71 (0.48–4.77)0.48
Histologytub1, tub2/por, muc1.07 (0.07–2.23)0.286
LocationC, A, T/D, S, R0.36 (0.27–6.47)0.738
Adverse events<Grade 3/≥Grade 30.39 (0.40–4.04)0.694
LN meta(−)/(+)0.49 (0.23–2.43)0.627
mFOLFOX6 (cycles) 1.5 (0.71–1.05)0.138
mtTFA expression(−)/(+)2.1 (0.08–0.92)0.036

Discussion

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Intrinsic resistance and acquired resistance are critical factors of Pt-based chemotherapy efficacy. Enhanced tolerance to Pt compounds and a decreased drug uptake may be due in part to increased Pt resistance.(28) Deficiency in mismatch repair activity (intrinsic resistance) and enhanced ability of the replication complex to synthesize DNA past the site of DNA damage (enhanced replicative bypass) were found to be responsible for the resistance to other Pt agents, for example, cisplatin and carboplatin, but were not found to contribute to oxaliplatin resistance.(29)

In the present study, we evaluated how mtTFA expression might be associated with characteristics of 59 colorectal tumors using an antihuman polyclonal mtTFA antibody. We found that immunohistochemical assessment of mtTFA expression in surgically resected specimens provided valuable prognostic information for patients with colorectal carcinoma. The outcome of patients whose tumors had strong mtTFA expression was worse than that of patients whose tumors had weak mtTFA expression. Furthermore, we identified that the OS and PFS of patients with colorectal cancer whose tumors had weaker mtTFA expression were significantly better than that of patients whose tumors had strong mtTFA expression, and multivariate Cox analysis identified mtTFA expression as an independent prognostic factor in patients with colorectal cancer. On the other hand, it appears that the p53 pathway only plays a minor role in oxaliplatin-induced cytotoxicity and that p53 is not involved in oxaliplatin resistance.(14) This result was confirmed in the present study (Fig. 2B).

It is unclear whether mtTFA may be useful for predicting the clinical outcome of metastatic colorectal cancer patients treated with 5-fluorouracil, leucovorin and irinotecan (FOLFIRI). However, because platinum is not included in FOLFIRI, the possibility that mtTFA becomes a predictive factor for FOLFIRI is low. A new prospective clinical trial has already been scheduled to solve this problem. If the clinical trial is completed, we want to publish the results.

To our knowledge, this is the first analysis of mtTFA as a predictive factor for FOLFOX therapy in patients with malignant disease. As with any tumor marker, the results vary depending on the threshold chosen for determining whether a tumor is positive or negative for the marker. To address this question, we classified 59 tumors semi-quantitatively as having 0–25%, 25–50%, 50–75% or 75–100% mtTFA expression. We then examined three different cut-off values: 25%, 50% and 75%. Subsequently, we found that mtTFA is a useful and independent marker of response rate, PFS and OS in patients with colorectal carcinoma only if the mtTFA cut-off value is set at 50%.

Although many clinical, biological and histological variables have been investigated as prognostic indicators, mtTFA may be one of the useful predictors. Molecular analyses as well as further correlative studies of mtTFA expression in patients with other types of adenocarcinoma need to be pursued.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

This paper was presented in part at the 45th Annual Meeting of the American Society of Clinical Oncology, Orlando, Florida in June 2009. This work was supported in part by the Osaka Medical Research Foundation for Incurable Diseases.

Disclosure Statement

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

The authors declare that they have no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References