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

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 theMLH1 gene, which is part of themismatch repair system (MMR), helping tomaintain genomic stability.Wehave found a higher rate ofD3S1611MSI in older patients, suggesting that thismarkermay be affected by aging processes in the DNA repair machinery.


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][8][9][10][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.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.

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  < 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.

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 ( = 0.273) and larynx ( = 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 ( = 0.339), probably due to the small sample size.Additionally, D3S1611 MSI was significantly associated with older age ( = 0.034, Table 3).
Genotypic frequencies for all markers showed no statistically significant distribution alteration, neither were they related to differentiation grade or patient age (Table 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).

Table 1 :
Primers used for STR amplification.

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

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

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