Non-steroidal anti-inflammatory drugs and the risk of oral cancer: a nested case-control study.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) seem to prevent several types of cancer, but could increase the risk of cardiovascular complications. We investigated whether use of NSAIDs was associated with a change in the incidence of oral cancer or overall or cardiovascular mortality. METHODS: We undertook a nested case-control study to analyse data from a population-based database (Cohort of Norway; CONOR), which consisted of prospectively obtained health data from all regions of Norway. People with oral cancer were identified from the 9241 individuals in CONOR who were at increased risk of oral cancer because of heavy smoking (15 pack-years), and matched controls were selected from the remaining heavy smokers (who did not have cancer). FINDINGS: We identified and analysed 454 (5%) people with oral cancer (279 men, 175 women, mean [SD] age at diagnosis 63.3 [13.2] years) and 454 matched controls (n=908); 263 (29%) had used NSAIDs, 83 (9%) had used paracetamol (for a minimum of 6 months), and 562 (62%) had used neither drug. NSAID use (but not paracetamol use) was associated with a reduced risk of oral cancer (including in active smokers; hazard ratio 0.47, 95% CI 0.37-0.60, p<0.0001). Smoking cessation also lowered the risk of oral cancer (0.41, 0.32-0.52, p<0.0001). Additionally, long-term use of NSAIDs (but not paracetamol) was associated with an increased risk of cardiovascular-disease-related death (2.06, 1.34-3.18, p=0.001). NSAID use did not significantly reduce overall mortality (p=0.17). INTERPRETATION: Long-term use of NSAIDs is associated with a reduced incidence of oral cancer (including in active smokers), but also with an increased risk of death due to cardiovascular disease. These findings highlight the need for a careful risk-benefit analysis when the long-term use of NSAIDs is considered.

COX-2 expression in striated muscle under physiological conditions.

OBJECTIVES: The role of cyclooxygenase-2 (COX-2) in disease has been extensively studied, (Annu Rev Med (2002) 53:35; N Engl J Med (2001) 345:433) but less information is available with respect to possible physiological functions of COX-2. Information on how and where COX-2 is expressed under physiological conditions may increase our understanding of its physiological role. Previous studies have revealed a COX-2 dependent production of prostanoids under physiological conditions, without entirely determining the source of this production. MATERIALS AND METHODS: To assess COX-2 expression under normal conditions, we analyzed tissue specimens that were removed from 30 healthy study subjects in conjunction with surgical procedure related to insertion of dental implants and from three patients which had muscle tissue from Quadriceps femoris muscle removed as part of surgical treatment of soft tissue sarcomas not directly affecting the muscle tissue. Immunohistochemistry and immunoblotting (Western blotting) was used to assess the presence of COX-2 protein. RESULTS: In 25 of 30 patients (83%), COX-2 protein was expressed in striated muscle, as assessed by immunohistochemistry. All cases had COX-2 expression verified by Western blotting. In none of the 25 subjects with COX-2 expression did we notice concomitant inflammation of the adjacent submucosal tissue. CONCLUSIONS: It is a novel finding that COX-2 is expressed in striated muscle under physiological conditions. COX-2 activity in striated muscle is a possible explanation for the hitherto unknown localization of prostanoids synthesis under physiological conditions.

Progress in determining the malignant potential of oral lesions.

The dilemma in managing patients with potentially malignant oral lesions and field change is of deciding which mucosal lesions or areas will progress to carcinoma. Although dysplasia may be predictive, this is not invariable, and there can be considerable inter- and intraexaminer variation in that diagnosis. Recent data on molecular and DNA changes in potentially malignant lesions suggest that it is now feasible to identify those lesions that are truly potentially malignant.

Abnormal DNA content predicts the occurrence of carcinomas in non-dysplastic oral white patches.

The majority of oral squamous cell carcinomas (OSCCs) are preceded by visible changes in the oral mucosa, most often white patches. Although the histological finding of dysplasia in oral white patches signals increased risk of developing OSCC, this may also occur in non-dysplastic lesions. However, no reliable markers exist to predict the occurrence of OSCC in these patients. From a total of 263 patients diagnosed with oral white patches, biopsies from 45 patients were selected on the criteria that the patients had lesions histologically proven to be non-dysplastic. The lesions were analyzed with respect to their DNA content. The clinical outcome of the patients was known from the Cancer Registry of Norway, and these data were compared to the DNA content of their lesions. Among the 45 patients, five cases (11%) later developed an OSCC. Four of the cases that subsequently developed an OSCC were among the five aneuploid (abnormal) cases (P=0.001). One aneuploid lesion did not develop a carcinoma during a follow-up time of 120 months. The fifth case that subsequently developed an OSCC was diploid (normal), and developed into an OSCC after an observation time of 73 months (P=0.001). In conclusion, aberrant DNA content reliably predicts the occurrence of OSCC in patients that otherwise would be regarded as at very low risk. Normal DNA content indicates low risk.

Comparison of histological grading and large-scale genomic status (DNA ploidy) as prognostic tools in oral dysplasia.

Approximately one in ten oral white patches (leukoplakia) are histologically classified as dysplasia, with a well-documented potential for developing into oral squamous cell carcinoma (OSCC). Histological grading in oral dysplasia has limited prognostic value, whereas large-scale genomic status (DNA ploidy, nuclear DNA content) is an early marker of malignant transformation in several tissues. Biopsies from 196 patients with oral leukoplakia histologically typed as dysplasia were investigated. Inter-observer agreement among four experienced pathologists performing a simplified grading was assessed by Cohen's kappa values. For 150 of the 196 cases, it was also possible to assess large-scale genomic status and compare its prognostic impact with that of histological grading. Disease-free survival was estimated by life-table methods, with a mean follow-up time of 103 months (range 4-165 months). The primary considered end-point was the subsequent occurrence of OSCC. For grading of the total of 196 cases, kappa values ranged from 0.17 to 0.33 when three grading groups (mild, moderate, and severe dysplasia) were considered, and from 0.21 to 0.32 when two groups (low grade and high grade) were considered (p=0.41). For the 150 cases in which large-scale genomic status was also assessed, kappa values for the histological grading ranged from 0.21 to 0.33 for three grading groups and from 0.27 to 0.34 for two grading groups (p=0.47). In survival analysis, histological grading was without significant prognostic value for any of the four observers (p 0.14-0.44), in contrast to DNA ploidy (p=0.001). It is concluded that DNA ploidy in oral dysplasia has a practical prognostic value, unlike histological grading of the same lesions.

DNA content as a prognostic marker in patients with oral leukoplakia.

BACKGROUND: Oral leukoplakia may develop into squamous-cell carcinoma, which has a poor prognosis. Risk factors for oral carcinoma have been identified, but there are no reliable predictors of the outcome in individual patients with oral leukoplakia. METHODS: We identified 150 patients with oral leukoplakia that was classified as epithelial dysplasia and measured the nuclear DNA content (ploidy) of the lesions to determine whether DNA ploidy could be used to predict the clinical outcome. Biopsy specimens obtained at annual follow-up visits were graded histologically and classified with respect to DNA content in a blinded fashion. Disease-free survival was assessed in relation to DNA ploidy and the histologic grade. The mean duration of follow-up was 103 months (range, 4 to 165). RESULTS: Among 150 patients with verified epithelial dysplasia, a carcinoma developed in 36 (24 percent). Of the 150 patients, 105 (70 percent) had diploid (normal) lesions, 20 (13 percent) had tetraploid (intermediate) lesions, and 25 (17 percent) had aneuploid (abnormal) lesions at the time of the initial diagnosis. A carcinoma developed in 3 of the 105 patients with diploid lesions (3 percent), as compared with 21 of the 25 patients with aneuploid lesions (84 percent), yielding a negative predictive value of 97 percent with respect to the diploid lesions and a positive predictive value of 84 percent with respect to the aneuploid lesions. Carcinoma developed in 12 of 20 patients with tetraploid lesions (60 percent). The mean time from the initial assessment of the DNA content to the development of a carcinoma was 35 months (range, 4 to 57) in the group with aneuploid lesions and 49 months (range, 8 to 78) in the group with tetraploid lesions (P=0.02). The cumulative disease-free survival rate was 97 percent among the group with diploid lesions, 40 percent among the group with tetraploid lesions, and 16 percent among the group with aneuploid lesions (P<0.001). CONCLUSIONS: The DNA content in cells of oral leukoplakia can be used to predict the risk of oral carcinoma.

[Diagnosis and treatment of oral precancerous lesions]. / Diagnostikk og behandling av forstadier til munnhulekreft.

BACKGROUND: Risk factors for oral carcinomas have been identified, but there are no reliable markers for assessing the clinical outcome in individual patients with oral precancerous lesions. DNA aneuploidy is now recognized as an early and significant event in carcinogenesis. METHODS: We identified 242 patients with oral red or white patches histologically verified as epithelial dysplasias and measured the nuclear DNA content (DNA ploidy) of the lesions to determine whether DNA ploidy could be used to predict the clinical outcome. Disease-free survival was assessed in relation to DNA ploidy and histological grade. The mean duration of follow-up was approximately eight years. RESULTS: Among 242 patients with verified epithelial dysplasia, a carcinoma developed in 48 (20%). 167 (69%) had diploid lesions, 20 (8%) had tetraploid lesions and 55 (23%) had aneuploid lesions. Of the 167 with diploid lesions, only four (1%) later developed an oral carcinoma. By contrast, 48 of 55 patients with aneuploid lesions (87%) later developed a carcinoma. INTERPRETATION: The DNA content (DNA ploidy) can be used to predict the risk for oral cancer in a wide range of oral precancerous lesions. By contrast, histological grading of the same lesions does not give any prognostic information. The clinical value of an early identification of oral lesions with malignant cell clones is substantiated by the fact that there are methods for early intervention.

[Digital image analysis in pathology--exemplified in prostatic cancer]. / Digital bildebehandling i patologifaget--eksemplifisert ved cancer prostatae.

Digital image analysis is increasingly used in medicine. This paper reviews image analysis methods currently used in pathology. To exemplify established and future use of these methods, we present our own research and available literature on prostate cancer. DNA ploidy examinations mean measuring nuclear DNA content. A review of available literature shows that DNA ploidy is a very good prognostic marker in early and localized prostate cancer. Nevertheless, request for such examinations is sparse, in contrast to what is the case for gynecological cancers. In addition to assessing nuclear DNA content, we have today the means of quantitatively assessing the chromatin organisation. Such nucleotyping and texture analysis of chromatin has been shown to give significant diagnostic and prognostic information both in localized and advanced prostate cancer. DNA ploidy and nucleotyping are methods that analyze features of single cells. New methods for describing tissue architecture in an objective and reproducible way have been developed in our department. Preliminary results demonstrate the ability of these methods to discriminate between groups of prostate cancer patients with good or poor diagnosis. Digital techniques in combination with new methods from molecular biology have a potential for reducing workload, with parallel assessment of a large number of markers on large populations. The combination of traditional, molecular and digital pathology already offers the possibility of improved diagnostic and prognostic activity. However, there is the challenge of actually employing these methods. DNA ploidy for localized prostate cancer is only one example of a reliable prognostic marker that is not used to its full potential.

Prognostic value of graph theory-based tissue architecture analysis in carcinomas of the tongue.

Several studies on oral squamous cell carcinomas (OSCC) suggest that the clinical value of traditional histologic grading is limited both by poor reproducibility and by low prognostic impact. However, the prognostic potential of a strictly quantitative and highly reproducible assessment of the tissue architecture in OSCC has not been evaluated. Using image analysis, in 193 cases of T1-2 (Stage I-II) OSCC we retrospectively investigated the prognostic impact of two graph theory-derived structural features: the average Delaunay Edge Length (DEL_av) and the average homogeneity of the Ulam Tree (ELH_av). Both structural features were derived from subgraphs of the Voronoi Diagram. The geometric centers of the cell nuclei were computed, generating a two-dimensional swarm of point-like seeds from which graphs could be constructed. The impact on survival of the computed values of ELH_av and DEL_av was estimated by the method of Kaplan and Meier, with relapse-free survival and overall survival as end-points. The prognostic values of DEL_av and ELH_av as computed for the invasive front, the superficial part of the carcinoma, the total carcinoma, and the normal-appearing oral mucosa were compared. For DEL_av, significant prognostic information was found in the invasive front (p < 0.001). No significant prognostic information was found in superficial part of the carcinoma (p = 0.34), in the carcinoma as a whole (p = 0.35), or in the normal-appearing mucosa (p = 0.27). For ELH_av, significant prognostic information was found in the invasive front (p = 0.01) and, surprisingly, in putatively normal mucosa (p = 0.03). No significant prognostic information was found in superficial parts of the carcinoma (p = 0.34) or in the total carcinoma (p = 0.11). In conclusion, strictly quantitative assessment of tissue architecture in the invasive front of OSCC yields highly prognostic information.

Caveats: numerical requirements in graph theory based quantitation of tissue architecture.

Graph theory based methods represent one approach to an objective and reproducible structural analysis of tissue architecture. By these methods, neighborhood relations between a number of objects (e.g., cells) are explored and inherent to these methods are therefore certain requirements as to the number of objects to be included in the analysis. However, the question of how many objects are required to achieve reproducible values in repeated computations of proposed structural features, has previously not been adressed specifically. After digitising HE stained slides and storing them as grey level images, cell nuclei were segmented and their geometrical centre of gravity were computed, serving as the basis for construction of the Voronoi diagram (VD) and its subgraphs. Variations in repeated computations of structural features derived from these graphs were related to the number of cell nuclei included in the analysis. We demonstrate a large variation in the values of the structural features from one computation to another in one and the same section when only a limited number of cells (100-500) are included in the analysis. This variation decreased with increasing number of cells analyzed. The exact number of cells required to achieve reproducible values differ significantly between tissues, but not between separate cases of similar lesions. There are no significant differences between normal and malignantly changed tissues in oral mucosa with respect to how many cells must be included. For graph theory based analysis of tissue architecture, care must be taken to include an adequate number of objects; for some of the structural features we have tested, more than 3000 cells.

New algorithms based on the Voronoi Diagram applied in a pilot study on normal mucosa and carcinomas.

An adequate reproducibility in the description of tissue architecture is still a challenge to diagnostic pathology, sometimes with unfortunate prognostic implications. To assess a possible diagnostic and prognostic value of quantitiative tissue architecture analysis, structural features based on the Voronoi Diagram (VD) and its subgraphs were developed and tested. A series of 27 structural features were developed and tested in a pilot study of 30 cases of prostate cancer, 10 cases of cervical carcinomas, 8 cases of tongue cancer and 8 cases of normal oral mucosa. Grey level images were acquired from hematoxyline-eosine (HE) stained sections by a charge coupled device (CCD) camera mounted on a microscope connected to a personal computer (PC) with an image array processor. From the grey level images obtained, cell nuclei were automatically segmented and the geometrical centres of cell nuclei were computed. The resulting 2-dimensional (2D) swarm of pointlike seeds distributed in a flat plane was the basis for construction of the VD and its subgraphs. From the polygons, triangulations and arborizations thus obtained, 27 structural features were computed as numerical values. Comparison of groups (normal vs. cancerous oral mucosa, cervical and prostate carcinomas with good and poor prognosis) with regard to distribution in the values of the structural features was performed with Student's t-test. We demonstrate that some of the structural features developed are able to distinguish structurally between normal and cancerous oral mucosa (P = 0.001), and between good and poor outcome groups in prostatic (P = 0.001) and cervical carcinomas (P = 0.001). We present results confirming previous findings that graph theory based algorithms are useful tools for describing tissue architecture (e.g., normal versus malignant). The present study also indicates that these methods have a potential for prognostication in malignant epithelial lesions.

The invasive front of carcinomas. The most important area for tumour prognosis?

Various molecular events of importance in tumour spread, like the gain and loss of adhesion molecules, secretion of proteolytic enzymes, increased cell proliferation, and the initiation of angiogenesis occur at the tumour-host interface (invasive front). We have hypothesised that molecular or morphological characteristics at the invasive front area of various carcinomas may reflect tumour prognosis better than other parts of the tumour. Consequently, we recently developed a simple malignancy grading system restricted to the deep invasive front area of head and neck squamous cell carcinomas. This grading system proved to have additional prognostic value over the established prognostic factors. All similar studies performed so far have confirmed the high prognostic significance of the invasive front grading in squamous cell carcinomas at different locations. In this review paper we describe the system and the hypothesis on which it has been developed. The reproducibility of the grading is acceptable for further extended studies. Interestingly, observations of similar invasive front alterations in different adenocarcinomas suggest that the invasive tumour front may underlie the biological aggressiveness of carcinomas of glandular origin, as well.

Diagnostic implications of p53 protein reactivity in nasal mucosa of nickel workers.

OBJECTIVE: To investigate whether the quantitation of p53 protein reactivity in nasal biopsies could be related to nickel exposure by comparing nickel workers with various control groups. STUDY DESIGN: Nasal biopsies taken from nickel workers (n = 93) were compared immunohistochemically to various controls, including office staff members (n = 34) and hospital attendants (n = 6). The material was studied immunohistochemically with p53 antibody DO-1. p53 Protein-positive cells were counted at 400x magnification. RESULTS: p53 Protein reactivity was found in 54% (49/93) of nickel workers, 50% (17/34) of office staff members, 67% (4/6) of hospital attendants. No differences were seen between roasting/smelting, electrolysis and other workers in the refinery. The positive cells were present predominantly in the basal layer of the epithelium. The number of positive nasal cells per field in the hospital attendants on the average was half of that in the workers and the office staff in the refinery. In no case in the control group were more than 10 cells per field seen. No significant differences in p53 protein positivity were observed between the three nickel worker groups and between production workers and office staff members. CONCLUSION: Accumulation of p53 protein in nickel workers seems not to be attributable to nickel exposure. The lack of p53 protein positivity in fetal tissues shows that the accumulation of p53 protein is an event taking place after birth. Stimuli in the natural environment during life may explain p53 protein positivity.

Diagnostic and prognostic value of nucleolar organizer regions in normal epithelium, dysplasia, and squamous cell carcinoma of the oral cavity.

BACKGROUND: Nucleolar organizer regions (AgNORs) are associated with proliferative activity and represent a diagnostic aid and prognostic marker in several neoplastic entities. METHODS: Sections from 51 T1-2 squamous cell carcinomas (SCC), 20 cases of dysplasia, and 8 specimens with normal epithelium were evaluated by 2 AgNOR counting methods: 1) the mean number of AgNORs per nucleus (mAgNOR) and 2) the percentages of nuclei with > 1, > 2, > 3, and > 4 AgNORs (pAgNOR > 1, pAgNOR > 2, pAgNOR > 3, and pAgNOR > 4, respectively). RESULTS: Both mAgNOR and pAgNOR counts enabled significant discrimination between normal epithelium and dysplasia (P < 0.0003) and between dysplasia and SCC (P < 0.0001). For SCC, no correlation was found between AgNOR counts and clinical classification. Univariate analysis using the log rank test showed that the overall means for mAgNOR and pAgNORs correlated with the disease free period and survival time (P < 0.0040). Patient age, gender, type of treatment, and T and N classification failed to predict the outcome. Multivariate analysis showed that pAgNOR > 1 (cutoff level of 88%) was the best discriminator regarding the disease free period and survival time (P = 0.0001 and P = 0.0076, respectively). CONCLUSIONS: AgNOR enumeration, in particular pAgNOR > 1, appears to be a useful tool in distinguishing between normal epithelium, dysplasia, and SCC of the oral cavity. In this study, AgNOR counts were strong prognostic markers for patients with SCC.

Corticopontine terminal fibres form small scale clusters and large scale lamellae in the cat.

We investigated whether terminal fibres in the pontine nuclei are arranged in a lamellar pattern like that demonstrated earlier for pontocerebellar neurones. Following tracer injections in visual and parietal cortices and subsequent computer-based 3-D analysis, we found that labelled corticopontine terminal fibres form numerous sharply delimited aggregates of variable shape. Several of the aggregates are cylindroids (diameter 200-300 microns, length 1-3 mm). The aggregates are confined to a lamellar subspace, the position of which depends on the anteroposterior location of the cortical injections. These findings suggest that the cerebroponto-cerebellar system may be organized according to fairly simple, topographical rules. We discuss the implications of our results in relation to the development of corticopontine topographical organization.