Co-expression of galectin-3 and vimentin in triple negative breast cancer cells promotes tumor progression, metastasis and survival.

BACKGROUND: Lack of druggable targets and complex expression heterogeneity of known targets is common among TNBC subtypes. An enhanced expression of galectin-3 in TNBCs has already been documented. We have observed a tumor progression-dependent galectin-3 expression in TNBCs compared to adjacent epithelium and non TNBCs. OBJECTIVE: To unravel the association of galectin- 3 in tumor progression, aggressiveness and drug resistance in TNBC patients. METHODS: Galectin-3 expression in 489 breast cancer tissues was correlated with clinicopathological features and the results were validated in cell lines and mouse model by silencing galectin-3 using shRNA and the proteins were profiled by western blot and qRT-PCR. Protein interaction was analyzed by GFP Trap and Mass spectrometry. RESULTS: Galectin-3 expression correlated with tumor stage in TNBC and a lower galectin-3 expression was associated with poor patient survival. The positive correlation between galectin-3, vimentin and CD44 expression, pinpoints galectin-3 contribution to epithelial to mesenchymal transition, drug resistance and stemness. Vimentin was found as an interacting partner of galectin-3. Duplexing of galecin-3 and vimentin in patient samples revealed the presence of tumor cells co-expressing both galectin-3 and vimentin. In vitro studies also showed its role in tumor cell survival and metastatic potential, elementary for tumor progression. In vivo studies further confirmed its metastatic potential. CONCLUSIONS: Tumor progression dependent expression pattern of galectin 3 was found to indicate prognosis. Co-expression of galectin-3 and vimentin in tumor cells promotes tumor dissemination, survival and its metastatic capability in TNBCs.

Galectin-3: A factotum in carcinogenesis bestowing an archery for prevention.

Cancer metastasis and therapy resistance are the foremost hurdles in oncology at the moment. This review aims to pinpoint the functional aspects of a unique multifaceted glycosylated molecule in both intracellular and extracellular compartments of a cell namely galectin-3 along with its metastatic potential in different types of cancer. All materials reviewed here were collected through the search engines PubMed, Scopus, and Google scholar. Among the 15 galectins identified, the chimeric gal-3 plays an indispensable role in the differentiation, transformation, and multi-step process of tumor metastasis. It has been implicated in the molecular mechanisms that allow the cancer cells to survive in the intravascular milieu and promote tumor cell extravasation, ultimately leading to metastasis. Gal-3 has also been found to have a pivotal role in immune surveillance and pro-angiogenesis and several studies have pointed out the importance of gal-3 in establishing a resistant phenotype, particularly through the epithelial-mesenchymal transition process. Additionally, some recent findings suggest the use of gal-3 inhibitors in overcoming therapeutic resistance. All these reports suggest that the deregulation of these specific lectins at the cellular level could inhibit cancer progression and metastasis. A more systematic study of glycosylation in clinical samples along with the development of selective gal-3 antagonists inhibiting the activity of these molecules at the cellular level offers an innovative strategy for primary cancer prevention.

Risk factors and prevalence of cervical squamous intraepithelial lesions among women in south India: A community-based cross-sectional study.

Background: ">Cervical cancer in India accounts for one-fifth of the global burden. Well-defined precancerous stages help early detection of the disease. Apart from human papillomavirus, the risk factors include age, education, occupation, early age at marriage and first delivery, abortions, and multiple sexual partners. Prevalence and risk factors for cervical squamous intraepithelial lesions (SIL) among women by Pap smear screening in south India were analyzed through a cross-sectional study. Methods: ">Women from rural and urban area were motivated by local accredited social health activists to attend pre-fixed Pap smear clinics in government hospitals. Pap smears collected in these clinics were taken to the Regional Cancer Centre, Thiruvananthapuram, processed, and cytology reports were prepared. Multiple logistic regression analysis was used to identify risk factors for SIL and high-grade SIL (HSIL). Results: ">The number of SIL was 67 out of 10,580 and HSIL was 39. Having higher education (Odds Ratio, OR:0.05(95% Confidence Interval, CI: 0.01-0.2), being married but living single (OR : 5.3, 95%CI:2.4-11.5), Having >2 abortions (OR:21, 95% CI:4.5-24), having younger age at delivery (OR : 0.1, 95% CI:0.01-0.3) and having unhealthy cervix (OR: 16.4, 95% CI:6.2-42.7) were the factors found to be the associated risk factors in multiple regression analysis. Conclusion: ">Pap smear screening can be focused on women with risk factors such as low education, married but living single, having >2 abortions, younger age at delivery, and unhealthy cervix on per speculum examination.

Immunoexpression of TTF1 and p63 Differentiates Lung Adenocarcinomas in Sputum Samples.

CONTEXT: Differentiating NSCLC as either adeno or squamous type and identification of Epidermal Growth Factor Receptor (EGFR) mutations is clinically relevant for lung cancer patients for selecting treatment. Thyroid transcription factor-1 (TTF-1) and p63 were demonstrated as useful markers for histologic typing of lung cancer. Mutation and overexpression of EGFR has been reported in a subset of non-small cell lung cancers. If these markers can be validated for the differential diagnosis of adenocarcinoma in a sputum sample itself, it will be highly beneficial for lung cancer patients. AIMS: To evaluate whether immunocytochemical expression of TTF-1, p63, and EGFR proteins in sputum samples can be used for differential diagnosis of lung adenocarcinoma by comparing with that of the corresponding tissue samples. SETTINGS AND DESIGN: Ninety sputum samples and matched tissue samples were used for the study. SUBJECTS AND METHODS: Monolayered smears and cell blocks of sputum and the corresponding tissue samples were immunostained with the standard ABC method. The expression patterns of these markers were analyzed statistically and compared with clinic-pathological parameters. STATISTICAL ANALYSIS USED: Chi-square test and paired t-test. RESULTS: The p63 protein had a positive expression in 73.9% of SCC whereas TTF1 had positive expression in 75.8% of ADC. The EGFR expression was positive in 27 cases of adenocarcinoma, 21 cases of SCC and 19 cases of NSCLC. CONCLUSIONS: Immunocytochemistry of the aforementioned antibodies in sputum samples can be used as supplementary evidence for the subtyping of NSCLC.

Immunocytochemistry on Sputum Samples Predicts Prognosis of Lung Cancer.

CONTEXT: Despite sputum cytology being accepted as a simple and noninvasive diagnostic method for lung cancer, the clinical usefulness of sputum for evaluation of prognosis is yet to be explored. Validation of some of the markers in sputum for prognosis prediction will be highly useful for selective therapy. AIMS: This study was aimed to evaluate a reliable panel of immunocytochemical markers for their significance to predict survival. MATERIALS AND METHODS: We have analyzed the expression of p53, p16, galectin-3, and epidermal growth factor receptor (EGFR) proteins in sputum samples processed in a mucolytic agent/cellblock and compared the same with that of the corresponding tissue samples. RESULTS: Overexpression of p16 and EGFR was found to have a better survival benefit, whereas positive p53 and galectin-3 expressions had shorter period of survival. Expression patterns of all these four proteins were more or less similar in smears, cellblocks of sputum, and tissue samples except for slight changes in staining intensity which was not found to be statistically significant. No significant difference was found in the association of these proteins with survival pattern between sputum and tissue samples. CONCLUSION: This is the first report of immunocytochemistry of a panel of markers on cells exfoliated in sputum samples which suggests that analysis of immunocytochemical markers in sputum samples can be attempted as a cost-effective and reliable predictor of prognosis and survival. Accumulation of mutated p53, overexpression of galectin-3, and lower expression of p16 and EGFR proteins were found to predict poor prognosis for lung cancer.

Significance of DNA Replication Licensing Proteins (MCM2, MCM5 and CDC6), p16 and p63 as Markers of Premalignant Lesions of the Uterine Cervix: Its Usefulness to Predict Malignant Potential

Cervical cancer continues to be a leading cancer among women in many parts of the world. Nation-wide screening with the Pap smear has not been implemented in India due to the lack of adequately trained cytologists. Identification of biomarkers to predict malignant potential of the identified low risk lesions is essential to avoid excessive retesting and follow up. The current study analyzed the expression patterns of DNA replication licensing proteins, proliferation inhibitor protein p16INK4A and tumor suppresser protein p63 in cervical tissues and smears to assess the ability of these proteins to predict progression. Methods: Cervical smears and corresponding tissues were immunostained using mouse monoclonal antibodies against MCM2, MCM5, CDC6, p16 and p63. Smears were treated with a non-ionic surfactant sodium deoxycholate prior to immuno-cytochemistry. The standard ABC method of immunohistochemistry was performed using DAB as the chromogen. The immunostained samples were scored on a 0-3+ scale and staining patterns of smears were compared with those of tissue sections. Sensitivity and specificity for each of these markers were calculated taking histopathology as the gold standard. Result: All the markers were positive in malignant and dysplastic cells. MCM protein expression was found to be up-regulated in LSIL, HSIL and in malignancies to a greater extent than p16 as well as p63. CDC6 protein was preferentially expressed in high grade lesions and in invasive squamous cell carcinomas. A progressive increase in the expression of DNA replication licensing proteins in accordance with the grades of cervical intraepithelial lesion suggests these markers as significant to predict malignant potential of low grade lesions in cervical smears. Conclusion: MCMs and CDC6 can be applied as biomarkers to predict malignant potential of low grade lesions identified in screening programmes and retesting / follow up might be confined to those with high risk lesions alone so that overuse of resources can be safely avoided.

Free Radical Scavenging Properties of Annona squamosa

Annona squamosa has extensively been used in the traditional and folkloric medicine and found to possess many biological activities. Different solvents, petroleum ether, chloroform, ethyl acetate and methanol extracts of Annona squamosa seeds (ASPE, ASCH, ASEA, ASME) have been used to prepare plant extracts. The present investigations dealt with the free radical scavenging activity of four extracts using various techniques such as total reducing power estimation, total phenolic count, 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging effect, evaluation of ABTS cation decolorisation capacity, FRAP assay, hdroxyl radical scavenging assay, super oxide assay and Nitric oxide radical scavenging assay of the extracts. The results showed that the four extracts of Annona squamosa showed significant reducing power in four extracts. The total phenolic contents in petroleum ether, chloroform, ethyl acetate, methanol extracts and positive control were 0.64±0.17, 0.54±0.27, 0.49±0.24, 0.57±0.22 and 0.66±0.33. The antioxidant capacity by ABTS assay of ASPE, ASCH, ASEA, ASME and positive control, trolox showed 77.75±0.5,73.25±1.7,78.5± 1.2 , 80 ± 0.8 µg/ml and 94.2 ± 0.9 respectively. The (50 % scavenging activity) SA50 of ASPE and ASCH, ASEA and ASME was found to be 34.4 µg/ml, 43.8 µg/ml 34.7 µg/m and 28.8 µg/ml respectively by DPPH assay. The percentage of hydroxyl radical scavenging increased with the increasing concentration of the extracts. ASPE, ASCH, ASEA and ASME showed superoxide radical scavenging activity, as indicated by their values 66 ± 0.5, 68 ± 1 ,63 ± 1 and 70 ± 0.5 µg/ml respectively compared to gallic acid which was 97 ± 0.5 µg/ml. The values for scavenging of nitric oxide for ASPE, ASCH, ASEA and ASME were 91.0 ± 1.0, 66.75 ± 0.5, 71.75 ± 1.1 and 75.75 ± 1.15 µg/ml while value for standard ascorbic acid was 91.0 ± 1.0 µg/ml. The results revealed strong antioxidants in four extracts may lead to the development of potent antioxidant agents from Annona squamosa seeds.

An Exploration of Phytochemicals from Simaroubaceae

Natural products such as plants, animals and minerals have been the basis of treatment of human diseases. Herbal remedies have been used for the treatment of many ailments. Many compounds have been derived from the plant species mentioned in the ancient texts of Indian system of medicine for the treatment of a number of ailments. The R and D thrust in the pharmaceutical sector is focused on development of new drugs, innovative/indigenous processes for known drugs and development of plant based drugs through investigation of leads from the traditional systems of medicine. The family Simaroubaceae is grouped in the order Rutales, is known to have a diverse range of secondary metabolites. Plants from this family are used as medicine to cure cancer and many other diseases. Isolation of diverse chemical compounds from Simaroubaceae on its stem bark, root bark and leaves have been reported. In this review, we are analysing with the chemical constituents of family Simaroubaceae.

DNA Replication Licensing Proteins for Early Detection of Lung Cancer

Background: To identify and characterize malignant and premalignant cells in sputum and matched tissue samples with reference to expression of minichromosome maintenance proteins (MCM2, MCM5) and cell division cycle protein 6 (CDC 6) and to assess their potential as biomarkers of premalignant and malignant lesions of the lung and associations with clinicopathological features. Methods: Expression of MCM2, MCM5 and 6 proteins in sputum samples and corresponding tissues was assessed by immunocytochemistry, and correlated with histological findings. Results: For characterization of malignant, metaplastic or dysplastic cells, CDC6 protein had the highest sensitivity of 87.7%. All the three markers together had a sensitivity of 94.4%. Furthermore these proteins could be employed to assess the proliferative potential of precancerous or atypical cells, as overexpression increasing with the stage of disease and degree of metastasis. Conclusion: The assessed markers can be utilized in routine cytopathology laboratories to supplement conventional morphological evaluation so that the sensitivity of sputum cytology can be enhanced. Potential applications in predicting the clinical behavior of lung lesions and predicting prognosis and survival deserve further attention.

Significance of silver-stained nucleolar organizer regions in early diagnosis and prognosis of oral squamous cell carcinoma: a multivariate analysis.

Argyrophilic nucleolar organizer regions (AgNORs) appear to be of diagnostic and prognostic significance in different types of human tumour. The present study examined the AgNOR counts in normal, premalignant and malignant oral mucosa to evaluate their potential as a biological marker for tumour progression and a prognostic predictor for treatment outcome in oral carcinomas. The Spearmann's rank correlation analysis between AgNOR counts and various stages of tumour progression in oral mucosa exhibited a highly significant positive coefficient (r(s) = 0.7969; p < or = 0.001), thus indicating the role of AgNORs in the early diagnosis of potentially malignant oral leukoplakia. When analyzing the prognostic significance of AgNOR in oral carcinomas, mean count > 2.8 concurred with poor prognosis in both univariate and multivariate analysis. Along with AgNOR counts, the T-status of disease was also found to be an independent predictor for treatment outcome in multivariate analysis. Thus T3 and T4 tumours, with mean AgNOR counts more than 2.8, are aggressive and may exhibit resistance to current treatment protocols.

Aggregation induced Raman scattering of squaraine dye: Implementation in diagnosis of cervical cancer dysplasia by SERS imaging.

The extent of squaraine dye aggregation that reflects on surface enhanced Raman signal scattering (SERS) intensity upon adsorption on nano-roughened gold surface has been investigated. Here we have synthesized a serious of six squaraine dyes consisting of two different electron donor moiety i.e. 1,1,2-trimethyl-1H-benzo[e]indole and 2-methylbenzo[d]thiazole which modulates the chemisorptions and hydrophobicity being designated as SQ1, SQ2, SQ3, SQ4, SQ5 and SQ6. Interestingly, SQ2 (mono lipoic acid appended), SQ5 and SQ6 (conjugated with hexyl and dodecyl side chain) squaraine derivatives having more tendency of aggregation in DMSO-water mixed solvent showed significant increase of Raman scattering in the fingerprint region when chemisorbed on spherical gold nanoparticles. Two sets of SERS nanotags were prepared with colloidal gold nanoparticle (Au-NPs size: 40 nm) by incorporating Raman reporters SQ2 and SQ5 followed by thiolated PEG encapsulation (SH-PEG, SH-PEG-COOH) denoted as AuNPs-SQ2-PEG and AuNPs-SQ5-PEG. Further conjugation of these nanotag with monoclonal antibodies specific to over expressed receptors, EGFR and p16/Ki-67 in cervical cancer cell, HeLa showed prominent SERS mapping intensity and selectivity towards cell surface and nucleus. The fast and accurate recognition obtained by antibody triggered SERS-nanotag has been compared with conventional time consuming immunocytochemistry technique which prompted us to extend further investigation using real patient cervical smear sample for a non-invasive, ultrafast and accurate diagnosis.

Argyrophilic nucleolar organizer regions in the evaluation of tumour progression in the oral mucosa: correlation with tissue pathology.

The present study has analysed the numbers of argyrophilic nucleolar organizer regions (AgNOR) in normal tissues and in premalignant and malignant lesions of the oral mucosa in order to assess their potential as a biological marker for tumour progression. On comparison of AgNOR numbers in different lesions, carcinomas showed the highest number (4.65 +/- 0.98) compared to leukoplakias (2.38 +/- 0.47) and normal tissues (1.53 +/- 0.39). Spindle cell carcinomas and poorly differentiated squamous cell carcinomas had higher AgNOR counts than well-differentiated carcinomas. In various clinically different types of oral leukoplakia, the lowest AgNOR counts were observed in homogenous leukoplakia and the highest in speckled leukoplakia. No significant difference in AgNOR number was observed between non-dysplastic and dysplastic leukoplakia, although a significant difference was evident between dysplastic leukoplakia and normal oral mucosa. Correlating the AgNOR count and tumour progression, a significantly high positive correlation coefficient (r = 0.7969, P = 0.0000) was observed.

Differential expression of jackfruit-lectin-specific glycoconjugates in metastatic adenocarcinoma and reactive mesothelial cells-a diagnostic aid in effusion cytology.

Distinguishing reactive mesothelial cells from adenocarcinoma cells in serous effusions on the basis of morphological criteria alone is often difficult. Interest has therefore been focused on identifying reliable methods to supplement the conventional cytological techniques. Plant lectins have been reported as diagnostic markers for malignant cells. We studied 51 aspirated samples of benign and malignant effusions using horseradish-peroxidase-conjugated jackfruit lectin. No significant difference was observed between the cells of pleural and peritoneal fluids. The reactively proliferated mesothelial cells of benign effusions showed a predominance of mild staining while moderate and intense staining was predominant in malignant effusions. Intense and irregular lectin binding was observed in macrophages irrespective of the cause of effusion. The lectin staining method therefore appears to have some clinical significance as an additional diagnostic aid for use in effusion cytology.

Jack fruit lectin-specific glycoconjugate expression during the progression of cervical intraepithelial neoplasia: a study on exfoliated cells.

The expression of glycoconjugates specific to Jack fruit lectin (JFL) was studied in the exfoliated squamous cells of different grades of intraepithelial and invasive neoplasia of the uterine cervix. It was observed that while normal cells showed almost negative binding, the lectin binding percentage of squamous cells significantly increased with increasing atypia of the epithelium. Correlation analysis between different groups revealed that mild lectin binding in cells had a negative correlation and intense binding had a positive correlation with various stages of tumor progression. These results indicate that the number of cells with aberrant expression of glycoconjugates increases as neoplastic transformation advances. The percentage of labeled and unlabeled cells also shows a continuous transition from low to severe grades of cervical intraepithelial neoplasia and invasive carcinomas. The present study therefore shows that JFL may be used as a probe for further elaboration of detection and grading of precancerous and cancerous lesions of the uterine cervix.

Significance of AgNOR count in differentiating malignant cells from reactive mesothelial cells in serous effusions.

OBJECTIVE: To distinguish reactive mesothelial cells from malignant cells in serous effusions using silver staining of nucleolar organizer regions (AgNOR) applied to ethanol-fixed cytologic preparations. STUDY DESIGN: One hundred aspirated samples of benign and malignant effusions were studied using the one-step silver staining method. Eight cytologically atypical samples were also included in the study. RESULTS: In malignant cells the mean AgNOR count was 4.72 +/- 0.76 (+/- SD), and the AgNORs were irregular in shape, while in benign mesothelial cells AgNORs were comparatively larger, single dots with a mean count of 1.92 +/- 0.23. Of the cytologically atypical samples, five were in the malignant range. The others were within benign limits. Repeat cytology of the second aspirate confirmed that finding. CONCLUSION: AgNOR study appears to be clinically useful as an additional diagnostic tool for use in ascitic and pleural fluid samples when the cytologic diagnosis is difficult.

Automated segmentation of free-lying cell nuclei in Pap smears for malignancy-associated change analysis.

This paper presents an automated algorithm for robustly detecting and segmenting free-lying cell nuclei in bright-field microscope images of Pap smears. This is an essential initial step in the development of an automated screening system for cervical cancer based on malignancy associated change (MAC) analysis. The proposed segmentation algorithm makes use of gray-scale annular closings to identify free-lying nuclei-like objects together with marker-based watershed segmentation to accurately delineate the nuclear boundaries. The algorithm also employs artifact rejection based on size, shape, and granularity to ensure only the nuclei of intermediate squamous epithelial cells are retained. An evaluation of the performance of the algorithm relative to expert manual segmentation of 33 fields-of-view from 11 Pap smear slides is also presented. The results show that the sensitivity and specificity of nucleus detection is 94.71% and 85.30% respectively, and that the accuracy of segmentation, measured using the Dice coefficient, of the detected nuclei is 97.30±1.3%.

Significance of a galactose specific plant lectin for the differential diagnosis of adenocarcinoma cells in effusion.

BACKGROUND: Distinguishing adenocarcinoma cells from reactively proliferated mesothelial cells and macrophages is one of the greatest challenges in the cytodiagnosis of effusions. Aberrant glycosylation of cell surface glycoconjugates is emblematic to malignancy, and lectins being an important class of probes to demonstrate these aberrations, lectin cytochemistry is of great interest to differentiate adenocarcinoma cells from reactive mesothelial cells. AIM: The present study analyzed the potential of a plant lectin to distinguish malignant cells from reactive mesothelial cells and macrophages. MATERIALS AND METHODS: Snake gourd lectin (SGL) was isolated, purified and conjugated to horse radish peroxidase (HRP) and incubated with the cells of benign (46) as well as malignant (39) effusions using the standard immunocytochemical method with diaminobenzidine as the chromogen. The lectin-bound areas were quantitatively assessed as mild, moderate and intense binding. STATISTICAL ANALYSIS: The mean score for benign and malignant effusions were statistically analyzed. Student's 't'-test was performed to assess the significance. RESULTS: The lectin HRP complex bind to the cytoplasm of benign and malignant cells as well as macrophages. A significantly higher score for intense binding (P = 0.001) was found to differentiate malignant cells from reactive mesothelial cells. Macrophages showed intense irregular binding. CONCLUSIONS: SGL binding assay can play a role in the differential diagnosis of metastatic adenocarcinoma in effusions.

Psammoma bodies in cervical smear in association with keratinizing squamous cell carcinoma of cervix: a case report.

The presence of psammoma bodies (PBs) in cervical smears is a rare finding. These structures have been identified in association with a wide range of benign and malignant conditions within the female genital tract. PBs in cervical smears have usually been associated with malignant serous epithelial ovarian tumors. However, many PBs associated with atypical squamous cells were detected in cervical smears of an 83-year-old woman with complaint of postmenopausal bleeding. Colposcopic examination revealed an ulceroinfiltrative growth in the cervix. Histological examination of the biopsy specimen from the growth revealed keratinizing squamous cell carcinoma with multiple and singly arranged PBs. This report suggests that cytologists should aware of the possibilities, on finding PBs associated with atypical cells in cervical specimens and report the cases accordingly.

Implications of gynaecological abnormalities in pre-selection criteria for cervical screening: preliminary evaluation of 3602 subjects in south India.

Early detection and eradication of cervical cancer and its precursor lesions through organized mass cytological screening programmes have recently gained considerable attention in developing countries. Strategies for both cost saving and effective implementation are however required for mass cervical screening in developing countries. In an early cancer detection programme conducted in South India, we analysed cytological abnormalities in 3602 women and correlated the results with other factors, including age, gynaecological complaints, number of years of married life and parity to see if pre-selection for cytologic screening was possible. Only lower grades of dysplasia were found in asymptomatic women below the age of 40 years. In asymptomatic women, malignancy and higher grades of dysplasia were confined to women with a clinically abnormal cervix only. Univariate analysis also revealed that subjects with a parity of more than 3 and a married life of more than 20 years had a significantly higher number of cytological abnormalities. However, on a multivariate analysis the increased number of marital years was not found to be an independent variable. These results suggest that asymptomatic women below the age of 40 years with a married life of less than 20 years and parity below 3, may be excluded from screening campaigns, and that pre-selection for cytologic screening is possible by introducing a programme of clinical and speculum examination of the cervix.

Expression of p53 in leukoplakia and squamous cell carcinoma of the oral mucosa: correlation with expression of Ki67.

Aim-To study p53 expression in relation to proliferative status in normal and nondysplastic, dysplastic and malignant lesions of the oral mucosa.Method-The standard avidin-biotin complex (ABC) immunohistochemical staining method was used to study the expression of p53 and Ki67 on frozen sections of oral leukoplakias and carcinomas.Results-Of the leukoplakia and carcinoma samples, 70% expressed p53 in over 5% of cells. In normal mucosa less than 5% of cells expressed p53. The proliferation index, as assessed by expression of Ki67, was highest in the malignant lesions (43%) and lowest in normal mucosa (11%). Statistical analysis revealed that expression of both p53 and Ki67 was correlated significantly with the histopathological stage of the tumour. However, expression of p53 was not correlated with that of Ki67. In leukoplakia lesions with proliferative features p53 immunostaining was less intense than in non-proliferative lesions; this difference was statistically significant.Conclusions-These results emphasise the potential of Ki67 and p53 as biomarkers of carcinogenesis in oral cancer and may also serve as intermediate points for cancer prevention programmes, such as the oral chemopreventive trials. Factors other than p53 may have a more important role in the deregulation of proliferation in pre-malignant oral lesions.