The January 15, 2002, article by Sudbø et al, entitled "Gross Genomic Aberrations in Precancers: Clinical Implications of a Long-Term Follow-Up Study in Oral Erythroplakias" (J Clin Oncol 20:456-462, 2002) has come under scrutiny. The corresponding author’s institution, the Norwegian Radium Hospital, is reviewing the data, and the Editorial office is awaiting the results of the investigation.

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Gross Genomic Aberrations in Precancers: Clinical Implications of a Long-Term Follow-Up Study in Oral Erythroplakias

From the Departments of Oncology and Pathology, the Norwegian Radium Hospital; Department of Pathology and Forensic Odontology, University of Oslo, Oslo; Department of Pathology, Gades Institute, University Hospital, Bergen; and Norwegian University of Science and Technology, Trondheim, Norway.

Address reprint requests to Jon Sudbø, DDS, MD, PhD, Department of Oncology, the Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway; email: jon.sudbo{at}rh.uio.no

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: Gross genomic aberrations are increasingly seen as a cause rather than a consequence of carcinogenesis. Carcinomas may be prevented by systemically acting agents when used in high-risk individuals. If gross genomic aberrations could be shown to be predictive markers in precancers, they could serve as a tool for identifying high-risk individuals to be included in chemopreventive trials.

PATIENTS AND METHODS: To investigate the predictive power of gross genomic aberrations in several types of oral premalignancies, we analyzed 57 biopsies from oral erythroplakias of 37 patients, both histologically and for DNA content. DNA content was measured by high-resolution image cytometry, and distribution histograms of DNA content were generated and interpreted according to established protocols. The primary end point was cancer-free survival.

RESULTS: Fifty-seven dysplastic oral red lesions from 37 patients were investigated. Forty-one lesions from 25 patients were classified with aberrant DNA content (DNA aneuploidy), of which 23 patients (92%) later developed an oral carcinoma (after a median observation time of 53 months; range, 29 to 79 months). Of 12 patients having altogether 16 lesions with normal DNA content, none developed a carcinoma (median observation time, 98 months; range, 23 to 163 months; P < .001). In multivariate analysis, DNA content was a significant prognostic factor (P < .001), whereas histologic grade, sex, use of tobacco, size and location of lesions, and the presence multiple of lesions were not.

CONCLUSION: Gross genomic aberrations are highly predictive for the subsequent occurrence of carcinomas from a wide range of oral premalignancies.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
THE ORAL CAVITY may serve as a model system for studies on cancer prevention because precancerous lesions in this site are readily identified and observed and are associated with subsequent carcinomas in other sites of the upper aerodigestive tract.¹ Despite the obvious opportunity for early intervention, survival from oral carcinomas is poor and incidence rates are rising, particularly among younger individuals.^2-7 Because no markers to distinguish between indolent and aggressive premalignancies are established, a wait-and-see approach is adopted, and patients "are often misdiagnosed and inappropriately treated leading to delay in definitive treatment."⁸ Although extensive data demonstrate the anticarcinogenic activity of retinoids in humans,⁹ studies on chemopreventive agents with a documented anticarcinogenic effect on epithelial linings of the upper aerodigestive tract, colon, prostate, and cervix^3,10-12 have not translated into reduced incidence rates of new carcinomas, other than in groups of patients with a well-defined high-risk profile,^13,14 which are not always possible to identify.¹⁵ Accordingly, in a recently published article, Lee et al¹⁶ sum up more than 10 years of research on chemoprevention by stating that, "with the identification of high risk individuals, more efficient chemoprevention trials and molecular targeting studies can be designed."

Compelling evidence now points to gross genomic aberrations as a cause rather than a consequence of malignant transformation.¹⁷ Recent studies indicate that mutations in genes controlling mitotic segregation of chromosomes give rise to chromosome instability in cancer,¹⁸ and the nonbalanced segregation of chromosome during mitosis have been shown to occur exclusively in DNA aneuploid tumor cell lines.¹⁹ The relationship between centrosome defects and genetic instability was also demonstrated in a study where centrosomes in nearly all tumors and tumor-derived cell lines were atypical in shape, size, and composition, were often present in multiple copies, and formed disorganized mitotic spindles, leading to missegregation of chromosomes.²⁰ Such abnormalities were not observed in nontumor cells. These findings indicate that centrosome defects are a common feature of malignancies and suggest that they may contribute to carcinogenesis.

Studies have demonstrated that retinoic acid beta–receptor expression, which may be modulated by chemopreventive agents, correlates with premalignant changes in mucous membranes,²¹ and the expression of retinoic acid ß–receptor has been linked to gross genomic aberrations.²² If it could be demonstrated that gross genomic aberrations constitute prognostically important findings in several types of oral epithelial premalignancies, this could pinpoint high-risk individuals who, to a particular extent, might benefit from treatment of their premalignant lesions with chemopreventive agents such as retinoids.

We have previously shown that assessing gross genomic aberrations in oral white patches (leukoplakias) predicts the subsequent occurrence of carcinomas with a considerable degree of certainty^23,24 and is superior to standard prognostic methods for oral precancers.²⁵ The present study was undertaken to assess whether such aberrations can be used to predict the clinical outcome in a wider range of precancers and presents the results of a long-term follow-up in 37 patients with erythroplakias, with a median follow-up time of more than 7 years.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Materials
Fifty-seven biopsies were obtained from 37 patients who had undergone complete excision of dysplastic oral erythroplakias between 1988 and 2000. To avoid skewness of data, the patients considered had no prior or concomitant history of frank malignancies in the upper aerodigestive tract because such patients are prone to developing second primary or secondary carcinomas in other locations of the upper aerodigestive tract. The location of red lesions and subsequent carcinomas were documented by the topographical codings in Systematized Nomenclature of Medicine (SNOMED).²⁶ Follow-up bioptic excisions had been performed only in cases where new red patches had occurred. Only carcinomas that developed in the same location or in the vicinity of previous red patches, as verified from SNOMED codings and drawings on standardized sketches, were regarded as progression of disease. From each analyzed tissue block, one section was first cut for hematoxylin and eosin (HE) staining and verification of dysplastic content, whereupon two thick sections (50-µm each) were cut for monolayer preparation of epithelial cells for analysis. Finally, one more section was cut for HE staining and verification of dysplasia, thereby serving as verification of dysplastic content within the thick sections. Only tissue blocks in which dysplastic content could be verified from both sections were used for the analysis. To ensure that mainly epithelial cells from dysplasias were analyzed, demarcations, guided by the inspection of the sections, were made with a microdissective technique, and surplus tissue was removed with a scalpel. Only tissue corresponding to epithelial dysplasias was included for processing and analysis.

Histologic Assessment
Only biopsies that were histologically verified as being dysplastic were included in the study. HE-stained sections of the dysplastic erythroplakias were further graded histologically by at least two oral pathologist, according to guidelines given by the World Health Organization,²⁷ into mild, moderate, and severe dysplasias.

Measurements of DNA Content
Two 50-µm sections were cut and enzymatically digested (SIGMA protease, type XXIV; Sigma Chemical Co, St Louis, MO) for the preparation of isolated nuclei (monolayers). Isolated epithelial-cell nuclei were cytospinned and mounted on a slide for staining according to the Feulgen-basic fuchsin method.²⁸ The DNA content of Feulgen-basic fuchsin–stained nuclei was measured and analyzed using the Fairfield DNA Ploidy System (Fairfield Imaging Ltd, Kent, United Kingdom) and assessed according to an established protocol.²⁹ The image analysis system consisted of a Zeiss Axioplan II microscope (Zeiss, Oberkochen, Germany) (x40/0.65) with a 546-nm green filter and modified for computer control of the stage (Prior HI52V2; Prior Scientific Instruments, Cambridge, United Kingdom). The microscope was equipped with a Single Chip digital camera (C4742 to 95; Hamamatsu Photonics KK, Hamamatsu, Japan). The final magnification was x1,600 at an estimated resolution of 170 nm per pixel, 1,024 x 1,024 pixels with 10-bit resolution (1,024 grey levels) per visual field. At least 300 epithelial-cell nuclei were measured and stored in galleries for each case, with lymphocytes included as internal (DNA diploid) controls in all 37 cases.

Criteria for the Classification of DNA Content
The protocol used for ploidy classification of the lesions²⁹ includes criteria for classification into a number of categories with respect to DNA content, but in the present study of 37 patients with dysplastic oral red patches, only lesions with clearly aberrant (DNA aneuploid) and normal (DNA diploid) DNA content were found. All specimens were coded, and distribution histograms of gross genomic content were classified in a blinded manner. In cases where multiple biopsies were obtained simultaneously, all biopsies were analyzed for DNA content and the most severe DNA ploidy classification was chosen if discrepancies existed. According to the employed protocol,²⁹ a lesion is classified as diploid if only one G0/G1 (2c) peak is present, if the number of nuclei in G2 (4c) peak did not exceed 10% of the total, or if the number of nuclei with a DNA content more than 5c did not exceed 1% of the total. A lesion is defined as aneuploid if peaks outside of 2c, 4c, or 8c are present or if the number of nuclei with a DNA content above 5c or 9c exceeded 1% (Fig 1).

View larger version (22K): [in this window] [in a new window]   Fig 1. (A-F) Distribution histograms from six patients. Euploid peaks represent cells with normal nuclear DNA content, and aneuploid peaks represent cells with aberrant DNA content. Patients C and D did not develop carcinomas during follow-up. Patients E and F developed carcinomas (after 78 and 103 months, respectively). 1c = haploid, 2c = diploid, 4c = tetraploid, and 8c = octaploid.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Clinical Summary
The baseline demographic characteristics of the patients are given in Table 1. Thirteen of the 37 patients had more than one biopsy taken (Table 2). To avoid skewness of data, only patients that exclusively had lesions clinically defined as erythroplakias were included in the study.

Gross Genomic Findings
Fifty-seven dysplastic red lesions of the oral cavity from altogether 37 patients (26 males [median age, 62.1 ± 15.0 years] and 11 females [median age, 67.0 ± 13.7 years]) were investigated. The age distribution in the aberrant group was 68.4 ± 15.0 years, whereas in the group with normal genomic content, it was 66.4 ± 13.9 years (P = .14). In 41 lesions from 25 patients (17 males [68%] v eight females [32%]), the DNA content was classified as aberrant. In 16 lesions from the other 12 patients (six males [50%] and six females [50%]), the lesions were classified as having a normal DNA content. Twenty-four patients had DNA aneuploid lesions, and 13 had DNA diploid lesions at baseline, whereas 25 patients had DNA aneuploid and 12 had DNA diploid lesions at follow-up (Table 2). With the exception of one patient, multiple lesions from one and the same patient were classified as having the same DNA at initial and subsequent follow-ups. In the one patient in whom a change was observed, it was from diploid (normal) to aneuploid (abnormal), after an observation period of 48 months. This patient had altogether four biopsies taken, and the change from DNA diploid to aneuploid was diagnosed in the third biopsy, obtained at 38 months of follow-up, whereas the fourth biopsy was obtained at 60 months follow-up. The patient was observed for a total of 87 months. This patient was one of the two among the 25 that had aberrant DNA content in their lesions without developing a carcinoma during follow-up. The majority of biopsies obtained (47 out of 57) were histologically typed and graded as moderate or severe dysplasia. There was no correlation between the histologic grading and the DNA ploidy status in the patient whose lesions had a change in ploidy status. The male fraction of the DNA aneuploid group was significantly higher than in the normal group (68% v 50%, respectively; P = .03). Of the 37 patients presenting with dysplastic oral red patches, 23 (61%) later developed a carcinoma in the oral cavity. The median duration of follow-up in these 23 patients was 57 months, ranging from 29 to 79 months. The subsequent carcinomas all appeared in 23 out of 25 patients with aberrant lesions (92%, P < .001). Two patients had aberrant lesions that did not develop into carcinomas, during a follow-up time of 87 and 143 months. With one exception, all the lesions with gross genomic aberrations displayed aberrations at the time of initial diagnosis (Table 2). None of the patients that were diagnosed as having DNA diploid lesions later developed a carcinoma (median observation time, 98 months; range, 23 to 163 months). Distribution histograms of DNA content from six representative patients are shown in Fig 1. Figure 1A and B show histograms from two patients with lesions having a normal DNA content, whereas Fig 1C through F show distribution histograms from four patients with lesions having an aberrant DNA content. There was a statistically significant difference between patients having lesions with aberrant and normal DNA content with respect to subsequent occurrence of carcinoma (Table 3, Fig 2A, P = .001). The Kaplan-Meier disease-free survival curves for the patients with normal and aberrant DNA content in their lesions are shown in Fig 2A. The corresponding Kaplan-Meier estimate for overall survival is shown in Fig 2B. Twelve (52%) of the 23 patients who developed a carcinoma later died of their disease (after a median follow-up time of 43 months; range, 32 to 81 months).

View larger version (11K): [in this window] [in a new window]   Fig 2. (A) Kaplan-Meier survival curves for the patients with normal (DNA diploid) and aberrant (DNA aneuploid) lesions with disease-free survival as the considered end point (P < .001); (B) overall survival from cancer-specific mortality is the considered end point (P < .001).

Multivariate Analysis
In univariate analysis, DNA ploidy (P .001), sex (P = .04), current use of tobacco (P = .05), lesion size > 3 cm in largest diameter, location of lesions on the floor of the mouth (P = .03), severe dysplasia (P = .05), and the presence of multiple lesions (P = .04) were significant prognostic factors. When these factors were fitted in a Cox multiple logistic regression analysis, only gross genomic content was a significant prognostic factor (P < .001). Histologic grade, sex, current use of tobacco, size and location of lesions, or the presence of multiple lesions did not reach statistical significance (P = .33, .12, .09, .11, .07, and .09, respectively).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
One of the most promising approaches to the difficult problem of reducing the incidence rates of cancers of the upper aerodigestive tract is to identify high-risk individuals at a precancerous stage of disease with subsequent subjection to preventive measures.³⁰ In this study, limited to 37 patients with oral erythroplakias, 23 (61%) later developed a carcinoma in the oral cavity (after a median observation time of 63 months; range, 29 to 78 months). Furthermore, only patients with aberrant genomic content in their lesions later developed a carcinoma. Thus, 23 (92%) out of 25 patients with aberrant lesions but none of the 12 patients with normal genomic content in their lesions later developed a carcinoma, suggesting that gross assessment of DNA content in these premalignancies reliably identifies persons at high risk for developing oral cancer.

The finding that histologic grade did not correlate with DNA content (normal or aberrant) and, accordingly, did not correlate with the clinical outcome in the study patients is in accordance with previous findings by our group.²⁵

The limited number of subjects included in this study may also limit the extent to which general conclusions may be drawn. Nevertheless, although our results pertain to a small cohort of patients, the data are highly significant and, moreover, consistent with previous findings by us on oral leukoplakias.^23,24 Although erythroplakias are less common than leukoplakias, they are biologically different and show a higher malignant transformation rate, giving rise to a substantial fraction of oral carcinomas.³¹ Accordingly, a large fraction of lesions clinically defined as erythroplakias will histologically be verified as carcinomas-in-situ or as frank carcinomas. As a result, only a limited number of these red lesions will turn out to be histologically verified as dysplastic and thus premalignant. Taking this into account, the analysis of 57 dysplastic erythroplakias in 37 patients with a follow-up time of more than 7 years may be seen as a considerable effort. In short, we feel that our results are sufficiently significant to warrant similar investigations on the prognostic impact of gross genomic aberrations in other epithelial linings.

The present study represents an extension of previous work by our group, restricted to leukoplakias only.^23,24 We now demonstrate that it is possible to predict the occurrence of carcinomas also from erythroplakias. Several lines of evidence indicate a key role of gross genomic aberrations in malignant transformation,¹⁷ and from our findings, one may further conjecture that gross genomic aberrations could also apply as a predictor of subsequent cancers from a wide range of precancers. The clinical value of an early and reliable identification of the risk profile in individuals with precancers is underlined by the fact that established protocols for treatment with anticarcinogenic agents exist.⁹ The finding that the majority of lesions with aberrant DNA content had so at initial visits underscores the early and incentive role of gross genomic aberrations, such as DNA aneuploidy, in carcinogenesis.¹⁷

Patients with head and cancers are at increased risk of a second primary cancer in other locations of the upper aerodigestive tract.^1,32,33 In this study, we found no such association to other cancers of the upper aerodigestive tract. However, the number of patients is limited, and therefore, inferences from our data must be drawn with caution. Furthermore, an extension of the follow-up time would possibly reveal an increased risk of a second primary cancer also in the study group. In addition, because of the limited number of patients and the fact that none of them developed a carcinoma during the follow-up period, our data do not allow us to draw any conclusions regarding a possible correlation between the DNA content of the investigated lesions and subsequent occurrence of carcinomas in other regions of the upper aerodigestive tract. Nevertheless, the fact that we found a high rate of progression to malignant disease, although in all cases the initial lesions had been completely excised with histologically confirmed free margins, indicates that the malignant transformation of the oral mucous membranes is a multilocalized process, which could either be explained through the model of field cancerization or it could be the result of clonal expansion through intraepithelial migration of aggressive cell clones.^34,35 Thus, our results indicate that the total excision of one or more visible lesions in the oral mucosa should not be viewed as a curative measure and indicates that treatment with systemically acting anticarcinogenic agents may be warranted. It is a well-established fact that patients who have had a carcinoma of the upper aerodigestive tract are prone to secondary or second primary carcinomas.¹ The fact that in this study no subsequent carcinomas occurred in other sites of the oral cavity or the upper aerodigestive tract may be due to chance because the number of study subjects was limited and the period of follow-up was less than 10 years.

It has been argued that the oral mucous membrane may be viewed as a surrogate marker site for other imminent malignancies of the upper aerodigestive tract.¹² In this perspective, the most promising treatment options for premalignant lesions of the oral cavity would be some systemic agents, with an established biologic effect on the epithelial cells of the oral mucosa.^12,36

One may further conjecture that as aberrant gross genomic content seems to be the cumulative expression of a series of pathologic molecular events, it may constitute a predictive marker in a wider range of precancers. Therefore, this marker could also serve as a tool for tailoring preventive measures in precancerous epithelia outside the oral cavity, at a stage of disease where treatment achieves better rates of cure than in manifest carcinomas. Ultimately, the long-term effect of such a strategy should be evaluated through a prospective randomized trial and evaluated by the only definitive end point for prevention of cancer, the incidence rates of new carcinomas.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Supported by grant no. 94042/001 from The Norwegian Cancer Society and by funding from the Research Foundation of the Norwegian Radium Hospital, University of Oslo, Oslo, Norway.

We thank Signe Eastgate and Ruth Punthervold for expert technical assistance in preparing monolayers and measuring the DNA content of epithelial cells. We thank Dr Tudor Barnard for critically reviewing and commenting on the manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted March 26, 2001; accepted August 29, 2001.

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