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

Authors: Elaine Stur; Eldamaria de Vargas Wolfgramm; Allan Kardec de Castro Neto; Lucas de Lima Maia; Lidiane Pignaton Agostini; Gabriela Tonini Peterle; Suzanny Oliveira Mendes; Marcelos dos Santos; Flávia de Paula; Iúri Drumond Louro
Journal: ISRN Biomarkers
 (2013)

---

## 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.


---

## Body

## 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
(
CA
)
n
(
CA
)
n
(
CA
)
n
(
CA
)
n
(
CA
)
n
(
CA
)
n
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 whenP<0.05). Statistical calculations were performed using the Epi Info v3.4.3, 2007 software.Samples that rendered unsuccessful amplification were excluded from the statistical analysis.


## 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
(
CA
)
n
(
CA
)
n
(
CA
)
n
(
CA
)
n
(
CA
)
n
(
CA
)
n
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 whenP<0.05). 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 (P=0.273) and larynx (P=0.279). 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 (P=0.339), probably due to the small sample size. Additionally, D3S1611 MSI was significantly associated with older age (P=0.034, 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 (Table4).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.



---

*Source: 474107-2013-12-14.xml*