Cancer Genetics and Cytogenetics 195 (2009) 175e178

Short communication

Role of mitochondrial DNA 4977-bp deletions in esophageal cancersusceptibility and prognosis in a northern Indian population

Rohit Upadhyaya, Meenu Jaina, Shaleen Kumarb, Uday ChandGhoshalc, Balraj Mittala,*aDepartment of Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareilly Road, Lucknow-226014, India

bDepartment of Radiotherapy, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareilly Road, Lucknow-226014, IndiacDepartment of Gastroenterology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareilly Road, Lucknow-226014, India

Received 24 February 2009; received in revised form 12 May 2009; accepted 17 June 2009

Abstract The mitochondrial DNA 4977-bp deletion (6m

* Corresponding

þ91-522-2668973.

E-mail addresse

(B. Mittal).

0165-4608/09/$ e see

doi:10.1016/j.cancerg

tDNA4977) has been explored in various cancers,but its predictive or prognostic role in esophageal cancer is poorly understood. The objective of thepresent study was to investigate a possible role of 6mtDNA4977 in susceptibility and prognosis ofesophageal cancer in a northern Indian population. The study was performed in 39 histopatholog-ically confirmed cases with esophageal cancer. Tumor, normal tissues, and intravenous bloodsamples were taken for detection of 6mtDNA4977 through a duplex polymerase chain reactiontechnique. 6mtDNA4977 was detected in two tumors and one adjacent normal tissue sample,but in none of the blood samples. All three patients with 6mtDNA4977 were male, with squamouscell carcinoma in the middle third of the esophagus. Survival analysis suggested a role of6mtDNA4977 in prognosis of esophageal cancer patients. Despite the low frequency of6mtDNA4977 in esophageal cancer patients of northern India, this feature may have a role inesophageal cancer progression and prediction of survival outcome. � 2009 Elsevier Inc. All rightsreserved.

1. Introduction

Mitochondrial DNA (mtDNA), is a circular, double-stranded extrachromosomal DNA of 16.5 kb in size,without introns, that replicates at a high rate without an effi-cient DNA repair mechanism. mtDNA is more prone toattacks by reactive oxygen species and free radicals,because mitochondria are the main endogenous source ofreactive oxygen species because of highly mutagenic sideproducts of oxidative phosphorylation [1]. The somaticmutation rate of mtDNA is therefore presumed to be 10to 20 times higher than that of nuclear DNA [2,3]. Mito-chondrial DNA has long been suspected of having a rolein tumorigenesis, and a large number of mtDNA mutationshave been detected in a variety of tumors [4,5].

One of the best-described mtDNA mutation is thecommon 4977-bp deletion (6mtDNA4977) [6], betweennucleotides 8,470 and 13,447 of the human mtDNA. Thedeletion affects several transfer RNA and respiratory chaingenes. The cells harboring a high proportion of

author. Tel.: þ91-522-2668004-8, Ext. 2322; fax:

s: balraj@sgpgi.ac.in or bml_pgi@yahoo.com

front matter � 2009 Elsevier Inc. All rights reserved.

encyto.2009.06.017

6mtDNA4977 cannot survive and lead to dropout fromthe population. The 6mtDNA4977 is known to be involvedin myopathies, Alzheimer disease, chronological aging, andphotoaging of the skin [6e10].

The prevalence of 6mtDNA4977 has been detected inseveral types of human tumors, including lung, breast,endometrial, gastric, hepatocellular, and thyroid cancers[11e16]. In esophageal cancer, there is a single reportshowing high prevalence of 6mtDNA4977 in a high-riskpopulation from China [15], but to date there has been nostudy investigating the prevalence and prognostic role of6mtDNA4977 in patients with esophageal cancer froma low-risk population. The present study was thereforeaimed at detecting the frequency and possible prognosticrole of 6mtDNA4977 in esophageal cancer patients fromnorthern India.

2. Materials and methods

2.1. Study subjects

A total of 39 histologically confirmed esophageal tumors,adjacent normal tissue and intravenous blood samples werecollected during endoscopy between July 2006 to October

Table 1

Demographic and clinical characteristics of patients with histologically

confirmed esophageal cancer

Characteristic Value

Sample size n 5 39

Median age, yr (range) 60 (30e84)

Sex, no. (%)

Male 31 (80)

Female 8 (20)

Histological type, no. (%)

Squamous cell cancer 33 (85)

Adenocarcinoma 6 (16)

Tumor location, no. (%)

Upper third 5 (13)

Middle third 23 (59)

Lower third 11 (28)

Tobacco use,* no. (%)

Smoker 7 (18)

Tobacco-chewer 13 (33)

Smoker þ tobacco-chewer 11 (28)

Tobacco nonuser 8 (21)

Alcohol consumption,** no. (%)

Drinker 20 (54)

Nondrinker 17 (46)

Mitochondrial 4977-bp deletion, no. (%)

Deletion in normal tissue 1 (3)

Deletion in tumor tissue 2 (5)

* Tobacco usage categorized in exclusive-Smokers, exclusive-tobacco

chewers, both smoker/tobacco chewers and nontobacco users (less than

100 cigarettes in lifetime where 1 cigarette 5 2 Bidi and never tobacco

chewers).

** Alcohol consumption was recorded in nonquantitative basis as daily,

occasional or never (nondrinker).

176 R. Upadhyay et al. / Cancer Genetics and Cytogenetics 195 (2009) 175e178

2008. Demographic data (age, sex, and smoking and alco-holic habits) and clinical characteristics (histopathologyand location of tumor) were obtained by questionnaire andfrom medical reports. All biopsy samples were collected inphosphate buffered saline (PBS) and kept at �80�C untilDNA extraction. Total cellular DNA was extracted usinga commercially available kit (Qiagen, Valencia, CA). Thequantification of DNA was done using a NanoDrop analyzerspectrophotometer (ND-1000; Thermo Fisher ScientificeNanoDrop Technologies, Wilmington, DE).

2.2. Detection of mitochondrial deletion

To assess the presence of the 6mtDNA4977, we useda duplex polymerase chain reaction (PCR) method asdescribed by Abnet et al. [15]. Briefly, two pairs of forwardand reverse PCR primers were used: Mitin2F 50-CTGAGCCTTTTACCACTCCAG-30, Mitin2R 50-GGGA-GACTCGAAGTACTCTG-30, Mitout2F 50-CCCAACTAAATACTACCGTATGG-30, Mitout2R 50-GGCTCAGGCGTTTGTGTATGAT-30. The Mitin primers amplified a 262-bpamplicon from intact mtDNA in the region of cytochromeoxidase subunit III as an amplification control, whereasMitout amplified a 214-bp fusion product only in thosesamples which had common 4977-bp deletion. The PCRconditions were as described previously [15]. The PCRproducts were separated on 10% PAGE (Fig. 1). All thesamples were re-analyzed by duplex PCR, and 100% simi-larity in results was found. The sequences of bands and thebreakage point of fusion products after deletion were recon-firmed by sequencing reactions using both sets of primers(supplementary material).

2.3. Statistical analysis

Fisher’s exact test was used to judge significant differ-ence for deletion in tumor and normal tissues (ScientificPackage for the Social Sciences, version 15.0; SPSS, Chi-cago, IL). A P-value of O0.05 was considered statisticallynonsignificant.

3. Results

Clinicodemographic characteristics are given in Table 1.We performed mutation analysis in biopsy samples from

Table 2

Characteristics of 3 esophageal cancer patients (n 5 39) with mutation for the c

Tob

Patient Tumor tissue Normal tissue Age, yr Sex Smo

EC29 Delþ No del 48 Male No

EC35 Delþ No del 64 Male No

EC40 No del Delþ 45 Male No

Characteristics in common for all three patients: male sex, squamous cell ca

blood sample. Occupational exposure included mainly outdoor fieldwork such as

sure to smoking fuels in case of females. This was recorded as presence or absea Patient EC29 was exposed to smoke throughout his job in a factory.

tumor and adjacent normal tissue, as well as from bloodsamples from same patient. Of the 39 patients, only 2patients (~5%) had the mitochondrial common deletion intheir tumor biopsies, and 1 other patient (!3%) had muta-tion in its normal biopsy; none of the 39 patients had themtDNA mutation in blood samples (Table 2). Thus, nosignificant difference was found in frequency of6mtDNA4977 between tumor and adjacent normal tissues(P O 0.05). Because of low frequencies of mitochondrialcommon deletions in the samples, we did not performany further subgroup analysis for the frequency differencesbetween tumor and adjacent normal tissues.

We further evaluated the association of 6mtDNA4977with demographic and clinical parameters of esophageal

ommon mitochondrial 4977-bp deletion

acco use

ker Chewer Alcohol consumption Occupational exposurea

No Drinker (daily) Yes

Yes Nondrinker No

Yes Nondrinker No

rcinoma histopathology, tumor location in middle third, and no deletion in

exposure to coal, smoke or petroleum products in males or household expo-

nce of exposure.

Fig. 1. Gel image of the common mitochondrial DNA 4977-bp deletion.

Lane 1, no deletion; lane 2, 50-bp DNA ladder; lane 3, deletion.

177R. Upadhyay et al. / Cancer Genetics and Cytogenetics 195 (2009) 175e178

cancer patients. The age of onset of cancer in the threepatients with the deletion was lower in two cases (ages48 and 45 years) but higher in one case (age 64 years),compared with in comparison to median age at onset of60 years for patients with no deletion. Shared characteris-tics for all three patients with 6mtDNA4977 were sex(male), histopathology (squamous cell carcinoma), differ-entiation status (moderate), and location of tumor (middlethird). Two of the three patients with 6mtDNA4977 weretobacco chewers, none were smokers, and one wasa frequent consumer of alcohol (Table 2).

Follow-up data were available from the radiotherapydepartment for two of the three patients with 6mtDNA4977(EC35 and EC40); the third patient (EC29) was lost tofollow-up. Overall survival was 24.4 months (EC35) and5.6 months (EC40), compared with median survival for allpatients of 11.2 months. Both patients had dysphagia gradeIII (i.e., they. could take food only in form of liquids).Dysphagia duration was 1 month (EC35) and 3 months(EC40). For one patient (EC35), tumor length was 12.5 cmwith involvement of nodes; the other patient (EC40) hadtumor of comparatively shorter length, 7.5 cm, with noinvolvement of nodes. Both patients had undergone radio-therapy with chemotherapy: 50 Gy with one cycle of cisplatin(EC35) and 45 Gy with four cycles of cisplatin (EC40) (Table3). One patient (EC35) had a family history of cancer; hisbrother and nephew had died with ‘‘head and neck’’ and‘‘blood’’ cancer, respectively.

4. Discussion

The frequency of common 6mtDNA4977 was low intissue (2/39 tumor and 1/39 normal) samples, and absent

Table 3

Survival details of patients having mitochondrial 4977bp deletion mutation

Patient

Overall

survival

(months) Status

Dysphagia

grade

Dysphagia

duration

(months)

Tumor

length

(cms)

EC29 Lost to

follow-up

EC35 24.4 Deceased 3 1 12.5

EC40 5.6 Deceased 3 3 7.5

in blood samples of the same subjects. This finding estab-lishes the mtDNA 4977-bp deletion as a sporadic mutation.Previously, Abnet et al. [15] reported high frequency of6mtDNA4977 deletion in esophageal cancer patients fromChina, but mutation analysis in whole mitochondrialgenome from northern India [5] did not find this deletion.It appears, therefore, that there is a significant differencebetween frequencies of 6mtDNA4977 common deletionin a high-risk Chinese population and a low-risk northernIndian population.

We did not find significant difference between thefrequencies of 6mtDNA4977 in tumor tissues and adjoiningnormal tissues. All the subjects with 6mtDNA4977 weremale, with squamous cell carcinoma histopathology and withmiddle-third location of tumor. This may be attributablesimply to the fact that ~80% of subjects were male and withsquamous cell carcinoma, or there may be an association ofmitochondrial deletion with squamous cell histopathologyand middle third location of tumor.

The role of environmental factors such as smoking [11]and alcohol consumption [17] has been documented forgeneration of defective copies of mtDNA. The present find-ings are in accord, in that all three subjects with6mtDNA4977 had a history of frequent usage of tobaccoor alcohol.

The next question is whether presence of 6mtDNA4977influences tumor progression or survival of the patients.The dividing tumor cells require an abundant supply ofenergy. The mtDNA 4977-bp deletion impairs respiratorychain, which leads to inhibition of proliferative cells. Inprevious studies, 6mtDNA4977 has been predominantlyreported to be present in normal tissues adjacent to tumors[15,18,19]. However, we did not find significant differencesin presence of deletion in normal and tumor tissue, prob-ably because of the low frequency of deletions in the studysample.

Considering the two patients with 6mtDNA4977 forwhom follow-up data were available, the patient (EC40)with comparatively shorter tumor length, positive familyhistory of cancer, and no involvement of lymph nodeshad lower survival, at 5.6 months, compared with the otherpatient (EC35), who had longer tumor length with involve-ment of lymph nodes had showed longer survival, at 24.4months. The explanation may involve presence of the mito-chondrial deletion, which appears during early stages ofcarcinogenesis and disappears in later stages of tumor

Tumor

differentiation

status TNM

Treatment

protocol

RT dose

given

Chemotherapy

dose/ cycles

Moderate

Moderate T2N1M0 CT+RT 50Gy CDDP/one

Moderate T2N0M0 CT+RT 45Gy CDDP/four

178 R. Upadhyay et al. / Cancer Genetics and Cytogenetics 195 (2009) 175e178

[20]. In case EC35, there was deletion in tumor tissue butnot in normal tissue, which suggests absence of early carci-nogenic changes in adjacent tissue in the patient. On theother hand, case EC40 had deletion in normal tissue butnot in tumor tissue, which shows initiation of early carcino-genic processes in adjacent tissues. Thus, overall survivalof patient EC35 was greater than that of EC40 even thoughboth cases had same grade of dysphagia (grade III).

Limitations of the present study include unavailability oftissue samples from normal esophagus, so that thefrequency of 6mtDNA4977 in normal esophagus couldnot be assessed. Similar studies with larger sample sizeand in different populations are warranted for furtherexploring the role of 6mtDNA4977 in esophageal cancer.

In conclusion, our study shows a low frequency of6mtDNA4977 in esophageal cancer in a northern Indianpopulation. However, the common deletion may have somerole in tumor progression and survival of patients withsquamous cell esophageal cancer.

Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.cancergencyto.2009.06.017.

References

[1] Fridovich I. Mitochondria: are they the seat of senescence? Aging

Cell 2004;3:13e6.

[2] Howell N, Kubacka I, Mackey DA. How rapidly does the human

mitochondrial genome evolve? Am J Hum Genet 1996;59:501e9.

[3] Taylor RW, Turnbull DM. Mitochondrial DNA mutations in human

disease. Nat Rev Genet 2005;6:389e402.

[4] Berneburg M, Kamenisch Y, Krutmann J. Repair of mitochondrial

DNA in aging and carcinogenesis. Photochem Photobiol Sci

2006;5:190e8.

[5] Gochhait S, Bhatt A, Sharma S, Singh YP, Gupta P, Bamezai RN.

Concomitant presence of mutations in mitochondrial genome and

p53 in cancer development: a study in north Indian sporadic breast

and esophageal cancer patients. Int J Cancer 2008;123:2580e6.

[6] Cortopassi GA, Arnheim N. Detection of a specific mitochondrial

DNA deletion in tissues of older humans. Nucleic Acids Res

1990;18:6927e33.

[7] Holt IJ, Harding AE, Cooper JM, Schapira AH, Toscano A, Clark JB,

Morgan-Hughes JA. Mitochondrial myopathies: clinical and

biochemical features of 30 patients with major deletions of muscle

mitochondrial DNA. Ann Neurol 1989;26:699e708.

[8] Corral-Debrinski M, Horton T, Lott MT, Shoffner JM, McKee AC,

Beal MF, Graham BH, Wallace DC. Marked changes in mitochon-

drial DNA deletion levels in Alzheimer brains. Genomics 1994;23:

471e6.

[9] Berneburg M, Gattermann N, Stege H, Grewe M, Vogelsang K,

Ruzicka T, Krutmann J. Chronically ultraviolet-exposed human skin

shows a higher mutation frequency of mitochondrial DNA as

compared to unexposed skin and the hematopoietic system. Photo-

chem Photobiol 1997;66:271e5.

[10] Berneburg M, Grether-Beck S, Kurten V, Ruzicka T, Briviba K,

Sies H, Krutmann J. Singlet oxygen mediates the UVA-induced

generation of the photoaging-associated mitochondrial common dele-

tion. J Biol Chem 1999;274:15345e9.

[11] Dai JG, Xiao YB, Min JX, Zhang GQ, Yao K, Zhou RJ. Mitochon-

drial DNA 4977 BP deletion mutations in lung carcinoma. Indian J

Cancer 2006;43:20e5.

[12] Ye C, Shu XO, Wen W, Pierce L, Courtney R, Gao YT, Zheng W,

Cai Q. Quantitative analysis of mitochondrial DNA 4977-bp deletion

in sporadic breast cancer and benign breast diseases. Breast Cancer

Res Treat 2008;108:427e34.

[13] Futyma K, Putowski L, Cybulski M, Miotla P, Rechberger T,

Semczuk A. The prevalence of mtDNA4977 deletion in primary

human endometrial carcinomas and matched control samples. Oncol

Rep 2008;20:683e8.

[14] Wu CW, Yin PH, Hung WY, Li AF, Li SH, Chi CW, Wei YH,

Lee HC. Mitochondrial DNA mutations and mitochondrial DNA

depletion in gastric cancer. Genes Chromosomes Cancer 2005;44:

19e28.

[15] Abnet CC, Huppi K, Carrera A, Armistead D, McKenney K, Hu N,

Tang ZZ, Taylor PR, Dawsey SM. Control region mutations and

the ‘common deletion’ are frequent in the mitochondrial DNA of

patients with esophageal squamous cell carcinoma. BMC Cancer

2004;4:30.

[16] Maximo V, Soares P, Lima J, Cameselle-Teijeiro J, Sobrinho-

Simoes M. Mitochondrial DNA somatic mutations (point mutations

and large deletions) and mitochondrial DNA variants in human

thyroid pathology: a study with emphasis on Hurthle cell tumors.

Am J Pathol 2002;160:1857e65.

[17] von Wurmb-Schwark N, Ringleb A, Schwark T, Broese T, Weirich S,

Schlaefke D, Wegener R, Oehmichen M. The effect of chronic

alcohol consumption on mitochondrial DNA mutagenesis in human

blood. Mutat Res 2008;637:73e9.

[18] Kamalidehghan B, Houshmand M, Ismail P, Panahi MS, Akbari MH.

Delta mtDNA4977 is more common in non-tumoral cells from

gastric cancer sample. Arch Med Res 2006;37:730e5.

[19] Eshaghian A, Vleugels RA, Canter JA, McDonald MA, Stasko T,

Sligh JE. Mitochondrial DNA deletions serve as biomarkers of aging

in the skin, but are typically absent in nonmelanoma skin cancers. J

Invest Dermatol 2006;126:336e44.

[20] Kamenisch Y, Wenz J, Metzler G, Bauer J, Neubauer H, Garbe C,

Rocken M, Berneburg M. The mitochondrial DNA common deletion

is present in most basal and squamous cell carcinoma samples iso-

lated by laser capture microdissection but generally at reduced rather

than increased levels. J Invest Dermatol 2007;127:486e90.