ISRN Biomarkers

Volume 2013 (2013), Article ID 474107, 5 pages

http://dx.doi.org/10.1155/2013/474107

Research Article

Microsatellite Instability in Head and Neck Squamous Cell Carcinoma: A Study of a Brazilian Population

Elaine Stur,¹ Eldamaria de Vargas Wolfgramm,¹ Allan Kardec de Castro Neto,² Lucas de Lima Maia,¹ Lidiane Pignaton Agostini,¹ Gabriela Tonini Peterle,¹ Suzanny Oliveira Mendes,¹ Marcelos dos Santos,¹ Flávia de Paula,¹ and Iúri Drumond Louro¹

¹Núcleo de Genética Humana e Molecular, Departamento de Ciências Biológicas, Centro de Ciências Humanas e Naturais, Universidade Federal do Espírito Santo, Avenida Marechal Campos, 1468, Maruípe, 29043-900 Vitória, ES, Brazil

²Hospital Santa Rita de Cássia, Setor de Patologia, Vitória, ES, Brazil

Received 9 September 2013; Accepted 28 October 2013

Academic Editors: H.-L. Chan, A. Debucquoy, and M. Lotfy

Copyright © 2013 Elaine Stur et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Squamous cell carcinoma (SCC) is the sixth most common solid tumor in the world. Apart from known risk factors for head and neck SCC (HNSCC), there is a lack of information about genetic susceptibility regions that may play pivotal roles in the tumorigenesis of these tumors. Therefore, we have aimed to analyze the presence of genetic instability in microsatellite markers distributed in the genome. Microsatellite instability (MSI) was found in 6 HNSCC patients, among which only one was detected by the D17S250 marker, whereas the other 5 occurrences (13.5%) were detected by the D3S1611 marker. No instability was found at markers D5S346, D10S197, D11S922, and D11S988. MSI detected by D3S1611 marker was present in 3 (14.3%) moderately differentiated tumors and in 2 (25.0%) poorly differentiated tumors, but no statistical significance was found. Genotypic frequencies for all markers showed no statistically significant distribution alteration, neither were they related to differentiation grade or patient age. Marker D3S1611 is located in the MLH1 gene, which is part of the mismatch repair system (MMR), helping to maintain genomic stability. We have found a higher rate of D3S1611 MSI in older patients, suggesting that this marker may be affected by aging processes in the DNA repair machinery.

1. Introduction

Head and neck cancer is a significant cause of mortality and morbidity worldwide, presenting approximately 600,000 new cases yearly [1], whereas tumors of the oral cavity contribute with 389,000 new cases per year, with a mortality rate of 50% [2]. Squamous cell carcinoma (SCC) is the most common histological variant, comprising 90% of all cases [3].

SCC is the sixth most common solid tumor in the world [4], found preferentially in 50–70-year-old individuals [5]. Nonetheless, some studies point towards a frequency shift towards younger ages, with several cases in men with less than 40 years of age [6].

The epidemiology of head and neck SCC (HNSCC) shows tobacco, alcohol, and Human Papilloma Virus (HPV) infection as the most important risk factors, as well as genetic susceptibility [7–11]. Therefore, the search for genetic markers has increased significantly over recent years.

Microsatellite regions are genomic 1–5 bp tandem repeats, generally noncoding, and the CA 2 bp repeat is the most common form [12]. These regions may present instability, especially caused by replication errors introduced by DNA polymerase, a phenomenon known as microsatellite instability (MSI) [13].

These replication mistakes may aid tumorigenesis through inactivation of tumor suppressor and repair genes, altering normal cell functions [12]. The fact that tumor cells harbor several genomic alterations and remarkable instability renders MSI markers a possible tool in the analysis of diverse tumor types. This study aimed to evaluate the MSI rate at six different genomic locations in HNSCC patients.

2. Materials and Methods

2.1. Samples

Samples were obtained from the Pathology Service of Santa Rita de Cássia Hospital (SRCH). Forty-two paraffin blocked HNSCC were analyzed (all from year 2009). Of all patients, 9 (21.4%) were women and 33 (78.6%) were men, with a mean age of 61 years (sd ± 13.7). This study was approved by the Ethics Committee of the Federal University of Espírito Santo on 30/04/2009 (Protocol no. 010/09).

2.2. Genotyping

DNA was extracted according to Goelz et al. [14]. MSI at six different genomic locations was tested by polymerase chain reaction (PCR) in Eppendorf Mastercycler, using D3S1611, D5S346, D10S197, D11S922, D11S988, and D17S250 markers (primers described in Table 1). These regions were selected due to previous descriptions in the literature of genetic instability occurring at these markers in different tumor types [15]. Genomic region and reaction conditions are described in Table 2. After amplification, PCR products were separated by vertical electrophoresis in 15% acrylamide gels and stained with 0.1% silver nitrate. Homozygozity or heterozygozity was characterized by the identification of one or two alleles in the normal tissue, respectively. Genetic instability was determined by the presence of different size bands or extra bands in the tumoral tissue, when compared to the normal tissue.

Table 1: Primers used for STR amplification.

Region
Sequence

D3S1611
5′-GCCGGGTACATTGGCCTGTAATC-3′ 

5′-AGCTGAGACTACAGGCATTTGCC-3′

D5S346
5′-ACTCACTCTAGTGATAAATCGGG-3′ 

5′-AGCAGATAAGACAGTATTACTAGTT-3′

D10S197
5′-ACCACTGCACTTCAGGTGAC-3′ 

5′-CTCAAGTGGCATTGTGAAATCTTCGAAC-3′

D11S922
5′-GGGGCATCTTTGGCTACACTGG-3′ 

5′-CTCTGACCGCCACCATGTATCC-3′

D11S988
5′-CAGAAAATAGTTCAGACCACCA-3′ 

5′-GGGACAAGAGAAAAGTTGAACA-3′

D17S250
5′-GGAAGAATCAAATAGACAAT-3′ 

5′-GCTGGCCATATATATATTTAAACC-3′

Table 2: Genomic characteristics and reaction conditions for MSI markers.

Characteristics
MSI marker

D3S1611
D5S346
D10S197
D11S922
D11S988
D17S250

Genomic

    Chromosomal location
3p21.3
5q21
10p12
11p15.5
11p15.5
17q11.2

    STR sequence

    Fragment size
140 pb
115 pb
141 pb
175 pb
120 pb
151 pb

Reaction conditions

    Platinum taq DNA polymerase
0.2 u
0.2 u
0.2 u
0.2 u
0.2 u
0.2 u

    25 µM primers F/R
0.3 µL
0.6 µL
0.6 µL
0.6 µL
0.6 µL
0.6 µL

    10 mM dNTP
0.2 mM
0.2 mM
0.2 mM
0.2 mM
0.2 mM
0.2 mM

    50 mM MgCl₂
1.5 mM
1.5 mM
1.5 mM
1.5 mM
1.5 mM
1.5 mM

    10X PCR buffer
1X
1X
1X
1X
1X
1X

    DMSO 100%

0.75 µL

    DNA
1.2 µL
1.2 µL
0.6 µL
1.8 µL
1 µL
0.6 µL

    Final volume
15 µL
15 µL
15 µL
15 µL
15 µL
15 µL

Cycling conditions

    Number of cycles
30
30
28
30
32
30

    Annealing
70°C for 15seg
55°C; 30seg
70°C; 30seg
68°C; 30seg
69°C; 30seg
50°C; 30seg

2.3. Statistical Analysis

The chi square and Fisher exact tests were used for association analysis and confirmation was obtained by the Lilliefors test (significance considered when ). Statistical calculations were performed using the Epi Info v3.4.3, 2007 software.

Samples that rendered unsuccessful amplification were excluded from the statistical analysis.

3. Results

MSI was found in 6 HNSCC patients, among which only 1 was detected by the D17S250 marker, whereas the other 5 (13.5%) were detected by the D3S1611 marker. No instability was found at markers D5S346, D10S197, D11S922, and D11S988.

MSI detected by D3S1611 marker was more frequent in SCC of the oropharynx, but without a significant difference when compared to tumors of the mouth () and larynx (). In relation to tumor differentiation, MSI detected by D3S1611 marker was present in 3 (14.3%) moderately differentiated tumors and in 2 (25.0%) poorly differentiated tumors, but no statistical significance was found (), probably due to the small sample size. Additionally, D3S1611 MSI was significantly associated with older age (, Table 3).

Table 3: D3S1611 MSI frequency according to clinical and epidemiological features.

Features
D3S1611 marker

Total
MSI
MSS
P

No.
(%)
No.
f
No.
f

Gender

    Female
8
(21.6)
1
0.125
7
0.875
—

    Male
29
(78.4)
4
0.138
25
0.862

Age range, yrs

    ≤50
8
(21.6)
0
0.0
8
1.0
0.034

    50–70
22
(59.5)
2
0.091
20
0.909

    >70
7
(18.9)
3
0.492
4
0.508

Tumor site

    Oral cavity
15
(40.5)
1
0.067
14
0.933
—

    Oropharynx
8
(21.6)
3
0.375
5
0.625

    Larynx
14
(37.9)
1
0.071
13
0.929

Differentiation

    Well
8
(21.6)
0
0.0
8
1.0
0.339

    Moderately
21
(56.8)
3
0.143
18
0.857

    Poorly
8
(21.6)
2
0.250
6
0.750

Total
37
(100.0)
5
0.135
32
0.865

MSI: microsatellite instability; MSS: microsatellite stable; f: frequency; P: significance value.

Genotypic frequencies for all markers showed no statistically significant distribution alteration, neither were they related to differentiation grade or patient age (Table 4).

Table 4: Marker genotypic frequencies, according to clinical and epidemiological features.

Features
Genotypic frequencies

D3S1611
P
D5S346
P
D10S197
P

HT
f
HM
f
HT
f
HM
f
HT
f
HM
f

Total
26
0.703
11
0.297
—
23
0.821
5
0.179
—
29
0.879
4
0.121
—

Gender

    Female
6
0.750
2
0.250
—
7
0.875
1
0.125
—
8
1.0
0
0.0
—

    Male
20
0.690
9
0.310

16
0.800
4
0.200

21
0.840
4
0.160

Age range, yrs

    ≤50
7
0.875
1
0.125
0.448
5
1.0
0
0.0
0.513
6
1.0
0
0.0
0.586

    50–70
14
0.636
8
0.364

14
0.778
4
0.222

16
0.842
3
0.158

    >70
5
0.714
2
0.286

4
0.800
1
0.200

7
0.875
1
0.125

Tumor site

    Oral cavity
9
0.600
6
0.400
—
11
0.786
3
0.214
—
12
0.857
2
0.143
—

    Oropharynx
6
0.750
2
0.250

6
1.0
0
0.0

7
0.875
1
0.125

    Larynx
11
0.786
3
0.214

6
0.750
2
0.250

10
0.909
1
0.091

Differentiation

    Well
8
1.0
0
0.0
0.115
7
1.0
0
0.0
0.334
7
1.0
0
0.0
0.518

    Moderately
13
0.619
8
0.381

11
0.786
3
0.214

15
0.833
3
0.167

    Poorly
5
0.625
3
0.375

5
0.714
2
0.286

7
0.875
1
0.125

Features
Genotypic frequencies

D11S922
P
D11S988
P
D17S250
P

HT
f
HM
f
HT
f
HM
f
HT
f
HM
f

Total
19
0.760
6
0.240
—
26
0.867
4
0.133
—
28
0.875
4
0.125
—

Gender

    Female
7
0.875
1
0.125
—
8
1.0
0
0.0
—
7
0.875
1
0.125
—

    Male
12
0.706
5
0.294

18
0.818
4
0.182

21
0.875
3
0.125

Age range, yrs

    ≤50
4
0.800
1
0.200
0.413
5
1.0
0
0.0
0.171
5
0.833
1
0.167
0.912

    50–70
11
0.688
5
0.313

13
0.765
4
0.235

17
0.895
2
0.105

    >70
4
1.0
0
0.0

8
1.0
0
0.0

6
0.857
1
0.143

Tumor site

    Oral cavity
9
0.692
4
0.308
—
11
0.917
1
0.083
—
12
0.923
1
0.077
—

    Oropharynx
6
1.0
0
0.0

7
0.875
1
0.125

8
1.0
0
0.0

    Larynx
4
0.667
2
0.333

8
0.800
2
0.200

8
0.727
3
0.273

Differentiation

    Well
6
0.857
1
0.143
0.586
7
1.0
0
0.0
0.436
6
0.857
1
0.143
0.986

    Moderately
10
0.769
3
0.231

12
0.800
3
0.200

15
0.882
2
0.118

    Poorly
3
0.600
2
0.400

7
0.875
1
0.125

7
0.875
1
0.125

HT: heterozygote; HM: homozygote; f: frequency; P: significance value.

4. Discussion

The present study has identified 13.5% MSI for samples analysed with D3S1611 marker. Our results are in agreement with Chakrabarti et al. [16], who identified even higher rates of MSI (40%). In contrast, a lack of MSI in HNSCC was reported by other studies [16, 17]. These discrepancies may be explained by the great variability among repeat regions throughout the genome [18]. Instability frequency is related to the repeat unit length and overall size of the short tandem repeat (STR), affecting the probability of error during DNA replication [19, 20]. Furthermore, individuals from diverse geographic locations may present differences in their STR characteristics [21], possibly affecting accuracy during replication. Therefore, it is possible that populations with a longer average repeat size are more prone to instability than the ones with a smaller repeat size.

Marker D3S1611 is located in the MLH1 gene (mutL homolog 1), coding for an enzyme responsible for aiding in the mismatch repair system (MMR), which is pivotal for the maintenance of genomic stability, repairing DNA heteroduplexes generated by replication [22, 23]. MLH1 gene alterations are related to the development of colorectal cancer, suggesting that this enzyme is needed for correct DNA repair at least in some tissues [21].

In addition, we have found a higher rate of D3S1611 MSI in older patients, suggesting that this marker may be affected by aging processes in the DNA repair machinery. According to Hardwick et al. repeat size expansion is an inevitable and progressive phenomenon along the life span of rodents [24].

In conclusion, the present study reports that D3S1611 marker can identify instability in a fraction of HNSCC patients, being related to older ages.

Ethical Approval

This study was approved by the Ethics Committee of the Federal University of Espírito Santo on 30/04/2009 ( Protocol no. 010/09).

Conflict of Interests

The authors declare that they have no competing interests.

Acknowledgments

Part of this study was sponsored by Fibria Celulose , Fundação de Amparo a Pesquisa do Espírito Santo (FAPES), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes) scholarships. The authors thank the Hospital Santa Rita de Cássia for providing the biological samples.

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