|Year : 2014 | Volume
| Issue : 1 | Page : 176-179
Absence of correlation between serum CRP levels and mitochondrial D-loop DNA mutations in gastro-oesophageal adenocarcinoma
Benjamin H. L. Tan1, Richard J. E. Skipworth1, Nicholas M Wheelhouse2, Kenneth C. H. Fearon1, James A Ross1
1 Clinical and Surgical Sciences (Surgery), University of Edinburgh, Royal Infirmary, Edinburgh, United Kingdom
2 Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, United Kingdom
|Date of Web Publication||23-Apr-2014|
Benjamin H. L. Tan
University of Edinburgh, Tissue Injury and Repair Group, Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB
Source of Support: None, Conflict of Interest: None
Introduction: Both inflammation and mitochondrial DNA (mtDNA) mutation are thought to play a role in the many human cancers. The aim of this study was to evaluate the relationship between inflammation and accumulation of mitochondrial DNA (mtDNA) mutations in the D-loop region in carcinogenesis of gastro-oesophageal adenocarcinomas.
Materials and Methods: Blood samples of 20 patients with gastro-oesophageal adenocarcinoma were taken for measurement of serum C-reactive protein (CRP) concentration. Direct sequencing of mtDNA in the D-loop region was done in the 20 adenocarcinoma samples and their corresponding surrounding non-cancerous tissue. Sequences were compared with existing mtDNA databases to identify mutations.
Results: mtDNA mutations in the D-loop region occur commonly with almost identical frequency in both non-cancerous tissue (3.0 1.6) and adenocarcinoma (3.1 1.9) (P = 0.916, paired t-test). CRP levels are not predictive of the number of D-loop mutations in both adenocarcinoma (β: -0.131; 95% CI: -2.354-1.364; P = 0.583) and non-cancerous tissue samples (β: 0.130; 95% CI: -1.125-1.933; P = 0.586). Five new mutations were identified that were not recorded previously in mtDNA databases.
Conclusion: D-loop mtDNA mutations are common in both gastro-oesophageal adenocarcinoma and surrounding non-cancerous tissue. However, the accumulation of such mutations appears to occur independent of systemic inflammation. The frequency of D-loop mutations is likely not useful as a marker for carcinogenesis in gastro-oesophageal adenocarcinoma.
结果：在D环区的mtDNA突变频率在非癌组织（3±1.6）和腺癌（3.1±1.9）中几乎相同（P = 0.916，配对t检验）。CRP水平不能预测D环突变数目，无论在腺癌（β：-0.131；95%可信区间：-2.354~1.364；P = 0.583），还是在非癌组织标本（β：0.130；95%可信区间：-1.125~1.933；P = 0.586）。有5个新的突变发现，先前并没有记录在线粒体DNA数据库。
Keywords: D-loop, gastro-oesophageal adenocarcinoma, inflammation, mitochondrial DNA mutations, serum CRP
|How to cite this article:|
Tan BH, Skipworth RJ, Wheelhouse NM, Fearon KC, Ross JA. Absence of correlation between serum CRP levels and mitochondrial D-loop DNA mutations in gastro-oesophageal adenocarcinoma. J Can Res Ther 2014;10:176-9
|How to cite this URL:|
Tan BH, Skipworth RJ, Wheelhouse NM, Fearon KC, Ross JA. Absence of correlation between serum CRP levels and mitochondrial D-loop DNA mutations in gastro-oesophageal adenocarcinoma. J Can Res Ther [serial online] 2014 [cited 2020 Aug 9];10:176-9. Available from: http://www.cancerjournal.net/text.asp?2014/10/1/176/131458
| > Introduction|| |
Gastro-oesophageal malignancies are the third leading cause of cancer-related deaths in the UK.  Despite advances in both staging techniques and treatments, five-year survival rarely exceeds 50%.  Systemic inflammation has been associated with a number of malignant diseases; in particular, adenocarcinomas of the stomach and oesophagus are frequently associated with preceding inflammatory alterations of normal mucosa.  Up to 50% of patients with cancer have an acute-phase protein response at the time of diagnosis, including patients with upper gastrointestinal malignancy.  The systemic inflammatory response, as evidenced by elevated circulating concentrations of C-reactive protein, has also been shown to be a disease-independent prognostic factor in gastro-oesophageal cancer. 
It has been suggested that reactive oxygen species are one of the most important factors in chronic inflammation-related carcinogenesis.  ROS tend to injure mitochondrial DNA more frequently than nuclear DNA because of a lack of histones, which protect against ROS-induced DNA injury, in the mitochondria. Alterations of mtDNA have been recognized to play an important role in the pathogenesis of cancer and mutations occur most frequently in the displacement loop region of mtDNA. ,
The D-loop region is the major control site for mtDNA expression because it contains the leading strand for the origin of replication and major promoters for transcription.  It has been suggested that the constitutive hypervariable areas such as the mtDNA D-loop are hot spots for somatic mutations in malignant tumors.  D-loop mutations have been detected in a wide variety of cancers. ,,,, In particular, the D310 mononucleotide repeat within the D-loop is a hotspot for somatic mutations in colorectal,  gastric,  lung,  and thyroid tumors.  Mutations in the D514 region have been associated with thyroid tumors. 
With a view to understanding the role of systemic inflammation and mtDNA mutations in the pathogenesis of gastro-oesophageal adenocarcinoma, this study examined the relationship between CRP and the accumulation of D-loop mutations in both malignant tissue and surrounding non-cancerous tissue. In addition, clinicopathological characteristics were analyzed to determine if they were predictors of the frequency of D-loop mutations.
| > Materials and Methods|| |
Serum was collected from each patient at the time of diagnosis. CRP was determined using an automated immunoturbidimetric assay (Abbott TDX, Abbott Laboratories, Maidenhead, UK). A level of 10 mg/L or above was considered as an evidence of systemic inflammation.
Tissue samples were collected from 20 patients with histologically confirmed gastro-oesophageal adenocarcinoma at the time of surgery with curative intent. Following surgical resection, paired samples of tumor tissue, and adjacent normal mucosa were dissected from each patient specimen by a consultant pathologist. The tissue samples were then snap-frozen in liquid nitrogen prior to storage at -80°C and, later, DNA analysis.
Tumors around the gastro-oesophageal junction were classified according to Siewert; those classified as type I and II were staged as oesophageal and type III were staged as gastric. All adenocarcinoma samples were examined prospectively by a consultant pathologist and histologically graded as well-differentiated, moderately differentiated, or poorly differentiated. All samples were obtained with approval from appropriate local ethics committee and informed consent was obtained from recruited patients.
DNA extraction from tissue
DNA was extracted using the Wizard Genomic DNA purification kit (Promega, Southampton, UK) according to the manufacturer's instructions. DNA concentration and quality was quantified by absorbance (A) readings taken at 260 nm and 280 nm using an Ultrospec 2000 UV/visible spectrophotometer (Pharmacia Biotech, Bucks, UK).
Sequencing of the D-loop region of mitochondrial DNA
Each DNA sample (100 ng) was subjected to amplification by polymerase chain reaction (PCR) of a 334-bp region spanning hypervariable region II (HVR II) of the D-loop (forward primer: 5'-gagctctccatgcatttggt-3'; reverse primer: 5'-tctttgtttttggggtttgg-3'). Cycle conditions were as follows: an initial denaturation step of 94°C for 5 min; two cycles of 94°C for 45 s, 64°C for 1 min 15 s, and 70°C for 30 s; 35 cycles of 94°C for 45 s, 58°C for 1 min 15s, and 70°C for 30 s; and final extension step of 70°C for 10 min. Sequencing was performed with an ABI 377 gel-based sequencing machine after labelling with the BigDye terminator cycle sequencing kit 3.1 on a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA), using the same PCR primers.
Analysis of mutations
Sequences were compared against an mtDNA sequence (Genbank accession no. J01415) using the 'Blast 2 Sequences' program. Identified sequence variants and mutations were compared with three different mitochondrial genome databanks: MITOMAP (http://www.mitomap.org), mtDB (http://www.genpat.uu.se/mtDB), and HmtDB (http://www.hmtdb.uniba.it).
Data are presented as mean ± standard deviation unless otherwise stated.
The significance of clinicopathological variables such as age, tumor stage, tumor differentiation, and systemic inflammation on D-loop mutations were analyzed using linear regression. Comparison of D-loop mutations in adenocarcinoma and its corresponding non-cancerous tissue was analyzed using the paired t-test. P < 0.05 was regarded as statistically significant. Statistical analysis was performed using SPSS 16.0 statistical package (SPSS Inc., Chicago, IL, USA).
| > Results|| |
[Table 1] presents the details of the 20 patients with gastro-oesophageal adenocarcinoma. The majority of patients were male (70%), and 60% of the patients had stage I/II disease. The primary tumor sites were gastric (n=11, 55%), oesophagus (n=6, 30%), and those arising from gastro-oesophageal junction (n=3, 15%). Histological confirmation and differentiation was obtained in all cases, and half of these were found to be well-differentiated adenocarcinomas. CRP concentrations were 20.1 ± 32.1 mg/L (mean ± S.D), and nine patients had systemic inflammation (CRP ≥ 10 mg/L). The number of D-loop mutations in adenocarcinoma samples was 3.1 ± 1.9. This was very similar to the number of D-loop mutations (3.0 ± 1.6) in non-cancerous samples (P = 0.916, paired t-test). Systemic inflammation is not a significant predictor of the number of D-loop mutations both in cancerous and non-cancerous tissue [Table 2]. Moreover, no significant relationships were observed between the number of D-loop mutations and age, tumor stage, or tumor differentiation in both cancerous and non-cancerous samples [Table 2].
|Table 2: Predictors of frequency of D - loop mutations in both gastro - oesophageal adenocarcinoma and non - cancerous tissue|
Click here to view
In total, 24 different mutations were identified in HVR II of the D-loop. Of these, five mutations have not been previously recorded in any mitochondrial genome databank [Figure 1]. These are all point mutations: 140C → T, 233T → C, 242C → G, 289A → T, and 299C → A. In addition, eight mutations were found only in adenocarcinoma specimens but not in non-cancerous tissue, and three mutations were found occurring only in non-cancerous tissue [Figure 1].
|Figure 1: Mutations identified in the D-loop region. Underlined mutations were not previously recorded in any mitochondrial database. Mutations with * were present only in adenocarcinoma but not in non-cancerous tissue. Mutations with ** were present only in non-cancerous tissue but not in adenocarcinoma|
Click here to view
| > Discussion|| |
Mitochondrial DNA is a small (16.6-kb) self-replicating molecule that encodes 13 essential proteins of the mitochondrial oxidative phosphorylation complexes, 2 rRNA, and 22 tRNA genes. The D-loop region is the major control site for mtDNA expression containing the origin of replication for the heavy DNA strand and the major promoters of transcription.  The hypervariable sites in D-loop may be hotspots in mtDNA for mutations, including those associated with cancer.  The present study showed that D-loop mutations occur commonly with almost equal frequency in both gastro-oesophageal adenocarcinoma and adjacent non-cancerous tissue, suggesting that mutagenic stressors may be already well established in the surrounding non-cancerous tissue.
The association between chronic inflammation and gastro-oesophageal adenocarcinomas is well known, and D-loop mutations have been found in both gastric and oesophageal malignancies. , The association between accumulation of mtDNA D-loop mutations resulting from chronic inflammation have been previously reported in a study on hepatocelluar carcinoma by Tamori and colleagues.  However, in this study, no correlation was observed between CRP, a marker of systemic inflammation, and the accumulation of D-loop mutations in both gastro-oesophageal adenocarcinoma as well as adjacent non-cancerous samples. Perhaps, local inflammation, which does not result in a rise in CRP, is the major determinant of mtDNA mutations. Another possible explanation is that accumulation of D-loop mutations secondary to inflammation may be tumor or tissue specific.
The frequency of mtDNA mutations have been known to increase with age,  but this association is not observed in D-loop mutations in the present study, although this may be skewed by the small number of patients below 65 years of age in this study. Furthermore, no discernable associations were found between the number of D-loop mutations and stage of disease or tumor differentiation.
Eight point mutations were found in adenocarcinoma samples but not in non-cancerous tissue, which may represent somatic mutations. Interestingly, a further three mutations were found in non-cancerous tissue only. Brandon et al.  have hypothesized that tumorigenic mutations, which are advantageous in the initial phases of tumor growth, may undergo selective loss once the tumor is well formed. These three mutations could be such examples. The current study, however, is not powered sufficiently to elucidate if these are potential biomarkers for carcinogenesis in gastro-oesophageal adenocarcinoma. In addition, five novel point mutations were identified in this study (140C → T, 233T → C, 242C → G, 289A → T, and 299C → A). Determining the functional consequences of these mtDNA D-loop region variants requires further investigation.
Overall, mtDNA D-loop mutations accumulate in both gastro-oesophageal adenocarcinoma and surrounding non-cancerous tissue independent of systemic inflammation. While specific D-loop mutations may have a role in carcinogenesis, our results suggest that the frequency of D-loop mutations is not useful as a marker for carcinogenesis in gastro-oesophageal adenocarcinoma.
| > References|| |
|1.||Deans C, Rose-Zerilli M, Wigmore S, Ross J, Howell M, Jackson A, et al. Host cytokine genotype is related to adverse prognosis and systemic inflammation in gastro-oesophageal cancer. Ann Surg Oncol 2007;14:329-39. |
|2.||Portale G, Peters JH, Hsieh CC, Tamhankar AP, Almogy G, Hagen JA, et al. Esophageal adenocarcinoma in patients < or = 50 years old: Delayed diagnosis and advanced disease at presentation. Am Surg 2004;70:954-8. |
|3.||Gomes LI, Esteves GH, Carvalho AF, Cristo EB, Hirata R Jr, Martins WK, et al. Expression profile of malignant and nonmalignant lesions of esophagus and stomach: Differential activity of functional modules related to inflammation and lipid metabolism. Cancer Res 2005;65:7127-36. |
|4.||Deans DA, Tan BH, Wigmore SJ, Ross JA, de Beaux AC, Paterson-Brown S, et al. The influence of systemic inflammation, dietary intake and stage of disease on rate of weight loss in patients with gastro-oesophageal cancer. Br J Cancer 2009;100:63-9. |
|5.||Crumley AB, McMillan DC, McKernan M, Going JJ, Shearer CJ, Stuart RC. An elevated C-reactive protein concentration, prior to surgery, predicts poor cancer-specific survival in patients undergoing resection for gastro-oesophageal cancer. Br J Cancer 2006;94:1568-71. |
|6.||Jackson AL, Chen R, Loeb LA. Induction of microsatellite instability by oxidative DNA damage. Proc Natl Acad Sci USA 1998;95:12468-73. |
|7.||Chatterjee A, Mambo E, Sidransky D. Mitochondrial DNA mutations in human cancer. Oncogene 2006;25:4663-74. |
|8.||Parsons TJ, Muniec DS, Sullivan K, Woodyatt N, Alliston-Greiner R, Wilson MR, et al. A high observed substitution rate in the human mitochondrial DNA control region. Nat Genet 1997;15:363-8. |
|9.||Taanman JW. The mitochondrial genome: Structure, transcription, translation and replication. Biochim Biophys Acta 1999;1410:103-23. |
|10.||Fliss MS, Usadel H, Caballero OL, Wu L, Buta MR, Eleff SM, et al. Facile detection of mitochondrial DNA mutations in tumors and bodily fluids. Science 2000;287:2017-9. |
|11.||Akouchekian M, Houshmand M, Hemati S, Ansaripour M, Shafa M. High rate of mutation in mitochondrial DNA displacement loop region in human colorectal cancer. Dis Colon Rectum 2009;52:526-30. |
|12.||Ding Z, Ji J, Chen G, Fang H, Yan S, Shen L, et al. Analysis of mitochondrial DNA mutations in D-loop region in thyroid lesions. Biochim Biophys Acta 2010;1800:271-4. |
|13.||Tamori A, Nishiguchi S, Nishikawa M, Kubo S, Koh N, Hirohashi K, et al. Correlation between clinical characteristics and mitochondrial D-loop DNA mutations in hepatocellular carcinoma. J Gastroenterol 2004;39:1063-8. |
|14.||Miyazono F, Schneider PM, Metzger R, Warnecke-Eberz U, Baldus SE, Dienes HP, et al. Mutations in the mitochondrial DNA D-Loop region occur frequently in adenocarcinoma in Barrett′s esophagus. Oncogene 2002;21:3780-3. |
|15.||Zhao YB, Yang HY, Zhang XW, Chen GY. Mutation in D-loop region of mitochondrial DNA in gastric cancer and its significance. World J Gastroenterol 2005;11:3304-6. |
|16.||Lievre A, Chapusot C, Bouvier AM, Zinzindohoue F, Piard F, Roignot P, et al. Clinical value of mitochondrial mutations in colorectal cancer. J Clin Oncol 2005;23:3517-25. |
|17.||Wu CW, Yin PH, Hung WY, Li AF, Li SH, Chi CW, et al. Mitochondrial DNA mutations and mitochondrial DNA depletion in gastric cancer. Genes Chromosomes Cancer 2005;44:19-28. |
|18.||Suzuki M, Toyooka S, Miyajima K, Iizasa T, Fujisawa T, Bekele NB, et al. Alterations in the mitochondrial displacement loop in lung cancers. Clin Cancer Res 2003;9:5636-41. |
|19.||Maximo V, Lima J, Soares P, Botelho T, Gomes L, Sobrinho-Simoes M. Mitochondrial D-Loop instability in thyroid tumours is not a marker of malignancy. Mitochondrion 2005;5:333-40. |
|20.||Wei YH, Lee HC. Oxidative stress, mitochondrial DNA mutation, and impairment of antioxidant enzymes in aging. Exp Biol Med (Maywood) 2002;227:671-82. |
|21.||Brandon M, Baldi P, Wallace DC. Mitochondrial mutations in cancer. Oncogene 2006;25:4647-62. |
[Table 1], [Table 2]