AJMB

Non-Invasive Detection of Esophageal Cancer using Genetic Changes in Circulating Cell-Free DNA

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Volume 4, Issue 1, January-March , Page 3 to 13
Saturday, December 24, 2011 :Received , Tuesday, January 31, 2012 :Accepted


  • - Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
  • Corresponding author Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran, Tel: 98 21 22432022 Fax: 98 21 22432021 E-mail: Ardekani@avicenna.ac.ir
    - Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran

Abstract: Cell free DNA (cfDNA) is a genetic biomarker that is present in serum or plasma in high concentration in many types of cancer. Identification of circu-lating cancer related DNA molecules in serum or plasma is a non-invasive tool for early diagnosis and prognosis in many cancer patients. For this review, study selection and data extraction were performed by the authors. Detection of point mutations, microsatellite alterations, DNA hypermethylations and losses of heterozygosity in circulating cell free DNA have been characterized in esophagus cancer. Application of circulating cell free DNA as a biomarker, provide the best opportunity for constructing non-invasive tests for early de-tection, prognosis and management of cancer patients, after therapy in many types of cancer.

 

 

Introduction :
The esophagus is a muscular tube that con-nects the throat to the stomach. Cancer of the esophagus, also called esophageal cancer, can occur any place along the liner of the tube. Among the diverse types of existent neo-plasms, esophageal carcinoma is the eighth deadliest malignancy worldwide. Adenocar-cinoma and squamous cell are liable for more than 95 percent of all esophagus cancers (1).
The predominant histological subtype in esophageal cancer is squamous cell carcinoma (SCC) which contributes to about 80% of all esophageal cancers in the world (2). SCC has the highest incidence in Western countries with traditions of alcohol consumption, smok-ing or tobacco, hot drinks and malnutrition (3-5). ADC is found in industrialized countries, with gastric esophageal reflux (which causes Barrett´s Esophagus), obesity, substances de-rived from the grain of moldy corn (fumo- nisins), alcoholism, and smoking (Table 1) (3,6,7).

 

Epidemiology and Pathology of Esophageal Cancer :
Epidemiology
Epidemiologic data have shown variability in determining attitude in incidence of esoph-ageal carcinoma malignancies worldwide. The ascending incidence of esophageal cancer over the past two decades conformed to the change in histological type and primary tumor location. An estimated 14,500 deaths from esophageal cancer occurred in the United States in 2010 (8).

The American Cancer Society's most recent estimates for esophageal cancer in the United States for 2011 are as follows: about 16,980 new esophageal cancer cases diagnosed (13,450 in men and 3,530 in women) and about 14,710 deaths from esophageal cancer (11,910 in men and 2,800 in women). This disease is 3-4 folds higher among men than women (9). The incidence of esophageal can-cer varies remarkably with geographic region,

especially in developing nations. ADC is the most common type of esophageal malignancy in the United States and Western Europe (10). In America, the reported incidence of esoph-ageal cancer in patients is 3.2-4.0 per 100,000 persons (11), while in Europe and Asia, reports are 0.5-7.0 and 0.02-0.4 per 100,000 persons, respectively (12,13).
The incidence of SCC among the Asian countries is higher than that of ADC, espe-cially in countries and areas of East Asia such as Korea and Japan with incidence of 8.2-21 per 100,000 persons (14,15). Noticeable is the observation that the highest rates occurred in northern China and northern Iran, with inci-dence of about 40 per 100,000 persons in 2003 (13,16).

Pathology
Esophageal cancers are usually found with signs and symptoms that a person is having. The most common symptoms of esophageal cancer include: weight loss, difficulty in swal-lowing, chest pain, constant cough, bone pain, hiccups and pneumonia (17). These symptoms are usually present for several months before medical treatment is sought and initially pre-sents itself by having difficulty in swallowing dry foods. Furthermore, weight loss of 10 per-cent of normal body weight occurs in less than six months. About half of esophageal cancer patients present with locally advanced unresectable disease or distant metastasis.
ADC spreads via transverse penetration through the full thickness of the wall, whereas SCC tends to spread linearly in a submucosal fashion (18). Esophageal cancer spreads through extensive lymphatic channels with a skip me-tastases pattern when observed in autopsy specimens (19).

Staging in esophageal cancer
The diagnostic evaluations of ADC and SCC are essentially identical. Several strat-egies and approaches are acceptable for diag-nosis, staging, chemoradiotherapy, treatment, and surveillance of patients with esophageal cancer. These include endoscopy (20), bron-choscopy (21), thoracoscopy (22), laparoscopy (23), computed tomography (24), and surgery (25). However, maybe some of these approaches and strategies are associated with painful ex-periences and cause discontent for cancer patients.
Recently, research focus has been in the development of non-invasive screening at the early stages of cancer. In most cases, early detection of cancers is extremely difficult be-cause there are multiple uncertainties related to the location of the disease but it is neces-sary for full treatment and recovery. Current-ly, among the various approaches used for screening cancer patients, circulating molecu-lar biomarkers have been found to be of the most convenient and useful non-invasive tool with esophageal carcinoma (26).

Molecular genetic changes in esophageal cancer
The most common genetic alterations in esophageal cancer involve genetic alterations in several oncogenes, tumor suppressor genes, and DNA repair genes. Furthermore, the most common alterations found in esophageal can-cers such as deletions, amplifications and Loss Of Heterozygosity (LOH) has been re-ported to occur on several chromosomes list-ed in table 2 (27).

Mutations in oncogenes and tumor suppressor genes
Tumor suppressor genes p53 and p16 have been introduced to the primordial G1 cell cycle regulatory genes (28). Alteration of these genes such as LOH and mutation can lead to the loss of regulation of cell growth, which is important in carcinogenesis (29). Mutation of p53 has been extensively investigated in both SCC and ADC (30,31). Previous studies have reported the incidence of p53 mutations (53 percent in ADC and more than 93 percent in SCC) (32) especially, in the exons 5-8 (encod-ing the DNA-binding domain of the protein) in SCC. In addition to mutations in p53, LOH at chromosome 17q13 is a significant event in both SCC and ADC.
In principle, the hypotheses were that p53 alteration occurs frequently as an early event in the tumor progression of esophagus carcin-oma (33). However, inactivation of the p16 gene, LOH, genetic mutation and aberrant DNA methylation in the coding and non-coding (promoter) regions also frequently occur in both SSC and ADC (34,35). Tarmin et al, has reported the incidence of LOH in about 50 to 65 percent of patients with SCC at the 9p21 chromosomal locus (36). However, aber-rant p16 hypermethylation in the promoter region is a common mechanism for the inacti-vation of this gene in SCC. Taghavi et al, showed the aberrant hypermethylation of this gene in 62 percent of SCC patients in Iran (37).
Previously, Abbaszadegan et al, had report-ed incidence of this aberration in SCC to be 73.3 percent in the northeastern Iran (38), while Hardie et al, reported that hypermethylation of the p16 promoter is 85 percent in ADC (39). Furthermore, inactivation of p14 and p15 genes has been observed in esophagus cancer. Aberrant methylation in the promoter regions of these genes was reported to be associated with loss of transcription in previous studies (40,41). Xing et al, reported alteration of methyl-ation patterns in the promoter of p14 and p15 genes and incidence of LOH in p14 and p15 genes in SCC tumor samples (42).
The Adenomatous Polyposis Coli (APC) gene is a tumor suppressor gene located at chromosome 5q21 and is involved in esoph-agus cancer (43). One of the alterations in this gene is LOH at 5q, occurring in about 55-80 percent of SCC and 20-55 percent of ADC (44,45) cases, but mutation of APC is under 10 percent in this cancer type (46). The most com-mon type of gene inactivation occurs via hypermethylation in the promoter region of the APC gene with an incidence of 92 percent in ADC and 50 percent in patients with SCC (47). Therefore, the hypermethylation of this gene has noticeable roles in both SCC and ADC.
The cyclin D1 gene, located at chromosome 11q13, encodes a protein that is required for controlling the cell cycle (48). Previous studies have analyzed cyclin D1 expression in patients with esophagus cancer and aberrant overex-pression of this gene in 23-73 percent of pa-tients with SCC has been reported (49,50). Furthermore, Metzger et al, showed an overe-xpression and gene amplification of cyclin D1 in 22-64 percent of patients with ADC (27).
The erbB-2 (HER2) is one of the members of Epidermal Growth Factor Receptor (EGFR) family, which acts as tyrosine kinase receptor. This receptor has an intracellular tyrosine ki-nase activity and extracellular binding do-mains. The erbB-2 oncogene encodes a trun-cated form of EGFR which contains continu-ous tyrosine kinase activity (51). The overex-pression and gene amplifications of erbB-2 has been demonstrated in patients with SCC and ADC with 20 to 60 percent frequency (52).

Microsatellite instability
Microsatellites are short tandem repeats of nucleotide sequences found at about 5000 base pair intervals. Microsatellite Instability (MSI) has been recognized as a length muta-tion that occurs especially in microsatellites. The two genes studied in this alteration in-clude MSH2 and MLH1 which are located at chromosomes 2p and 3p, respectively. The functions of these genes are essential in DNA mismatch repair and reduction of genomic replicative error rate. Several studies have reported 10 to 20 percent frequency of MSI in patients with ADC and SCC, with a higher frequency in SCC patients (53-55).

 

Molecular Biomarkers :
Circulating molecular biomarkers
Currently, progress of proteomics has open-ed the door to cancer-related biomarker dis-covery. Proteomics is the complete descript-tion of all proteins encoded by the genome, called the proteome (56). Advances in proteo-mic technologies such as the development of quantitative proteomic methods, high-reso-lution and high-throughput methods have been used to identify and understand patho-physiology of carcinomas. Rapid advances in the field of proteomics promise discovery of biomarkers which could potentially aid in early diagnosis, prognosis and accurate pre-diction of outcomes during cancer treatments and management (57-62).
Fortunately, biological fluids such as urine, blood, serum or plasma contain many bio-markers originating in many different tissues of the body. In the past few decades different biomarkers have been tested as screening tools for cancer patients. Proteins are prob-ably the first-generation of biomarkers to have been investigated and discovered in the biological fluids. For example Prostate Spe-cific Antigen (PSA), CA19-9, CA125, squa-mous cell carcinoma antigen (SCC) and cyto-keratin 19 fragment (CYFRA) are clinically applied as biomarkers (63-66). However, these conventional biomarkers have low sensitivity and insufficient specificity. Thus, researchers have looked toward the other types of tumor biomarkers (67).
Recent advances in analytical assay tech-nologies have allowed development of ampli-fication techniques that are based on circu-lating nucleic acids (RNA and DNA) as bio-markers in serum or plasma of cancer patients (68). Over the past decade, tremendous amount of information has been accumulated which are related to cancer-specific changes such as gene mutation, Single Nucleotide Polymorph-ism (SNP), gene deletion, LOH, epigenetic al-terations (69), genome instability (70) and aber-rant at the expression level at RNAs and pro-tein levels (71,72).
In recent years, efforts in many laboratories throughout the world have been focused on the utilization of genetic and expression ab-normalities in circulating biomarkers for early detection, prognosis and assessment of cancer patients after therapy (73).

Biological characteristics of cfDNA
In 1948, Mandel and Metais reported the existence of circulating extracellular nucleic acids in human blood (74). Thereafter, Stroun et al, showed biological characteristics of circulating cell free DNA (75). Circulating cfDNAs are small fragments of genomic DNA present in the plasma or serum (76). The mechanisms of the occurrence of cfDNA in blood under normal and pathological condi-tions remain unknown. Two main mechan-isms of release cfDNA in the plasma or serum have been postulated: (a) cells detach and extravasate into the blood stream where they undergo lysis and (b) cells undergo apoptosis or necrosis in cancer tissues in vivo and their DNA is released in the blood stream (77). Both molds simultaneously contribute to cfDNA production in cancer patients.
In the programmed enzymatic apoptosis, low molecular weight DNA fragments of about 185-200 base pairs are released in the necrosis products, much of the high molecular weight DNA fragments of about 400 base pairs were detected in serum and plasma (78,79). The role of macrophages in degradation of DNA fragments and then release into the bloodstream has been suggested to probably occur through engulfing process of necrotic or apoptotic cells (80).

Isolation and quantification of cfDNA
The cfDNA is extracted using phenol/ chloroform or standard commercially avail-able DNA kits such as glass-milk-based methods, nucleospin blood kit and PureGene DNA isolation kit from serum or plasma tumor patients. Furthermore, a variety of DNA quantification methods is utilized to measure including DipStick (Invitrogen), fluorometry with SYBR-green and real-time PCR (76). Diehl et al, developed a technique called BEAMing (beads, emulsion, amplification and magnetics) for quantification of circu-lating mutant cfDNA in serum or plasma of tumor patients (81). However, with the appear-ance of novel approaches incorporating auto-mated DNA isolation and amplification with multiplex quantitative fluorescent PCR of circulating cfDNA, the potential to utilize cfDNA as a screening tool for many types of cancer exists.

 

Non-Invasive Screening of Esophagus Cancer using cfDNA :
Alterations at the cfDNA level
Leon et al reported that the average cfDNA concentration in the serum or plasma of can-cer patients is higher than in normal subjects (82). This is because the increase of cfDNA levels in the serum or plasma of tumor pa-tients are contributed by both tumor DNA and non-tumor DNA. However, meta-analysis data has shown high discrepancy in the cfDNA concentrations between studies. And this probably depends on variables such as number of patients in a given study, cancer subtypes, tumor size, stages, location, and other risk-related factors (75).
Unfortunately, high variability in the abnor-mal cfDNA concentrations has prevented it from becoming more than a critical biomarker in each of malignancies (83). One of the com-plicating facts is that an increased plasma cfDNA level has been also observed due to inflammation (84), trauma (85), premalignant states (86), after exercise (87), and in patients suffering from acute or chronic illnesses (88,89).
Several investigators have reported circu-lating cfDNA levels to be significantly higher in patients with malignancies such as lung, breast, colon, hepatocellular, ovarian, pro-state, and melanoma than in healthy individ-uals (90-93). In another study, Banki et al, re-ported that cfDNA level in patients with esophageal cancer was significantly higher and after complete resection of the tumor, the cfDNA level returned to normal (94). Chikawa et al, reported similar results in which the average circulating cfDNA concentrations from esophageal cancer patients were shown to be significantly higher than those in healthy controls (95). An earlier study had shown that the average concentration of cfDNA in serum was 13 ng/mL in a healthy individual and
180 ng/mL in various cancer patients (80). De-tection of differences in cfDNA levels may be a good approach and potentially a valuable tool for early detection and for the evaluation of the prognosis of patients with esophagus carcinoma.

Methylation analysis

Aberrant DNA methylation patterns as a measurement of epigenetic changes are fre-quently found in several types of cancer. The epigenetic changes are described as changes in hypomethylation and hypermethylation levels in special gene regions (e.g. promoter regions). For example, tumor suppressor genes have been demonstrated to be hyper-methylated at an early stage of tumorigenesis and hypermethylation process is known to silence regulatory genes involved in the cel-lular pathways related to cancer. Esteller et al, proposed detection of aberrant methylation changes of cfDNA in the serum or plasma in tumor patients and suggested it can be used as a tool for early detection and monitoring of the efficacy of therapy in tumor patients (96).

Kawakam et al, observed hypermethylation in APC DNA, with incidence of 6.3 percent in the plasma of SCC patients and 25 percent in plasma of ADC patients (97). Furthermore, Hibi et al, detected aberration in methylation level of the promoter of p16 gene in serum DNA of 18 percent of SCC cancer patients (98). Liu et al, assessed the methylation status of Wnt antagonist family of genes in esoph-ageal cancer patients and found that hyper-methylation of promoters for SFRP-1, WIF-1, DKK-3, and RUNX-3 genes could be detected in plasma DNA using Methylation-Specific PCR method (99). Therefore, detection of aber-rant promoter hypermethylation of cancer re-lated genes in serum may be useful for esoph-ageal cancer early diagnosis and detection of recurrence.

Microsatellite analysis
The presence of microsatellite alterations in the serum or plasma could be used as a prog-nostic indicator. Microsatellite analysis of cir-culating cfDNA represents an appearing group of biomolecular tumor markers, where as control subjects show no serum alterations (100). Claus et al, analyzed several micro-satellite markers which are commonly altered in serum of patients with SCC of the esoph-agus. These markers were located at chromo-somes 9p (p16), 17p (p53) and 5q (APC gene) and he observed that in 96.4 percent of cases, at least one alteration was found in the serum DNA specimen of esophagus cancer patients (101). Identification and analysis of microsatel-lite alterations in the serum DNA from SCC patients may be a valuable tool for evaluation at early stage and follow-up studies.

 

Conclusion :
Early diagnosis of cancer provides much promise for full recovery of patients. Unfortu-nately, conventional methods of cancer scre-ening are often invasive and expensive. Sensi-tivity and specificity of these methods are also insufficient for diagnosis of cancer at an earlier stage. For this reason, many research-ers are attempting to increase sensitivity and specificity of methods for early detection and monitoring of tumor recurrence.
In the past decade, there has been a revo-lution in the number of studies analyzing cfDNA of tumor, as a reliable substitute for tissue analysis and a possible tool for the prognosis and diagnosis of cancer patients. At the present time, the major advantage of cfDNA as a biomarker, is the easy accessibil-ity and its stability in plasma or serum speci-mens.
Another advantage is the use of minimally invasive method for obtaining blood to ana-lyze cfDNA and this may have the potential to replace the existing cancer tissue biomark-ers in the future. The combined observations that a correlation exists between molecular al-terations such as mutations, microsatellite in-stability and aberrant methylations in cfDNA and clinical data in esophageal cancer pa-tients, is a strong indicator that cfDNA would likely play a role in early diagnosis and prog-nosis of esophageal cancer patients in the future.
Furthermore, recent developments of se-quencing methods and characterization of cir-culating cfDNA obtained from a variety of cancer patients in different conditions would increase our knowledge about the mechan-isms and functions of circulating cfDNA. Ex-ploration for novel cancer biomarkers with improved diagnostic sensitivity and specifi-city should help clinicians to apply therapeut-ics more efficiently and effectively during the management of the cancer disease.

 



Table 1. The most common risk factors affecting the development of esophageal carcinomas SCC, Squamous cell carcinoma; ADC, Adenocarcinoma. Information from references (3-7)
Table 1. The most common risk factors affecting the development of esophageal carcinomas SCC, Squamous cell carcinoma; ADC, Adenocarcinoma. Information from references (3-7)




Table 2. Summary of the most common alterations in esophageal cancers SCC, Squamous cell carcinoma; ADC, Adenocarcinoma; LOH, loss of heterozygosity; TSG, tumor suppressor gene; APC, adenomatous polyposis coli; MLH1, mutL homolog1
Table 2. Summary of the most common alterations in esophageal cancers SCC, Squamous cell carcinoma; ADC, Adenocarcinoma; LOH, loss of heterozygosity; TSG, tumor suppressor gene; APC, adenomatous polyposis coli; MLH1, mutL homolog1




References :
  1. Kamangar F, Dores GM, Anderson WF. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. J Clin Oncol 2006;24(14):2137-2150.
  2. Ya W, Wistuba II, Emmert-Buck MR, Erickson HS. Squamous cell carcinoma-similarities and dif-ferences among anatomical sites. Am J Cancer Res 2011;1(3):275-300.
  3. World Cancer Research Fund & American Institute for Cancer Research (1997). Food, nutrition, and the prevention of cancer: A global perspective. American Institute for Cancer Research Washing-ton, DC.
  4. Abedi-Ardekani B, Kamangar F, Hewitt SM, Hai-naut P, Sotoudeh M, Abnet CC, et al. Polycyclic aromatic hydrocarbon exposure in oesophageal tissue and risk of oesophageal squamous cell car-cinoma in north-eastern Iran. Gut 2010;59(9):1178- 1183.
  5. Stewart BW, Kleihues P. Esophageal cancer. World cancer report. Lyon: International Agency for Re-search on Cancer (IARC); 2003. 5p. Report No.: ISBN 9283204115.
  6. Claudia AM, Regiane CJ P, Aureliano CC. Fumon-isin as a risk factor to esophageal cancer: a Review. Appli Canc Rese 2009;29(3):102-105.
  7. Sorriano JM, Gonzalez L, Catala AI. Mechanism of action os sphin¬8. golipids and their metabolites in the toxicity of fumonisin B1. Prog Lipid Res 2005; 44(6):345-356.
  8. Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin 2010; 60(5):277-300.
  9. Cancer facts and figures 2011. Atlanta, GA: Ameri-can Cancer Society, 2011.
  10. Blot WJ, McLaughlin JK. The changing epidemic-ology of esophageal cancer. Semin Oncol 1999;26 (5 Suppl 15):2-8.
  11. Kubo A, Corley DA. Marked multi-ethnic variation of esophageal and gastric cardia carcinomas within the United States. Am J Gastroenterol 2004;99(4): 582-588.
  12. Corley DA, Buffler PA. Oesophageal and gastric cardia adenocarcinomas: analysis of regional vari-ation using the cancer incidence in five continents database. Int J Epidemiol 2001;30(6):1415-1425.
  13. Hongo M. Review article: Barrett's esophagus and carcinoma in Japan. Aliment Pharmacol Ther 2004; 20(Suppl 8):50-54.
  14. Shibata A, Matsuda T, Ajiki W, Sobue T. Trend in incidence of adenocarcinoma of the esophagus in Japan, 1993-2001. Jpn J Clin Oncol 2008;38(7): 464-468.
  15. Son JI, Park HJ, Song KS, Kim KJ, Lee CY, Lee SI, et al. A single center's 30 years' experience of esophageal adenocarcinoma. Korean J Intern Med 2001;16(4):250-253.
  16. Sadjadi A, Marjani H, Semnani S, Nasseri Mo-ghaddam S. Esophageal cancer in Iran: A review. Mid East J Can 2010;1(1):5-14.
  17. Ojala K, Sorri M, Jokinen K, Kairaluoma M. Symptoms of carcinoma of the oesophagus. Med J Aust 1982;1(9):384-385.
  18. Danoff B, Cooper J, Klein M. Primary adenocar-cinoma of the upper esophagus. Clin Radiol 1978; 29(5):519-522.
  19. Hosch SB, Stoecklein NH, Pichlmeier U, Rehders A, Scheunemann P, Niendorf A, et al. Esophageal cancer: the mode of lymphatic tumor cell spread and its prognostic significance. J Clin Oncol 2001; 19(7):1970-1975.
  20. Jacobson BC, Hirota W, Baron TH, Leighton JA, Faigel DO. The role of endoscopy in the assess-ment and treatment of esophageal cancer. Gastro-intest Endosc 2003;57(7):817-822.
  21. Nishimura Y, Osugi H, Inoue K, Takada N, Taka-mura M, Kinosita H. Bronchoscopic ultrasono-graphy in the diagnosis of tracheobronchial inva-sion of esophageal cancer. J Ultrasound Med 2002; 21(1):49-58.
  22. Levy RM, Wizorek J, Shende M, Luketich JD. Laparoscopic and thoracoscopic esophagectomy. Adv Surg 2010;44:101-116.
  23. De Graaf GW, Ayantunde AA, Parsons SL, Duffy JP, Welch NT. The role of staging laparoscopy in oesophagogastric cancers. Eur J Surg Onco 2007; 33(8):988-992.
  24. Choi J, Kim SG, Kim JS, Jung HC, Song IS. Com-parison of endoscopic ultrasonography (EUS), pos-itron emission tomography (PET), and computed tomography (CT) in the preoperative locoregional staging of resectable esophageal cancer. Surg Endosc 2010;24(6):1380-1386.
  25. Mariette C, Seitz JF, Maillard E, Mornex F, Tho-mas PA, Raoul J, et al. Surgery alone versus chemoradiotherapy followed by surgery for local-ized esophageal cancer: analysis of a randomized controlled phase III trial FFCD 9901. J Clin Oncol 2010;28(Suppl 15):4005.
  26. Vallbohmer D, Lenz HJ. Predictive and prognostic molecular markers in outcome of esophageal can-cer. Dis Esophagus 2006;19(6):425-432.
  27. Metzger R, Schneider PM, Warnecke-Eberz U, Brabender J, Holscher AH. Molecular biology of esophageal cancer. Onkologie 2004;27(2):200-206.
  28. Tsuda H, Hashiguchi Y, Nishimura S, Kawamura N, Inoue T, Yamamoto K. Relationship between HPV typing and abnormality of G1 cell cycle regulators in cervical neoplasm. Gynecol Oncol 2003;91(3): 476-485.
  29. Dong M, Ma G, Tu W, Guo KJ, Tian YL, Dong YT. Clinicopathological significance of p53 and mdm2 protein expression in human pancreatic cancer. World J Gastroenterol 2005;11(14):2162-2165.
  30. Putz A, Hartmann AA, Fontes PR, Alexandre CO, Silveira DA, Klug SJ, et al. TP53 mutation pattern of esophageal squamous cell carcinomas in a high risk area (Southern Brazil): role of life style factors. Int J Cancer 2002;98(1):99-105.
  31. Robert V, Michel P, Flaman JM, Chiron A, Martin C, Charbonnier F, et al. High frequency in esoph-ageal cancers of p53 alterations inactivating the regulation of genes involved in cell cycle and apop-tosis. Carcinogenesis 2000;21(4):563-565.
  32. Wang LD, Zhou Q, Hong JY, Qiu SL, Yang CS. p53 protein accumulation and gene mutations in multifocal esophageal precancerous lesions from symptom free subjects in a high incidence area for esophageal carcinoma in Henan, China. Cancer 1996;77(7):1244-1249.
  33. Shi ST, Yang GY, Wang LD, Xue Z, Feng B, Ding W, et al. Role of p53 gene mutations in human esophageal carcinogenesis: results from immuno-histochemical and mutation analyses of carcinomas and nearby non-cancerous lesions. Carcinogenesis 1999;20(4):591-597.
  34. Abbaszadegan MR, Raziee HR, Ghafarzadegan K, Shakeri MT, Afsharnezhad S, Ghavamnasiry MR. Aberrant p16 methylation, a possible epigenetic risk factor in familial esophageal squamous cell carcinoma. Int J Gastrointest Cancer 2005;36(1): 47-54.
  35. Ishii T, Murakami J, Notohara K, Cullings HM, Sasamoto H, Kambara T, et al. Oesophageal squa-mous cell carcinoma may develop within a back-ground of accumulating DNA methylation in nor-mal and dysplastic mucosa. Gut 2007; 56(1):13-19.
  36. Tarmin L, Yin J, Zhou X, Suzuki H, Jiang HY, Rhyu MG, et al. Frequent loss of heterozygosity on chromosome 9 in adenocarcinoma and squamous cell carcinoma of the esophagus. Cancer Res 1994; 54(23):6094-6096.
  37. Taghavi N, Biramijamal F, Sotoudeh M, Khademi H, Malekzadeh R, Moaven O, et al. p16INK4a hypermethylation and p53, p16 and MDM2 protein expression in esophageal squamous cell carcinoma. BMC Cancer 2010;10:138.
  38. Abbaszadegan MR, Raziee HR, Ghafarzadegan K, Ghavamnasiry MR. p16/INK4a promoter hyper-methylation in serum, blood and tumor of patients with esophageal squamous cell carcinoma. IJBMS 2004;6(4):4-9.
  39. Hardie LJ, Darnton SJ, Wallis YL, Chauhan A, Hai-naut P, Wild CP, et al. p16 expression in Barrett's esophagus and esophageal adenocarcinoma: associ-ation with genetic and epigenetic alterations. Can-cer Lett 2005;217(2):221-230.
  40. Xing EP, Nie Y, Wang LD, Yang GY, Yang CS. Aberrant methylation of p16INK4a and deletion of p15INK4b are frequent events in human esoph-ageal cancer in Linxian, China. Carcinogenesis 1999;20(1):77-84.
  41. Sarbia M, Geddert H, Klump B, Kiel S, Iskender E, Gabbert HE. Hypermethylation of tumor suppres-sor genes (p16INK4A, p14ARF and APC) in adeno-carcinomas of the upper gastrointestinal tract. Int J Cancer 2004;111(2):224-228.
  42. Xing EP, Nie Y, Song Y, Yang GY, Cai YC, Wang LD, et al. Mechanisms of inactivation of p14ARF, p15INK4b, and p16INK4a genes in human esoph-ageal squamous cell carcinoma. Clin Cancer Res 1999;5(10):2704-2713.
  43. Fearnhead NS, Britton MP, Bodmer WF. The ABC of APC. Hum Mol Genet 2001;10(7):721-733.
  44. Boynton RF, Blount PL, Yin J, Brown VL, Huang Y, Tong Y, et al. Loss of heterozygosity involving the APC and MCC genetic loci occurs in the major-ity of human esophageal cancers. Proc Natl Acad Sci USA 1992;15;89(8):3385-3388.
  45. Dolan K, Garde J, Walker SJ, Sutton R, Gosney J, Field JK. LOH at the sites of the DCC, APC, and TP53 tumor suppressor genes occurs in Barrett's metaplasia and dysplasia adjacent to adenocarcin-oma of the esophagus. Hum Pathol 1999;30(12): 1508-1514.
  46. Powell SM, Papadopoulos N, Kinzler KW, Smoli-nski KN, Meltzer SJ. APC gene mutations in the mutation cluster region are rare in esophageal can-cers. Gastroenterology 1994;107(6):1759-1763.
  47. Wu DL, Sui FY, Jiang XM, Jiang XH. Methylation in esophageal carcinogenesis. World J Gastroente-rol 2006;12(43):6933-6940.
  48. Montesano R, Hollstein M, Hainaut P. Genetic al-terations in esophageal cancer and their relevance to etiology and pathogenesis: a review. Int J Cancer 1996;69(3):225-235.
  49. Kawakubo H, Ozawa S, Ando N, Kitagawa Y, Mukai M, Ueda M, et al. Alterations of p53, cyclin D1 and pRB expression in the carcinogenesis of esophageal squamous cell carcinoma. Oncol Rep 2005;14(6):1453-1459.
  50. Izzo JG, Luthra R, Wu TT, Correa AM, Luthra M, Anandasabapathy S, et al. Molecular mechanisms in Barrett's metaplasia and its progression. Semin Oncol 2007;34(2 Suppl 1):S2-6.
  51. Friess H, Fukuda A, Tang WH, Eichenberger A, Furlan N, Zimmermann A, et al. Concomitant analysis of the epidermal growth factor receptor family in esophageal cancer: over expression of epidermal growth factor receptor mRNA but not of c-erbB-2 and c-erbB-3. World J Surg 1999;23(10): 1010-1018.
  52. Andolfo I, Petrosino G, Vecchione L, De Antonel-lis P, Capasso M, Montanaro D, et al. Detection of erbB2 copy number variations in plasma of patients with esophageal carcinoma. BMC Cancer 2011;11: 126.
  53. Ikeguchi M, Unate H, Maeta M, Kaibara N. Detec-tion of loss of heterozygosityat microsatellite loci in esophageal squamous cell carcinoma. Oncology 1999;56(2):164-168.
  54. Muzeau F, Flejou JF, Belghiti J, Thomas G, Hame-lin R. Infrequent microsatellite instability in oe-sophageal cancers. Br J Cancer 1997;75(9):1336-1339.
  55. Fitzgerald RC, Triadafilopoulos G. Recent develop-ments in the molecular characterization of Barrett's esophagus. Dig Dis 1998;16(2):63-80.
  56. Scaros O, Fisler R. Biomarker technology roundup: from discovery to clinical applications, a broad set of tools is required to translate from the lab to the clinic. Biotechniques 2005;Suppl:30-32.
  57. Cho WC. Proteomics–leading biological science in the 21st century. Sci J 2004;56:14-17.
  58. Ardekani AM, Petricoin EF 3rd, Hackett JL. Mo-lecular diagnostics: an FDA perspective. Expert Rev Mol Diagn 2003;3(2):129-140.
  59. Ardekani AM, Liotta LA, Petricoin EF 3rd. Clinic-al potential of proteomics in the diagnosis of ovar-ian cancer. Expert Rev Mol Diagn 2002;2(4):312-320.
  60. Petricoin EF, Ardekani AM, Hitt BA, Levine PJ, Fusaro VA, Steinberg SM, et al. Use of proteomic patterns in serum to identify ovarian cancer. Lancet 2002;359(9306):572-577.
  61. Ardekani AM, Akhondi MM, Sadeghi MR. Appli-cation of genomic and proteomic technologies to early detection of cancer. Arch Iran Med 2008;11 (4):427-434.
  62. Petricoin EF 3rd, Ornstein DK, Paweletz CP, Arde-kani A, Hackett PS, Hitt BA, et al. Serum pro-teomic patterns for detection of prostate cancer. J Natl Cancer Inst 2002;94(20):1576-1578.
  63. Steinberg W. The clinical utility of the CA 19-9 tumor-associated antigen. Am J Gastroenterol 1990; 85(4):350-355.
  64. Moore RG, McMeekin DS, Brown AK, DiSilvestro P, Miller MC, Allard WJ, et al. A novel multiple marker bioassay utilizing HE4 and CA125 for the prediction of ovarian cancer in patients with a pel-vic mass. Gynecol Oncol 2009;112(1):40-46.
  65. Leman ES, Getzenberg RH. Biomarkers for pro-state cancer. J Cell Biochem 2009; 108(1):3-9.
  66. Quillien V, Raoul JL, Laurent JF, Meunier B, Le Prise E. Comparison of Cyfra 21-1, TPA and SCC tumor markers in esophageal squamous cell carcin-oma. Oncol Rep 1998;5(6):1561-1565.
  67. Chatterjee M, Wojciechowski J, Tainsky MA. Dis-covery of antibody biomarkers using protein micro-arrays of tumor antigens cloned in high throughput. Methods Mol Biol 2009;520:21-38.
  68. Swarup V, Rajeswari MR. Circulating (cell-free) nucleic acids-a promising, non-invasive tool for early detection of several human diseases. FEBS Lett 2007;581(5):795-799.
  69. Chin L, Gray JW. Translating insights from the can-cer genome into clinical practice. Nature 2008;452 (7187):553-563.
  70. Eden A, Gaudet F, Waghmare A, Jaenisch R. Chro-mosomal instability and tumors promoted by DNA hypomethylation. Science 2003;300(5618):455.
  71. Hanash SM, Pitteri SJ, Faca VM. Mining the plasma proteome for cancer biomarkers. Nature 2008;452 (7187):571-579.
  72. van't Veer LJ, Bernards R. Enabling personalized cancer medicine through analysis of gene expres-sion patterns. Nature 2008;452(7187):564-570.
  73. Zhu J, Yao X. Use of DNA methylation for cancer detection: promises and challenges. Int J Biochem Cell Biol 2009;41(1):147-154.
  74. Mandel P, Bieth R. Les acides nucléiques du plas-ma sanguin chez l‘homme. C R Seances Soc Biol Fil 1948;142(3-4):241-243.
  75. Stroun M, Anker P, Lyautey J, Lederrey C, Mau-rice PA. Isolation and characterization of DNA from the plasma of cancer patients. Eur J Cancer Clin Oncol 1987;23(6):707-712.
  76. Van der Vaart M, Pretorius PJ. Is the role of circu-lating DNA as a biomarker of cancer being prema-turely overrated? Clin Biochem 2010;43(1-2):26-36.
  77. Jahr S, Hentze H, Englisch S, Hardt D, Fackel-mayer FO, Hesch RD, et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necro-tic cells. Cancer Res 2001;61(4):1659-1665.
  78. Giacona MB, Ruben GC, Iczkowski KA, Roos TB, Porter DM, Sorenson GD. Cell-free DNA in human blood plasma: length measurements in patients with pancreatic cancer and healthy controls. Pancreas 1998;17(1):89-97.
  79. Suzuki N, Kamataki A, Yamaki J, Homma Y. Char-acterization of circulating DNA in healthy human plasma. Clin Chim Acta 2008;387(1-2):55-58.
  80. Choi JJ, Reich CF, 3rd, Pisetsky DS. The role of macrophages in the in vitro generation of extracel-lular DNA from apoptotic and necrotic cells. Im-munology 2005;115(1):55-62.
  81. Diehl F, Schmidt K, Choti MA, Romans K, Good-man S, Li M, et al. Circulating mutant DNA to assess tumor dynamics. Nat Med 2008;14(9):985-990.
  82. Leon SA, Shapiro B, Sklaroff DM, Yaros MJ. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res 1977;37(3):646-650.
  83. Wu TL, Zhang D, Chia JH, Tsao KH, Sun CF, Wu JT. Cell-free DNA: measurement in various carcin-omas and establishment of normal reference range. Clin Chim Acta 2002;321(1-2):77-87.
  84. Fatouros IG, Destouni A, Margonis K, Jamurtas AZ, Vrettou C, Kouretas D, et al. Cell-free plasma DNA as a novel marker of aseptic inflammation severity related to exercise overtraining. Clin Chem 2006;52 (9):1820-1824.
  85. Lo YM, Rainer TH, Chan LY, Hjelm NM, Cocks RA. Plasma DNA as a prognostic marker in trauma patients. Clin Chem 2000;46(3):319-323.
  86. Fournié GJ, Martres F, Pourrat JP, Alary C, Ru-meau M. Plasma DNA as cell death marker in eld-erly patients. Gerontology 1993;(39):215-221.
  87. Atamaniuk J, Vidotto C, Tschan H, Bachl N, Stuhl-meier KM, Muller MM. Increased concentrations of cell-free plasma DNA after exhaustive exercise. Clin Chem 2004;50(9):1668-1670.
  88. Jung K, Stephan C, Lewandowski M, Klotzek S, Jung M, Kristiansen G, et al. Increased cell-free DNA in plasma of patients with metastatic spread in prostate cancer. Cancer Lett 2004;205(2):173-180.
  89. Laktionov PP, Tamkovich SN, Rykova EY, Bryz-gunova OE, Starikov AV, Kuznetsova NP, et al. Extracellular circulating nucleic acids in human plasma in health and disease. Nucleosides Nucleo-tides Nucleic Acids 2004;23(6-7):879-883.
  90. Silva JM, Garcia JM, Dominguez G, Silva J, Miral-les C, Cantos B, et al. Persistence of tumor DNA in plasma of breast cancer patients after mastectomy. Ann Surg Oncol 2002;9(1):71-76.
  91. Sozzi G, Conte D, Leon M, Ciricione R, Roz L, Ratcliffe C, et al. Quantification of free circulating DNA as a diagnostic marker in lung cancer. J Clin Oncol 2003; 21(21):3902-3908.
  92. Ren N, Ye QH, Qin LX, Zhang BH, Liu YK, Tang ZY. Circulating DNA level is negatively associated with the long-term survival of hepatocellular car-cinoma patients. World J Gastroenterol 2006;12 (24):3911-3914.
  93. Boddy JL, Gal S, Malone PR, Harris AL, Wains-coat JS. Prospective study of quantitation of plasma DNA levels in the diagnosis of malignant versus benign prostate disease. Clin Cancer Res 2005;11 (4):1394-1399.
  94. Banki F, Mason RJ, Oh D, Hagen JA, DeMeester SR, Lipham JC, et al. Plasma DNA as a molecular marker for completeness of resection and recurrent disease in patients with esophageal cancer. Arch Surg 2007;142(6):533-539.
  95. Tomita H, Ichikawa D, Ikoma D, Sai S, Tani N, Ikoma H, et al. Quantification of circulating plasma DNA fragments as tumor markers in patients with esophageal cancer. Anticancer Res 2007;27(4C): 2737-2741.
  96. Esteller M, Sanchez-Cespedes M, Rosell R, Sidran-sky D, Baylin SB, Herman JG. Detection of aber-rant promoter hypermethylation of tumor suppres-sor genes in serum DNA from non-small cell lung cancer patients. Cancer Res 1999;59(1):67-70.
  97. Kawakami K, Brabender J, Lord RV, Groshen S, Greenwald BD, Krasna MJ, et al. Hypermethylated APC DNA in plasma and prognosis of patients with esophageal adenocarcinoma. J Natl Cancer Inst 2000;92(22):1805-1811.
  98. Hibi K, Taguchi M, Nakayama H, Takase T, Kasai Y, Ito K, et al. Molecular detection of p16 promoter methylation in the serum of patients with esoph-ageal squamous cell carcinoma. Clin Cancer Res 2001;7(10):3135-3138.
  99. Liu JB, Qiang FL, Dong J, Cai J, Zhou SH, Shi MX, et al. Plasma DNA methylation of Wnt antag-onists predicts recurrence of esophageal squamous cell carcinoma. World J Gastroenterol 2011;17(44): 4917-4921.
  100. Johnson PJ, Lo YM. Plasma nucleic acids in the diagnosis and management of malignant disease. Clin Chem 2002;48(8):1186-1193.
  101. Eisenberger F, Knoefel T, Peiper M, Merkert P, Yekebas EF, Scheunemann P, et al. Squamous cell carcinoma of the esophagus can be detected by microsatellite analysis in tumor and serum. Clin Can Res 2003;(15):4178-4183.