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1 Dept of Pathological Anatomy, Gregorio Marañón General University Hospital, Madrid, Spain. 2 , LABIEMET, Los Andes University, Venezuela, and 3 Dept of Pathological Anatomy, La Raza National Medical Centre, Mexico.
CORRESPONDENCE: N. Sánchez-Mora, Hospital General Universitario Gregorio Marañón, C/ Dr. Esquerdo No. 46, Dpto. de Anatomía Patológica, CP. 28007 Madrid, Spain. Fax: 34 915868162. E-mail: histonora{at}yahoo.es
Keywords: Histo-blood group, squamous dysplasia, squamous metaplasia
Received: January 23, 2006
Accepted September 14, 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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In total, 100 bronchial biopsies were included in the present study. The cases were classified according to the World Health Organization grading system. Immunohistochemical stains for histo-blood groups A and B, and reactivity tests to p53 and the cellular proliferation index were performed.
A total of 56 (56%) patients belonged to blood group A. Among them, six (10.7%) patients who did not express antigen in squamous metaplasia, showed carcinoma at the moment of the biopsy (n = 3) or developed synchronous lung carcinoma (n = 3). A total of nine (9%) patients belonged to blood group B. Loss of antigenic expression was observed in five cases. All of them developed synchronous lung carcinoma. The patients with low- and high-grade dysplasia developed lung cancer in 71 and 100% of the cases, respectively.
In conclusion, the findings of the present study suggest that the loss of histo-blood antigens expression is an event in the carcinogenesis of bronchial mucosa and it is usually associated with high-grade lesions and hyperproliferative activity.
Lung carcinogenesis is a multiple-step process characterised by accumulation of successive molecular genetic and epigenetic abnormalities, resulting in epithelial cell malignant transformations 1, 2, such as expression of oncogenes, and loss of tumour-supressor genes 2, 3. Increasing information is currently available on the genetic alterations leading to the final invasive stage of bronchial carcinomas. However, the natural history of the pre-invasive lesions is poorly understood. Currently, it is clear that high-grade pre-invasive lesions will develop into invasive carcinoma in 30–50% of the cases, whereas the majority of low-grade pre-invasive lesions remain stable or regress during follow-up 4, 5. Nevertheless, increased efforts to better characterise lesions are needed.
Histo-blood group antigens are a group of glycoproteins and glycolipids whose antigenic specificity is determined by a variation in their constituent carbohydrate chains 6. These antigens are involved in various biological processes, such as cellular differentiation, maturation, proliferation, malignant transformation and intercellular signalling 7. The prognostic significance of a loss of or a modified histo-blood group antigen (ABH) expression has been suggested in carcinomas of various sites, including the urinary bladder 8, the gastrointestinal tract 9 and the lungs 10–12. Davidsohn and Ni 10 demonstrated a loss of ABH determinants in pulmonary carcinomas. Subsequently, Lee et al. 11 and Moldvay et al. 13 demonstrated that a reduction or deletion of antigenic expression correlated with a reduced survival rate. Matsumoto et al. 14 concluded that this loss of ABH blood group antigens in lung carcinomas correlated with their metastatic potential, especially with recurrence and haematogenous metastasis. Similar findings were reported by Ichikawa et al. 15.
The aim of the present study was to explore the significance of the expression of histo-blood antigens in squamous metaplasia and its association to other prognostic factors, such as oncoprotein p53 expression and cellular proliferation index (Ki67) according to immunohistochemical techniques.
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MATERIAL AND METHODS |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Demographic information was obtained from the clinical records. Smokers were defined as patients who smoked at the time of biopsy. Ex-smokers were defined as those who had given up smoking >1 yr before the time of biopsy. Nonsmokers were defined as those patients who never acquired the habit. Tobacco consumption was evaluated as a pack-yr index. In the follow-up, synchronous carcinoma was defined as a carcinoma diagnosed within 1 yr after a biopsy, demonstrating squamous metaplasia. Metachronous carcinoma was defined as occurring 
1 yr after the biopsy.
Preparation of tissues
Tissues were fixed in a neutral buffered solution of 10% formalin, embedded in paraffin, sectioned at a thickness of 4 µm and stained with haematoxylin and eosin.
Immunohistochemical studies
Sections 4-µm thick from paraffin-embedded biopsy specimens were cut and mounted on glass slides. The specimens were de-paraffinised in xylene and rehydrated step by step with descending concentrations of ethanol. The sections were incubated at 37°C with 0.3% H2O2 in absolute methanol for 10 min, to block endogenous peroxidase. After being washed for 20 min with PBS of pH 7.2, the specimens were incubated with the primary antibodies for 45 min in a moist chamber at room temperature (RT). The primary antibodies used were: anti-blood group A (Clon: T36; Signet Laboratories, Dedham, MA, USA; 1:60 dilution), anti-blood group B (Clon: CLCP-19B; Signet Laboratories; 1:60 dilution), anti-p53 (Clon: DO7; Novocastra, Newcastle, UK; 1:100 dilution) and the antibody against the Ki67 (Clon: MIB-1; DAKO Corporation, Carpinteria, CA, USA; 1:200 dilution). These were used after pretreatment in a microwave oven (15 min, 500 W) in citrate buffer (10 mM, pH 6.0).
The sections were subsequently incubated with biotinylated anti-mouse and anti-rabbit immunoglobulin G and ligand-binding assay (DAKO Corporation) for 25 min at RT, then rinsed in PBS for 5 min and immersed in avidin peroxidase complex for 25 min. Finally, the peroxidase was localised by treatment of the samples with a fresh mixture of diaminobenzidine and substrate for 10 min. After washing in distilled water, the sections were lightly counterstained with haematoxylin, dehydrated in ethanol, cleared in xylene and coverslipped using permount.
Immunostaining to A and B anti-groups was defined as positive when the cells showed a granular staining in the cytoplasm and/or the cytoplasmic membrane, regardless of its intensity. Inmunohistochemical reactivity to p53 and Ki67 was recorded as the percentage of stained nuclei. The test was deemed positive when >10% of the nuclei were stained. The intensity was divided into five groups according to the percentage of stained cells as follows: 0 = 0–10%, + = 10–25%, ++ = 26–50%, +++ = 51–75% and ++++ = 76–100%.
Statistical analysis
The p-values were calculated using Chi-squared according to Yates' test and finally corrected using the exact Fisher's test when values were less than five. The association of variable strength, as well as the possible risk to develop any respiratory neoplasia, was performed using odds ratios and 95% confidence intervals.
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RESULTS |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Bronchial lesions were graded according to the WHO grading system. One case showing angiogenic squamous dysplasia (ASD) was classified as presenting severe dysplasia. All the patients had respiratory symptoms related with lung cancer, but not yet lung cancer diagnosis.
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). Among the remaining cases without loss of expression of histo-blood group A, 22 (39.3%) patients developed synchronous lung carcinoma, (12.5%) seven cases developed metachronous lung carcinoma, 10 cases (17.9%) did not develop neoplasia and 11 (19.6%) cases were lost to follow-up. The case with ASD was associated with loss of histo-blood group A antigen expression, showed a positive stain to oncoprotein p53 and Ki67 and developed synchronous lung carcinoma (fig. 1).
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). Concerning the four remaining cases without loss of expression of histo-blood group B (45%), three (33.3%) cases developed metachronous lung carcinoma and one (11.1%) case was lost to follow-up.
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Follow-up was available for 82 cases. The mean (range) follow-up period was 24.7 (6–48) months. A total of 56 (68.3%) cases developed synchronous carcinoma. Squamous lung cancer was the most frequent histological type in 28% of the cases. Small cell lung carcinoma developed in 6.1% of the cases, lung adenocarcinoma developed in 3.7% of the cases and 8.5% of the cases disclosed carcinoma in other localisations (six (7.3%) cases laryngeal carcinoma and one (1.2%) case breast carcinoma). Approximately 12.2% of the cases developed metachronous carcinomas; six squamous lung carcinoma, one case lung adenocarcinoma and three cases with carcinoma in other localisations (two cases in the larynx and one ureteral). In total, 18 (21.9%) patients with clinical and radiological criteria of lung cancer, died without histological diagnoses. There was no development of neoplastic lesions in 16 (19.5%) cases in the 48 months of follow-up (table 4).
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DISCUSSION |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the risk of lung cancer increases with the presence of pre-invasive lesions, the molecular determinants predicting the irreversible progression to lung cancer have not yet been identified.
The demonstration of variations in the immunohistochemical profile of expression of histo-blood groups A and B antigens, similar to those seen in carcinoma, would be of clinical significance not only to prognosticate lung carcinomas but also as a useful tool for early diagnosis. Deletion or reduction of histo-blood A or B epitopes has been extensively studied in human cancer. After an initial report in gastric cancer 18, this phenomenon was also described and correlated with histological grade and metastatic potential in other gastrointestinal 9, lung 10–12, cervical 19, oral 20 and bladder carcinomas 8. As far as bronchial carcinoma is concerned, previous reports have suggested that patients belonging to blood groups A or B and bearing tumours that do not express the corresponding histo-blood groups, show a shorter survival time after diagnosis 13, 14, 21. Regarding histo-blood antigen expression, the present results suggest that antigenic loss occurs in the cancerisation process of bronchial mucosa, as it is a rare event in low-grade squamous lesions but a frequent one in high-grade lesions associated with developed synchronous lung carcinoma 10–12, 14, 15. The present authors considered that, in spite of the fact that the size of the present series is too short to discuss predictive or diagnostic value, the trends found suggest that the loss of histo-blood antigen expression in pre-invasive bronchial lesions could reveal a severe field cancerisation process of bronchial mucosa and this can be a feature shown by pre-invasive bronchial lesions associated with synchronous carcinoma. It must be emphasised that the present study included patients with clinical suspicion of lung cancer.
The data analysed in the present study showed a high percentage (68.3%) of synchronous cancer associated with pre-invasive bronchial lesions. This fact can be explained by two different hypotheses. First, it could support the theory that the sequential step-wise histological progression of pre-invasive bronchial lesions does not always occur 5. Secondly, it may reflect the fact that field cancerisation may randomly affect any site in the bronchial tree and, therefore, concomitant lesions can be of different age and progress toward invasion at different rates 22. Unfortunately, the results of the present study cannot clarify this point.
The alteration of tumour suppressor genes plays a critical role in the development of many forms of cancer. The present authors have tried to correlate this factor with histo-blood antigen expression. The disturbance of the normal function of the p53 oncoprotein has been suggested to reflect an important event during malignant transformation. Some authors have reported data showing p53 overexpression in squamous bronchial metaplasia 23 and p53 mutations associated to high-grade dysplasia 1. Other authors have studied metaplastic lesions from patients with coexisting carcinomas, showing p53 overexpression 23, 24. The results of the present study are in accordance with these data.
All cases with loss of expression of the histo-blood antigen also show a high staining to Ki67 and p53 in the present series. These results confirm that the loss of antigenic expression and other factors, such as p53 expression and increased cellular proliferation, are at least simultaneous events, rendering a larger series of cases to evaluate its statistical significance necessary. These findings support the progressive and step-wise accumulation of genetic/epigenetic abnormalities leading to cell transformation 1, 2, 25, 26.
ASD is characterised by small capillaries growing underneath the epithelium in a micropapillary pattern, indicating abnormal vascularisation of the bronchial mucosa. This is reflected by both increased microvascular density and aberrant morphology of the bronchial capillary bed. Possible mechanisms of angiogenesis in ASD may involve small populations of dysplastic squamous cells harbouring pre-malignant mutations transmitting angiogenic signals over very short distances 27. In the present study only one case with ASD was observed. It was associated with loss of histo-blood group A antigen expression, oncoprotein p53 and Ki67 positivity and developed synchronous carcinoma. This finding gives additional support to the opinion that it must be classified as a high-grade lesion.
In conclusion, the present findings suggest that the loss of histo-blood antigen expression is an event in the carcinogenesis of bronchial mucosa and is usually associated with high-grade lesions and hyperproliferative activity. Further studies are needed to better characterise the alterations that define the malignancy of the bronchial epithelium, as well as to identify critical factors, or association of factors, that would be predictive of the progression of pre-invasive bronchial lesions to invasive cancers. Together with very sensitive and specific methods and acceptable reproducibility to endobronchial detection of the malignant cell clones, such studies may help develop a more effective strategy for the management of this highly fatal disease.
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