Mutagen sensitivity and environmental exposures as contributing causes of chromosome 3p losses in head and neck cancers
Stimson P. Schantz5,
Qiang Huang,
Kinner Shah,
V.V.V.S. Murty2,
T.C. Hsu3,
Guopei Yu,
Peter E. Andersen4,
Andrew G. Huvos1 and
Raju S.K. Chaganti2
Department of Surgery,
1 Department of Pathology and
2 Department of Human Genetics, Memorial Sloan-Kettering Cancer Center, New York,NY 10021,
3 Department of Cell Biology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030 and
4 Department of OtolaryngologyHead and Neck Surgery, Oregon Health Sciences University, Portland, OR 97201, USA
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Abstract
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The interaction between environmental exposures and host susceptibility may lead to specific mutational events within head and neck squamous cell carcinoma (HNSCC). Furthermore, this interplay may determine not only the probability of cancer development but also the biologic characteristics of the tumor once it occurs. To better understand the relationship of mutagen sensitivity and tobacco and/or alcohol consumption on HNSCC carcinogenesis, we examined loss of heterozygosity on chromosome 3p in 58 HNSCCs using 10 microsatellite markers. Mutagen sensitivity was determined in vitro by quantitating bleomycin-induced chromatid breaks utilizing peripheral blood lympocytes from respective patients. Forty-six of the 58 invasive cancers showed allelic loss at one or more loci. Consistent with previous investigations, three discrete regions of deletions were identified: 3p1314.2, 3p21.121.2, and 3p25.126.1. The frequency and types of deletions were dependent upon tobacco and alcohol exposures. The distal region of 3p but not the remaining two regions was most frequently influenced by tobacco exposure. In contrast, heavy alcohol use when combined with tobacco use was associated with whole-arm loss of 3p rather than identifiable site-specific damage. Furthermore, this combined influence of alcohol and tobacco exposures on whole-arm loss was most apparent in those patients who expressed mutagen-sensitivity; the odds ratio of whole-arm loss increasing from 2.67 (95% CI 0.2133.49) in those individuals who were mutagen resistant to 13.5 (95% CI 1.3136.0; P = 0.02 by Fisher's exact test) in those who were mutagen sensitive. An assessment of clinical parameters in this population demonstrated that patients with whole-arm loss were more likely to present with cervical lymph node metastases and advanced stage disease than patients with partial losses. Results indicate that various environmental exposures as well as the expression of mutagen sensitivity will influence the types of chromosome 3p allelic losses in head and neck cancers as well as the behavior of disease once it develops.
Abbreviations: HNSCC, head and neck squamous cell carcinoma; LOH, loss of heterozygosity.
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Introduction
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The development of head and neck cancer may depend upon the interaction between host susceptibility factors and environmental carcinogens. Tobacco and alcohol have been implicated as the etiologic agents in ~80% of individuals (1). Host susceptibility factors are only now being elucidated and may include abnormalities in carcinogen metabolism, factors linked to blood group antigen expression, as well as deficiencies in DNA repair. Furthermore, exposure to tobacco carcinogens may lead to specific mutational events commonly identified in head and neck cancers, the most common of which involves the p53 gene. This gene is observed to be mutated in 4269% of head and neck cancers, dependent upon the extent of a patient's tobacco use (25). Mutations commonly involve a G
A transversion which can be duplicated in the laboratory with benzo[a]pyrene (6).
One of the more frequently identified genetic alterations in head and neck cancers involves the short arm of chromosome 3 in which chromosomal rearrangements and deletions predominate. Indeed, deletion mapping of chromosome 3p shows losses involving at least one locus in >50% of the patients. Using allelotype analysis, Maestro et al. and others found three regions of chromosomal loss, 3p14-cen, 3p21.3 and 3p24-ter (reviewed in ref. 7). Though not universally confirmed, Roz et al. identified similar losses and demonstrated that such mutations commonly occurred early in the progression from dysplasia to invasive disease (reviewed in ref. 7). The high frequency of similar losses in other tobacco-related diseases such as the lung and esophagus would suggest a causal relationship; an observation reinforced in the study by Sozzii et al. (8) in which the probability of mutations involving the FHIT gene at 3p14.2 is significantly increased within lung cancers from smokers as compared with non-smokers, but no point mutations or deletions of this gene were found in head and neck squamous cell carcinoma (HNSCC) (9).
A premise in this study is that damage to chromosome 3p in head and neck cancers may be influenced by the interaction between carcinogen exposures and host susceptibility factors. One such factor within head and neck cancer patients may be reflected in the mutagen-sensitivity assay (1012). The assay makes use of peripheral blood lymphocytes in order to test for bleomycin-induced chromosomal breakage in vitro. Bleomycin induces chromosomal damage through the generation of free radical oxygen and, thus, is reflective of one measure of tobacco-induced damage. Previous studies have demonstrated that head and neck cancer patients may be abnormally sensitive to bleomycin-induced chromosomal damage as compared with age- and sex-matched healthy controls (11,1320). Furthermore, risk assessments in these studies suggest an interaction between carcinogen exposure and mutagen-sensitivity measures, risk estimates being highest in those individuals who both consume tobacco and express sensitivity to free radical oxygen damage in vitro (14,16). The basis of this mutagen sensitivity may reflect either an underlying DNA repair deficiency or factors which control susceptibility to initial clastogenic influences (19).
Relevant to our presented study, Dave et al. (21) has also suggested that chromatid break sites induced by bleomycin in vitro were not random but rather were predetermined by both host-susceptibility factors as well as specific mechanisms related to free radical oxygenchromatid interactions. In support of this hypothesis, lymphocytes obtained from head and neck cancer patients more frequently demonstrated breaks on the short arm of chromosome 3 following bleomycin exposure in vitro than similarly treated lymphocytes from either healthy controls or patients with melanoma. In light of these observations, we present here additional evidence of deletional events involving chromosome 3p with three discrete regions of deletion. Furthermore, we were able to extend previous 3p allelotypic studies by assessing for the relationship of specific deletional events to the duration and intensity of tobacco and alcohol exposures; the latter information obtained as part of a prospective casecontrol epidemiologic study (16). The above results were then examined with respect to quantitative measures of mutagen sensitivity expressed in vitro by peripheral blood lymphocytes from the respective patients. The data presented here will demonstrate that 3p deletional events will vary, dependent upon the level of tobacco and alcohol exposure and their overall frequency will be influenced by the patient's expression of mutagen sensitivity.
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Materials and methods
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Patient population
Fifty-eight patients with previously untreated squamous cell carcinoma of the upper aerodigestive tract formed the basis of this study. Information regarding carcinogen exposures was obtained using a self-administered questionnaire prior to treatment, mutagen-sensitivity assessment and analysis of 3p deletions in the patient's respective cancer. This questionnaire included information regarding the duration and intensity of tobacco and alcohol exposure. History regarding tobacco exposure included type of tobacco consumed, years of smoking and the age at initiation. Alcohol consumption patterns included the types of alcohol, frequency and quantities of alcohol consumption, and years of alcohol ingestion. The validation of this self-administered, comprehensive questionnaire has been reported previously (22). Of note, in three patients no questionnaire information was provided and information regarding tobacco and alcohol exposures were obtained from the medical chart without knowledge of mutagen-sensitivity and allelotype assessments. Informed consent was obtained from all patients prior to questionnaire administration using a protocol approved by the institutional review board of Memorial Sloan-Kettering Cancer Center.
Mutagen-sensitivity assay
The assay utilized in this study has been described in detail previously (16,23). A peripheral blood sample was collected from each donor in a heparinized tube prior to the initiation of lymphocyte culture. The standard lymphocyte culture procedure utilized RPMI-1640 medium, supplemented with 15% fetal calf serum and phytohemagglutinin in a ratio of blood to medium of 1:9. At 67 h of incubation, cultures are treated with bleomycin (0.03 U/ml) for 5 h. Colcemid (0.04 mg/ml) is added in the last hour to induce mitotic arrest prior to harvesting. Conventional cell harvesting procedure follows: the cells are treated with hypotonic KCl (0.06 M) solution for 1520 min, fixed, washed with a freshly prepared mixture of methanol and acetic acid (3:1) and air-dried on wet slides. The slides are stained with Giemsa solution without banding.
Fifty well-spread metaphases are examined from coded slides. Chromatid aberrations are recorded as frank chromatid breaks or exchanges. Bleomycin tends to induce few chromatid exchanges (which if present, are considered as two breaks). Chromatid gaps or attenuated regions are disregarded. The frequency of breakage was expressed as breaks per cell (b/c) for purposes of comparison.
The reliability of cytogenetic scoring has previously been evaluated by comparing four separate blood samples from a respective donor with a minimum interval between samples of 1 week (23). Using a random effect one-way analysis of variance model, significance within group variation was noted, suggesting that sensitivity appeared to be stable and representative.
Microdissection and DNA isolation
Normal tissue, dysplasia and frankly invasive cancer were identified by a pathologist (A.G.H.) using a 5 µm H&E stained section. Four to eight additional 8 µm serial sections were obtained from the same specimens above for microdissection. Using the 5 µm stained sections as a guide, suitable areas for tumor, dysplasia and normal non-epithelial tissue contained within the corresponding 8 µm sections were microdissected using a microcapillary pipette that had been reconfigured into a fine pipette tip. The microdissected cells, deparaffinized in xylene, were collected in a 1.5 ml Eppendorf tube containing 100 µl of digestion buffer (50 mM TrisHCl pH 8.5, 1 mM EDTA and 0.5% Tween-20) and then subjected to proteinase K (at a final concentration of 400 µg/ml) digestion at 50°C for 72 h. The digested samples, which were incubated at 95°C for 10 min to inactivate the proteinase K, were centrifuged and the supernatant was extracted with phenolchloroform and ethanol precipitation before being used for PCR analysis.
Loss of heterozygosity (LOH) analysis
Ten microsatellite markers mapping to chromosome 3p were used (Table I
). All primer pairs were obtained from Research Genetics (Huntsville, AL). A standard PCR reaction was carried out in a toal volume of 15 µl containing 2550 ng of DNA, 10 pmol of each primer, 0.3 µCi of [
-32P]dCTP (3000 Ci/mmol; DuPont New England Nuclear, Boston, MA). The PCR products were denatured in sequencing stop solution and subjected to electrophoresis in 6% denaturing polyacrylamide gels and the dried gels were autoradiographed for 472 h. Either the absence or the decrease in signal intensity by >50% of one allele in tumor or dysplasia DNA compared with the same allele in normal DNA by visual examination was considered as LOH. Alterations were judged as microsatellite instability when additional bands appeared in tumor or dysplasia DNA. All samples showing LOH or microsatellite instability were subject to repeat analysis and in all cases, the replicate test gave identical results.
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Results
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Allelic deletions on 3p
The frequency of LOH in tumor DNA among the tested loci ranged from 36% (10 of 28 cases) at D3S656 to 78% (31 of 40 cases) at D3S1038 (Table I
). Forty-six of the 58 invasive carcinoma specimens (79%) showed allelic loss at one or more loci. Of these, 17 (37%) tumors showed LOH at all informative loci suggestive of entire 3p deletions. The remaining 29 tumors exhibited interstitial deletion. The boundaries for minimal deletion were defined by retention of heterozygosity of markers on either side or on one side of a loss. The patterns of LOH in this group identified three common regions of deletions at 3p25.126.1, 3p21.121.2, and 3p1314.2. The distal 3p25.126.1 region spanning the markers D3S1038 and D3S1110 was deleted in 22 (76%) tumors with interstitial deletions. The tumors p-19, p-6, o-36, y-14, p-9, y-10, p-3, y-16 and p-22 define the boundaries. The proximal 3p1314.2 region containing the markers D3S659 and D3S1228 was deleted in 20 (69%) tumors with interstitial deletions. The boundaries of the 3p1314.2 were defined by the tumors y-20, y-6, y-5, y-9, p-33, p-6, y-2, y-17, p-10, o-36, y-10, p-3 and y-11. The third region, 3p21.121.2, spanned by the marker D3S1076 was deleted in 13 (45%) tumors with the boundaries identified by y-17, y-14, p-26 and y-8 (Figure 1
). Of the 29 tumors identified with common regions of deletion, 10 showed deletions at all three regions, 11 at two regions, and eight tumors exhibited deletions at one region. Thus, these data have identified three specific regions of deletions on 3p in head and neck cancer.

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Fig. 1. Patterns of LOH and replication errors on chromosome 3p in 46 head and neck cancers.Vertical lines on right of the idiogram indicate sites of three discrete regions of deletion. Solid, hatched and blank squares represent LOH, retention of heterozygosity and homozygous/non-informative cancers, respectively. Solid circles within squares indicate replication error-type microsatellite instability. ND, not done.
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To determine the relative timing of chromosome 3p alterations in head and neck cancer progression, we analyzed LOH on this chromosome in 18 paired dysplasia and tumor DNA samples. In all instances, the dysplasia from each patient was identified as adjacent to the respective individual's cancer. All samples were graded as mild to moderate dysplasia with no evidence of severe dysplasia or carcinoma in situ. As a comparison, we similarly assessed for losses involving the 9p21 locus using the D9S171 primer. This region has characteristically been identified as deleted in dysplastic oral lesions and is thereby considered an early event (24,25). Although five of 18 (28%) dysplastic samples in which DNA had been obtained showed LOH in at least one locus, the overall frequency of loci showing LOH was very low (five of 115 informative loci; 4%). In contrast, the corresponding tumor samples adjacent to the dysplastic lesions showed a high incidence of LOH. Eleven (61%) of the 18 tumors showed evidence of loss in at least one informative locus. Overall, 55 (45%) of the 121 informative loci within the cancers displayed LOH. Assessment of mutational events at chromosome 9p21 showed LOH in 15% (two of 13 informative cases) and 54% (seven of 13 informative cases) in the same dysplasias and cancers, respectively. Of note, the regions of LOH within the dysplastic lesions were similar to those within tumors and included three at 3p25.126.1, one at 3p21.121.2 and one at 3p1314.2.
Tobacco, alcohol and LOH
Fifty-six patients had information available regarding tobacco and alcohol consumption as well as allelotype assessment. The relationship between the intensity of tobacco and alcohol exposure and deletions at any of the three discrete regions on chromosome 3p is detailed in Tables II and III
. Patients were categorized as either non-users or categorized by increasing use of each of the two substances. Of the 56 patients, 55 had information regarding tobacco use and 47 (86%) patients gave a history of tobacco consumption. Among cigarette smokers, the probability of LOH within any of the three discrete regions increased with increasing tobacco use (Table II
). The probability of LOH at a particular region among smokers was most evident in the most distal region of chromosome 3p, i.e. 3p25.126.1. Within this latter group, the probability of a deletion approximated 91% as compared with 30% probability in those who were non-users (
2 for trend = 4.96, P = 0.03).
Alcohol history could be obtained in 54 of the 56 patients and its use was identified in 36 (67%) of these individuals. It is of note that 83% of these individuals who used alcohol also gave a history of tobacco use. In contrast to tobacco consumption, increasing alcohol exposure was not associated with an increase in the probability of an interstitial deletion at a particular site (Table III
). Indeed, the highest probability of loss was noted in non-users of alcohol at the 3p21.121.2 locus. Losses were identified in all informative patients within this latter group.
We then assessed for the relationship between whole-arm loss, i.e. loss of all informative loci on 3p in a respective individual's tumor, and alcohol and tobacco exposure. As seen in Table IV
, the most significant relationship between whole-arm loss and exposure was seen in those individuals who consumed alcohol. This was most evident in individuals who consumed more than 12 drinks per week either unadjusted or after adjusting for age (
2 = 6.33; P-value for trend = 0.01). When assessing for interactions, the greatest probability of whole-arm loss was seen in those individuals who used both tobacco and alcohol, after adjusting for age [odds ratio (OR) = 28.76, 95% confidence interval (95% CI) 2.0417.6; P = 0.01] with the greatest probability occurring in the younger populations. Of note, just as we examined for the presence of LOH at any of the loci within dysplastic lesions, we also examined for the presence of whole-arm loss. None of the dysplastic lesions demonstrated evidence of a whole-arm loss of chromosome 3p.
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Table IV. The relationship of whole-arm loss of chromosome 3p in head and neck cancers to tobacco and alcohol usea
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Mutagen sensitivity and LOH
Mutagen-sensitivity values were obtained from 43 HNSCC patients in whom respective tumors were allelotyped for LOH. Mutagen-sensitivity results of these 43 patients had been published previously as part of a larger case- control study (16). The use of one break per cell as a cut-off value to classify individuals as mutagen sensitive or non-sensitive has previously been reported by us (16). Overall, 26 (60%) of these 43 patients demonstrated mutagen sensitivity, i.e. >1.0 break/cell. The relationship between the expression of mutagen sensitivity and both the interstitial deletions as well as whole-arm loss was examined. In individuals whose tumors demonstrated interstitial deletions, the probability of a deletion at a particular region was increased slightly but not significantly if the patient expressed mutagen sensitivity. This was true for all three regions of interstitial deletion (Table V
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Table VI
shows the relationship between whole-arm loss in head and neck cancers and both tobacco and alcohol exposures and mutagen sensitivity. The greatest probability of demonstrating whole-arm loss was in those individuals who used both substances and were mutagen sensitive [OR = 13.5, P = 0.02 by Fisher's exact test (two-tailed)].
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Table VI. The relationship of whole-arm loss of chromosome 3p in head and neck cancers to tobacco and alcohol use and mutagen sensitivity
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Taken together, the patterns of deletional events on the short arm of chromosome 3 were dependent on the type of exposure and the presence of mutagen sensitivity. Increasing alcohol exposure when combined with tobacco use was more likely to be associated with whole-arm loss while increasing tobacco usage alone induced changes related to specific loci. The presence of mutagen sensitivity within the respective head and neck cancer patient was more likely to enhance certain genetic events; specifically, whole-arm loss in those who were heavy alcohol users. As noted above, the vast majority of this latter group also smoked cigarettes, suggesting that it was the combined presence of these three factors which contributed to the whole-arm loss (Table VI
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Clinical characteristics and allelic losses
Results suggest that two types of mutational events are likely to occur in head and neck cancer patients after accounting for tobacco and alcohol use and mutagen sensitivity: (i) allelic losses limited to specific regions on 3p in patients who used tobacco and (ii) whole-arm loss on chromosome 3p, i.e. the loss of all informative loci within an individual patient's tumor in those who consumed heavy alcohol (the majority of whom used tobacco) and who were also mutagen sensitive. It should be noted again that though losses at particular regions could be identified in dysplastic mucosa adjacent to a particular cancer, no dysplastic lesion demonstated whole-arm loss.
The clinical characteristics of those who demonstrated whole-arm loss versus those with either partial or no loss was assessed. As noted above, patients with whole-arm loss tended to be younger, i.e. more likely <50 years of age [six of 16 individuals (38%)] than those with partial or no losses [five of 40 individuals (12%)] (
2 = 5.38, P < 0.05). No other significant relationship could be identified when assessing for either of these mutational events with patient sex and race. When assessing for significant tumor characteristics, patients with whole-arm loss were more likely to present with advanced stage disease than the remaining population (Table VII
). Indeed, no cases of stage one disease could be identified in the group with whole-arm loss. Patients with whole-arm loss, in contrast to those with partial or no losses, were also more likely to present with clinical evidence of cervical lymph node metastases (P < 0.05).
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Discussion
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The identification of three discrete regions of loss on the short arm of chromosome 3, the frequency of these lesions, as well as the timing, i.e. their presence within dysplastic mucosa adjacent to invasive head and neck cancer, are mostly similar in this study with that of previous reports (2630). Some variability in frequency can be identified, however, when examining particular loci. For instance, though Maestro et al. (26) found a similarly high rate of loss at 3p25 using the microsatellite marker D3S1038 (53 versus 78% in our analysis, respectively), Wu et al. found somewhat lower rates (19%) (29). The results of our study benefited from a larger patient sample in order to determine areas of loss (58 cases, over twice that used in previous reports). Furthermore, as we will describe below, our larger patient sample size was important when considering the influence of identifiable host and environmental factors such as mutagen sensitivity, tobacco and alcohol use. These variables had not been accounted for in previous studies and will influence results.
Also similar to other reports, we identified the presence of 3p allelic losses within dysplastic lesions adjacent to the invasive cancer suggesting an early event in the carcinogenic process (2931). The frequency of loss in our dysplastic lesions were lower, however, than those reported previously (2931). This may reflect the degree of dysplasia as none of our samples demonstrated severe epithelial dysplasia or carcinoma-in-situ. The report of Roz et al. (30), for instance, demonstrated LOH within 3p regions to be as high as 67% in carcinoma in situ and as low as 33% in less-severe intraepithelial lesions. The probability of allelic loss anywhere on 3p was also lower within intraepithelial tumors in our study as compared with the matched invasive tumors (28 versus 68%, respectively). The probability of invasive disease is unlikely to be influenced by any single allelic loss. Rather, accumulated damage to multiple genes on the chromosome 3 short arm may be a prerequisite. Only eight of the 58 cancer samples had loss confined to a single region and these eight cases had losses equally distributed throughout the three identifiable regions (Figure 1
). Indeed, in support of this concept as we will later discuss, the most aggressive disease was found in patients whose cancers demonstrated losses of all informative loci.
As mentioned above, one critical part of this investigation was to expand past analyses to account for the influence of tobacco and alcohol use to account for chromosome 3p allelic losses. Using data gained from questionnaires in a prospective casecontrol study (16), we have shown that the probability of loss within any of the three discrete regions increased with increasing tobacco use. The region most significantly affected was 3p25.126.1 in which LOH increased >2-fold as compared with non-smokers. Given that most tumors which show allelic losses at at these three regions are tobacco-related (3236), it is not surprising that a quantitative relationship should be identified here. An unexpected finding in this study regarding chromosomal 3p damage, however, related to alcohol exposure. Interestingly, damage associated with alcohol was not site specific but related to 3p losses in cancers which involved all informative loci, a process we termed whole-arm loss. Alcohol has been considered to contribute to the carcinogenic process by principally promotional means and not through a direct carcinogenic effect (37). Of note, the vast majority of alcohol users in this study, likewise, used tobacco. Thus, any process other than promotional cannot be inferred. A limitation in this study, however, is the number of patients analyzed. Categorical groupings comprised of small populations increase the risk of random findings.
A particular focus of this study was the influence of mutagen sensitivity on chromosomal 3p deletional events. Significantly, the probability of the combined tobacco- and alcohol-related deletional losses was further increased in those patients who expressed this sensitivity phenotype. The ORs of whole-arm loss in those individuals who used both tobacco and alcohol increased nearly 6-fold in those individuals who were mutagen sensitive as compared with non-sensitive individuals. It is relevant that Dave et al. (21) examined for specificity of bleomycin-induced chromosomal damage within peripheral blood lymphocytes from various populations including patients with head and neck cancer. Bleomycin was shown to induce breaks within all chromosomes. However, the short arm of chromosome 3 was most commonly effected. Thus, our results provide additional evidence, when viewed in concert with the study by Dave et al. (21), to suggest that 3p may have a constituitively expressed predisposition site for mutations within head and neck cancer patients. Distinct from the study of Dave et al. in which 3p21 was most often affected, we found no allele that was more commonly involved in mutagen-sensitive individuals. It should be emphasized that limited markers were utilized at the 3p21 region; only one at 3p21.3. Caution should thus be taken in interpreting these results. Furthermore, more direct evidence to support the presence of constitutively expressed fragility sites would have to come by assessing site-specific damage induced within chromosomes of peripheral blood lymphocytes from the respective patients. Are the patients with losses at this 3p chromosomal site within their tumor also the same patients who show chromatid breaks following bleomycin exposure? Likewise, further evidence that this represents a host site predisposition to free radical oxygen damage would come from analyzing for site-specific damage within normal mucosal cells from the upper aerodigestive tract in a method similar to Dave et al. (21). These studies remain to be performed. Whether or not sensitivity to bleomycin is a heritable host susceptibity factor to tobacco-induced cancer also needs to be established. In support of that contention, Li et al. (38) have reported that first degree relatives of mutagen-sensitive head and neck cancer patients also show sensitivity to bleomycin. Bondy et al. (36) have related that the risk of cancer among first degree relatives is also increased in those head and neck cancer patients who are mutagen sensitive.
The influence of mutagen-sensitivity assessments on the frequency of alcohol-related 3p deletions provides a potential clue as to underlying mechanisms. For instance, in vitro studies by Hsu et al. (39) have shown that the repair of free radical chromosomal damage is impaired in the presence of alcohol, which could account for our findings. The end result would be increased clastogenic processes in individual patients, dependent upon level of tobacco exposure, a process which would be enhanced by mechanisms which relate to decreased DNA repair, such as potentially reflected by alcohol use and the mutagen-sensitivity assay. The greater the clastogenic effect, the more likely the end result would be the loss of an entire chromosomal arm. Our two previous casecontrol epidemiologic studies would support this interaction as a contributing influence to head and neck cancer development (13,16). These two separate studies demonstrated that the OR of head and neck cancer was most influenced by the presence of both alcohol exposure and mutagen sensitivity. The OR of disease increased >40-fold in those who both consumed alcohol and were mutagen sensitive, and indicated a multiplicative effect. Either variable alone was associated with an OR of disease which did not exceed 7-fold.
Numerous genes relevant to carcinogenesis have been identified within the three identified regions of loss on 3p, including genes involved in growth factor control, DNA repair and cell-cycle regulation (4045). It would stand to reason that the loss of all these genetic regulatory elements would contribute to a more progressive tumorigenic process. Our results would support that contention. It is of note that the von HippelLindau tumor suppressor gene, localized at chromosome 3p2526 and responsible for tumorigenesis in clear cell renal cell carcinoma (46), is not likely involved in the pathogenesis of HNSCC (47). Mutagen-sensitive patients with heavy environmental exposures were more likely to lose the entire short arm of chromosome 3. The loss of the entire short arm was associated with a greater probability of presenting with advanced stage disease and clinically evident lymph node metastases as compared with those whose losses were limited to a specific locus, such as 3p21. Also in contrast to both our results here as well as previous findings involving allelic losses at 3p21, none of the dysplastic mucosa adjacent to invasive cancers showed evidence of whole-arm loss (30). It would suggest that intensive levels of environmental exposures, potentially influenced by host susceptibility factors, would lead to a more rapidly progressive process, one in which early disease is unlikely to be identified. The clinical relevance of this hypothesis may relate to the problem of head and neck cancer among certain ethnic populations. Numerous studies have suggested that head and neck cancer is a worse disease among black males (4852). Previous epidemiologic studies have suggested that this population can also be characterized by both more intensive alcohol exposure as well as a greater probability of expressing mutagen sensitivity (48,52). Further research should confirm more direct evidence of whole-arm losses within head and neck cancers, such as through the use of comparative genomic hybridization analysis, and relate findings to environmental exposures and other ethnic and socioeconomic factors. The identification of a more virulent disease influenced by these factors may impact upon screening strategies as well as the use of multimodalities therapies designed to improve quality of life as well as survival.
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Notes
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5 To whom correspondence should be addressed at Department of Otolaryngology, New York Eye and Ear Infirmary, New York Medical College, 310 East 14th Street, New York, Email: sschantz{at}nyee.eduNY 10003, USA 
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Acknowledgments
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This work was partially supported by National Cancer Institute Grant RO1 CA-57155
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Received December 9, 1999;
revised February 17, 2000;
accepted February 29, 2000.