1 Molecular Genetics and Oncology Group, Clinical Dental Sciences, The University of Liverpool, Liverpool L69 3BX,
2 Department of Medicine, The University of Liverpool, Liverpool L69 3GA, UK,
3 Deparment of Biochemistry, Faculty of Science, Annamalai University, Tamilnadu, India,
4 Department of Otorhinolaryngology, The University of Liverpool, Liverpool L69 3BX, UK,
5 Maxillofacial Unit, Fazakerley Hospital, Liverpool,
6 Department of Pathology, The University of Liverpool, Liverpool L69 3BX and
7 The Roy Castle International Centre for Lung Cancer Research, Liverpool L3 9TA, UK
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Abstract |
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Abbreviations: FAL, fractional allele loss; HGI, high genomic instability; LGI, low genomic instability; LOH, loss of heterozygosity; NSCLC, non-small cell lung cancer; SCCHN, squamous cell carcinoma of the head and neck; SSCP, single-strand conformation polymorphism; TSGs, tumour suppressor genes.
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Introduction |
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SCCHN arise as a result of the accumulation of genetic events. Statistical analysis based on age-specific incidence suggests that SCCHN arise following 610 independent genetic events (5). This is in agreement with the multistep carcinogenesis model of SCCHN development which investigators have proposed in recent years. Genetic alterations involved in SCCHN include activation of proto-oncogenes and inactivation of tumour suppressor genes (TSGs) (69).
Allelic imbalance is probably the most common genetic factor of cancer. To date, there are a large number of reports demonstrating that a significant proportion of the genome of human tumours contains losses or duplications of certain loci. Allelic imbalance resulting in loss of one allele has also been termed loss of heterozygosity (LOH). It is still unclear whether allelic imbalance is the cause or the result of carcinogenesis. Nevertheless, it is a direct indication of the cancer cell's genomic instability. The existing model of carcinogenesis indicates that all human tumours have an unstable genome and that allelic imbalance is a very useful tool in assessing the level of genetic damage in the early stages of cancer. A number of LOH studies have been undertaken in head and neck cancer (1012). The largest of these studies analysed 80 SCCHN using 145 microsatellite markers on 39 chromosomal arms (12). Fractional allele loss (FAL) was calculated for all tumours for which data on nine or more chromosomal arms were available and the median value was found to be 0.22. A correlation was found between FAL > median value and positive lymph nodes at pathology and poor survival.
Allelic imbalance on the short arms of chromosomes 3, 9 and 17 has received a great deal of attention and it has been argued that these events are associated with the early stages of carcinogenesis in SCCHN and non-small cell lung cancer (NSCLC) (1026). We have recently demonstrated that FAL values calculated from the analysis of allelic imbalance on chromosome arms 3p, 9p and 17p in NSCLC specimens provides a basis on which one may subgroup these tumours into two different genetic pathways (14).
Gupta et al. have investigated the mechanism of LOH and argue that mitotic recombination may be the primary mechanism for the high frequency of LOH in normal somatic cells in vivo (27). Thus LOH caused by mitotic recombination may trigger one of the early steps in carcinogenesis. Furthermore, the role of p53 in the maintenance of genetic instability has been demonstrated by allelic imbalance (28,29), chromosome stability (30), ploidy (31) and suppression of homologous recombination (32). It is clear that the interaction of genetic instability with p53 alterations most likely plays an important role in carcinogenesis. A study undertaken by Edington et al. on LOH in senescent and immortal SCCHN cell lines demonstrated that high FAL was associated with p53 alterations, indicating that loss of the normal p53 gene may be the rate limiting step in carcinogenesis (29). These authors argued that their results demonstrated two mechanisms were involved in the development of SCCHN, based on FAL and p53 data.
It is unclear if allelic imbalance on chromosomes 3p, 9p and 17p are solely involved in the pathogenesis of all SCCHN and whether these changes are related to the degree of genetic damage. LOH has been observed in 6275% of SCCHN on chromosome 9p, with the majority of loss being minimised to the chromosomal region 9p2123 (11,12,16,17,21). The range of allele loss is most likely due to differing pathology and stages of tumours studied. Van der Reit et al. observed similar frequencies of loss on chromosome 9p in both preinvasive and invasive lesions, suggesting that the loss of chromosome 9p is an early event in SCCHN progression (21). A second, frequently deleted locus occurs on chromosome 3p, but several studies have suggested that this region of loss is complex because of the possible overlap of three distinct suppressor regions (3p2425, 3p2114 and 3p1214) juxtaposed to one another (10,11,13,15,16,19,20). Loss of chromosomal region 17p has been demonstrated in 3150% of SCCHN (1012,17,18), with previous reports finding the highest loss at 17p1211.1, which is distinct from the p53 region (17p13.1) (17,18). p53 mutations have been shown to occur in 19% of non-invasive oral lesions which rises to 3869% of invasive lesions (3338). This suggests that p53 mutations may precede invasion of a primary SCCHN.
In this study we have addressed the question whether allelic imbalance at 3p, 9p and 17p, together with p53 alterations, are associated with the pathogenesis of SCCHN and thus indicative of the degree of genetic damage seen in the whole genome.
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Materials and methods |
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Microsatellite primers were obtained from Isogen (Amsterdam, The Netherlands) and Research Genetics (Huntsville, AL). PCRs were performed in a 25 µl reaction volume which contained 100 ng of genomic DNA, 200 mM each dNTP, 10 pmol of each forward and reverse primer, 2.5 µl of 10x PCR buffer (670 mM TrisHCl, pH 8.5, 166 mM ammonium sulphate, 67 mM magnesium chloride, 1.7 mg/ml bovine serum albumin, 100 mM ß-mercaptoethanol and 1% w/v Triton X-100) and 0.2 U Taq polymerase (Bioline, London, UK). Reactions were denatured at 94°C for 5 min, followed by 25 cycles of 94°C for 30 s, 4560°C for 30 s and 72°C for 30 s, with a final extension of 72°C for 5 min. A 10 µl volume of the PCR product was electrophoresed on a 10% polyacrylamide gel (Accugel; National Diagnostics, Hull, UK) and visualized by silver staining.
LOH, or allelic imbalance, was scored by direct visual comparison of the allelic ratios of the normal and tumour DNA. A reduction of at least 50% in the intensity of one allele in the tumour was considered as LOH (Figure 1). In addition, demonstration of a shift of one or both of the alleles in the tumour DNA as compared with the corresponding normal DNA was recorded as a microsatellite alteration.
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SSCP analysis
For SSCP 24 µl of PCR product were mixed with 10 µl of denaturing solution, consisting of 80% formamide, 100 mM NaOH, 10 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol. Samples were then denatured at 95°C for 3 min, snap frozen on ice and loaded onto an 810% polyacrylamide (29:1) gel (Accugel; National Diagnostics). Gels were run at 15°C and visualized by silver staining after electrophoresis.
DNA sequencing
Samples that showed altered mobility by SSCP analysis were re-amplified and cleaned up using Promega Wizard PCR cleanup kits (Promega, Madison, WI). These samples were cycle sequenced (39) using the appropriate sequencing primers and BIG DYETM terminator kits from PE Biosystems (Perkin-Elmer Corp., Foster City, CA). The resultant reaction was precipitated with 75% isopropan-1-ol to remove excess fluorescent dyes and salts. The pellets were re-suspended in Template Suppression ReagentTM from PE Biosystems and denatured for 5 min and chilled on ice. The samples were loaded into an ABI 310 Genetic Analyzer (PE Biosystems). The samples were electrophoresed in the analyzer's capillary, through Performance Optimized Polymer 6 (PE Biosystems) at 15 kV for 25 min. The resultant data were analysed using ABI Sequence Analysis software.
To reduce the possibility of missing mutations during SSCP analysis, five SSCP-negative tumour DNA samples for each exon were randomly picked and sequenced. No mutations were revealed by sequencing in any of the SSCP-negative specimens examined.
Statistical analysis
Microsatellite markers on chromosomal arms other than 3p, 9p and 17p have been analysed, which has allowed the genomic FAL value for each tumour to be recalculated. FAL was calculated by dividing the number of chromosomal arms showing LOH by the number of informative chromosomal arms. FAL was only calculated for those tumours in which nine informative chromosomal arms were available. Ten of the previously studied 45 samples produced the same FAL value (12) while the remaining samples altered their FAL value, due to the further analysis that had been undertaken.
Specimens were included for analysis, in Table II, if at least three of the microsatellite markers used on each of the three chromosomal arms demonstrated informative results. Quantitative data were analysed by
2 or Fisher's exact test where appropriate. Survival analysis was performed using the log rank test and Cox's proportional hazards multiple regression, using SPSS software for Windows. A P value < 0.05 was considered statistically significant.
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Results |
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FAL analysis
The allelic imbalance data for these tumours were re-examined on the basis of their FAL scores and the tumours subdivided into low FAL (LFAL, 0.000.19), medium FAL (MFAL, 0.200.32) and high FAL (HFAL, 0.330.88) groups using the total allelotype data (i.e. allelic imbalance on at least nine chromosomal arms) to calculate the FAL score. This subdivision was into three equal sized groups after ranking the FAL score, rather than using exogenously determined cut-off points that might have better supported our a priori hypothesis. The data from the SCCHN allelotype was also incorporated into this analysis in conjunction with all of the new specimens and markers analysed in this study. It was found that these FAL value subgroups were based symmetrically around the median value of 0.25. The results of this analysis demonstrated a very clear grouping of allelic imbalance data on chromosomes 3p, 9p and 17p depending on FAL subgroup. There was no significant difference in the number of markers used in analysis of the LFAL (median 81 markers, range 30166), MFAL (median 63.5 markers, range 35142) and HFAL (median 72 markers, range 38158) subgroups. In all sections of Figure 2 the allelic imbalance data are diagramatically represented for each of the three main FAL groups. Each FAL group is categorized according to the scale of allelic imbalance, i.e. no allelic imbalance on 3p, 9p or 17p up to individuals containing allelic imbalance on all three chromosomal arms.
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Similarly, when the allelic imbalance data for 9p were subgrouped on the basis of FAL, it was observed that 5/15 (33%) of the LFAL tumours had allelic imbalance compared with 15/16 (94%) of the HFAL SCCHN group (Table II and Figure 2
). The majority of the markers used in this analysis were located in the 9p23p21 region.
This relationship between HFAL and a high percentage of allelic imbalance at specific chromosomal locations was further demonstrated by the allelic imbalance data on chromosome 17p. On 17p, the LFAL tumours had 43% (3/7) allelic imbalance compared with 100% (16/16) for HFAL tumours (Table II and Figure 2
).
Statistical analysis of these results demonstrated that there was a significantly higher allelic imbalance on 3p, 9p and 17p in the HFAL subgroup compared with the LFAL and MFAL subgroups, individually and combined (Table II). In addition, the chromosomal loci 3p26p24 and 3p14.2p12 were found to contain a significantly higher level of allelic imbalance in the HFAL subgroup compared with the LFAL and MFAL subgroups.
Of the 16 tumours in the LFAL subgroup, seven had allelic imbalance on 3p (0310, 0359, 0339, 0204, 0361, 0358 and 0336), five had allelic imbalance on 9p (0353, 0359, 0339, 0361 and 0336) and three had allelic imbalance on 17p (0302, 1171 and 0336). Four LFAL tumours had allelic imbalance on both 3p and 9p (0359, 0339, 0361 and 0336) and one LFAL tumour, which had the highest FAL value within the LFAL group, had allelic imbalance on 3p, 9p and 17p. Thus, six of the 16 tumours (1176, 128, 345, 1228, 1087 and 1101) showed no allelic imbalance on chromosomes 3p, 9p and 17p in our analysis; allelic imbalance was observed at other chromosomal locations in all of these tumours, including 2p25p24, 5q2122, 7pterp22, 8q13q22.1, 11q23.3, 13q32, 17q, 18q21.31, 18p11.21 and 19q12q13.1. All but three of the MFAL tumours had allelic imbalance at 3p, 9p or 17p; these three tumours (0340, 0348 and 0202) with FAL values of 0.22, 0.25 and 0.28, respectively, had allelic imbalances on other chromosomal arms.
Microsatellite alterations
The 52 SCCHN were additionally assessed for microsatellite alterations; 24/52 (46%) of the tumours demonstrated microsatellite alterations at more than two loci. When divided into FAL subgroups, 8/16 (50%) LFAL, 7/18 (39%) MFAL and 9/19 (47%) HFAL tumours showed microsatellite alterations and no statistically significant differences were found. No relationship between p53 mutations and genomic instability was found.
p53 mutational profile in SCCHN
In this study, a number of the p53 mutational results for exons 59 in 39 samples have been reported previously by Liloglou et al. (9). This study incorporates those results, together with eight new specimens. Forty-seven samples have been investigated for the presence of mutations within exons 59 of the p53 gene, using SSCP and sequencing analysis. SSCP analysis was used to rapidly screen for p53 mutations in exons 59 and all samples presenting abnormal electrophoretic mobilities were sequenced. Sequence analysis revealed 24 mutations in 15 of the 47 SCCHN specimens (32%; Table III). Sequencing demonstrated 13 missense, two nonsense, two frameshift and five silent mutations, as well as one affecting splicing. The mutational profile found was four deletions and 20 base substitutions. The base substitutions consisted of 12 transitions (10 GC
AT and two AT
GC) and eight transversions (five GC
TA, two AT
TA and one GC
CG).
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Clinico-pathological correlations with FAL
Statistically significant correlations were demonstrated between FAL and the node status of the SCCHN specimen (P = 0.04); no other correlations were found between FAL and any of the clinico-pathological parameters
p53 alterations and FAL values
In this group of 47 SCCHN patients, 15 patients had p53 mutations. It should be noted that the tumours with p53 mutations fell evenly over the FAL subgroups, with six in the LFAL group, five in the MFAL group and four in the HFAL group. No correlation was found between the p53 mutations and the FAL score. The allelic imbalance details of patients were analysed to ascertain whether specific genetic loci were associated with p53 mutations, however, none were found.
In addition, when the patients were divided into two groups, FAL < 0.25 (median value) [low genomic instability (LGI); 8/26] and FAL 0.25 [high genomic instability (HGI); 7/26], there was no difference in the distribution of p53 mutations in the two groups.
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Discussion |
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On examination of SCCHN with FAL scores, based on total allelotype data, tumours were subdivided into three groups based symmetrically around the median value of 0.25, low FAL (LFAL, 0.000.19), medium FAL (MFAL, 0.200.32) and high FAL (HFAL, 0.330.88) groups. The combination of allelic imbalance and FAL data demonstrated a predominance of allelic imbalance on chromosomes 3p, including 3p14.212 and 3p26p24, 9p and 17p in the HFAL group rather than the LFAL and MFAL groups (P < 0.005). Thus genetic alterations on these chromosome arms are mainly observed in human SCCHN tumours that also demonstrate high levels of allelic imbalance across the remainder of the genome. Conversely, LFAL tumours develop with a minimum amount of genetic damage and a lower frequency of allelic imbalance of chromosomes 3p, 9p and 17p. This study agrees with findings in NSCLC, in which a clear grouping of allelic imbalance on chromosomes 3p, 9p and 17p was also seen in HFAL rather than LFAL tumours (14). This considerably increases the strength of our conclusions in this paper. Thus similar chromosomal alterations are apparently involved in the pathogenesis of SCCHN and NSCLC, which may reflect the common risk factor, tobacco consumption, for these tumours.
The finding that 6/16 (38%) LFAL SCCHN did not demonstrate allelic imbalance at 3p, 9p and 17p suggests that a second genetic pathway is involved in the initiation and progression of these tumours. Allelic imbalance was observed at other chromosomal locations in these six tumours, including 2p2425, 5q2122, 7pterp22, 8q13q22.1, 11q23.3, 13q32, 17q, 18q21.31, 18p11.2 and 19q1213.1. Previous studies of SCCHN have found allelic imbalance on these chromosomal arms in a variable proportion of these tumours: 023% at 2p, 2243% at 5q, 823% at 7p, 838% at 8q, 2361% at 11q, 062% at 13q, 2349% at 18q, 1627% at 18p and 040% at 19q (1012,16,4043). TSGs identified in these regions include the adenomatous polyposis coli, APC (5q2122) gene and the deleted in colorectal cancer, DCC (18q21.1) gene (42) and the mismatch repair gene hPMS2 is located at 7p22 (44). Thus our findings in LFAL tumours raise the possibility that alterations on chromosomes other than 3p, 9p and 17p play a specific role in the development and progression of these SCCHN in certain tumours.
The CDKN2A gene, which encodes for p16, has been considered to play an important role in SCCHN carcinogenesis. We have found that 23% (6/26) of informative individuals demonstrated allelic imbalance (MSI or LOH) at D9S171 (a microsatellite marker near to CDKN2A), while Wu et al. found 31% (45). They showed that 87% of oral pharyngeal tumours analysed lost CDKN2A expression and that there was a direct correlation between allelic imbalance and loss of p16 expression. However, p16 inactivation appears to be a complex interaction between p16 mutations, methylation status, allelic imbalance and homozygous deletion.
In this study we have investigated exons 59 of the p53 gene for mutations, as this area had been reported to contain >95% of all the published p53 mutations (46,47). While some groups have examined exons 24, 10 and 11 of the p53 gene (4850), only one group has found any mutations in these exons (50) and these were all found in oral SCC cell lines. In the present study, we detected 15 specimens containing mutations within exons 59 of the p53 gene (15/47, 32%). This figure is in agreement with previous studies which have reported p53 mutations in the range 1869% within head and neck carcinomas (9,3338). The published data on p53 mutations in SCCHN have a broad range, however, even though we have found 32% with mutations, it is unlikely that we have missed any mutations within exons 59. We sequenced a selection of SSCP negatives and found no sequence abnormalities. However, we cannot completely exclude mutations in exons 24 or 1011 or other methods of p53 inactivation, such as HPV or MDM2. We have previously reported an association between p53 mutational status and FAL score (9) based on 34 SCCHN patients. We have now enlarged this study to include 52 patients and this association no longer stands. Our current data indicate that 8/26 patients with LGI had p53 mutations and 7/26 patients with HGI had p53 mutations. The reason for this is most likely due to a larger and more complete data set, as well as an increased number of microsatellite markers used in the analysis. It is important to note that even with the increased number of microsatellite markers used only three of the patients (342, 318 and 1062), previously reported in Field et al. (12), with LGI have moved into the HGI group.
In this study there are eight patients with p53 mutations in their SCCHN which have a median FAL value 0.25. This result is important when considered in the context of previous publications. It is of note that the hypothesis put forward by Edington et al. (29) that p53 mutational status was associated with FAL score in SCCHN is not supported by these results. Edington et al. investigated immortal and senescent cell cultures and demonstrated that immortal SCCHN cell lines were more prevalent in advanced or recurrent tumours and that those with a high FAL value were found to harbour p53 mutations (29). It was argued that the loss of normal p53 function is a rate limiting step in carcinogenesis and that after p53 is altered SCCHN are destined to become immortal and exhibit high FAL (29). Edington et al. based their hypothesis on a relatively limited number of cell lines, which may have led to a biased result, a factor we have had to take into account with our new data set.
The results presented here and by Ligloglou et al. (9) disagree with the finding of an association between p53 mutations and high FAL and that the loss of wild-type p53 contributes to the high genetic damage seen in tumour cells. Furthermore, in a recent study on oral squamous cell carcinomas, no correlation was found between p53 mutations and the FAL score, however, overexpression of p53, as measured by immunohistochemistry, did correlate with the FAL score (37). Our results indicate that there is more than one mechanism leading to tumourigenesis. In this study 7/26 of the HGI group contain p53 mutations and thus it may be argued that there is a subset of patients where there may be an interaction between alterations in the p53 gene and genetic instability. However, it is clear that there is another group of patients with LGI that do have p53 mutations. Furthermore, the allelic imbalance data in the LFAL group indicates that LOH on 3p, 9p and 17p is not the trigger for carcinogenesis in all of these patients. These results argue strongly for the hypothesis that there are two distinct subgroups of patients in the development of SCCHN.
Liloglou et al. (9) found that as half of individuals who were heavy or former smokers contained p53 mutations and that the majority of these patients had minimal amounts of genetic damage (LGI) and thus argued that p53 mutations were early initiating events in SCCHN and accelerate their progression through carcinogenesis. Our results support this theory, as 10/22 (45%) of the heavy/former smokers contained p53 mutations, with 6/10 having a FAL score below the median value. Furthermore, in agreement with Liloglou et al. (9), it was observed that a significantly higher number of present and former smokers contained p53 mutations compared with non-smokers (P = 0.05).
The role of p53 mutations in the generation of genetic instability in SCCHN is unclear. To date this is the largest study of p53 mutations with allelic imbalance data and the lack of any correlation between p53 and FAL status argues against the hypothesis put forward by Edington et al. (29). Various chromosomal mechanisms have been suggested which may explain the loss of an allele in a tumour DNA specimen compared with a corresponding normal DNA specimen (51,52). These include: (i) deletion of the wild-type chromosome resulting in hemizygosity at all loci near the tumour suppressor gene; (ii) loss followed by duplication resulting in two copies of one allele and loss of the other; (iii) mitotic recombination between homologues, resulting in heterozygosity at loci in the proximal region and homozygosity throughout the rest of the chromosome, including the TSG locus; (iv) localized events such as point mutations, small deletions and gene conversions (51,52).Thus it may be argued that the HFAL group are gaining or losing whole chromosomes, which may explain the lack of a correlation between p53 status and FAL. However, the results of this investigation strongly support the statement that allelic imbalance on 3p, 9p and 17p does separate the SCCHN tumours into two distinct genetic populations. We have also provided evidence for this hypothesis in NSCLC (14).
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Acknowledgments |
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Notes |
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References |
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