Identification of site-specific prognostic biomarkers in patients with oral squamous cell carcinoma.

The purpose of the present study was to identify site-specific prognostic biomarkers in patients with oral squamous cell carcinoma (OSCC). For this purpose, Epidermal growth factor receptor (EGFR), Stat3, H-ras, c-myc, p53, cyclin D1, p16, Rb and Bcl-2 were localized immunohistochemically in buccal mucosa carcinoma (n=74) and tongue carcinoma (n=61) patients. Expression of markers was compared between buccal mucosa and tongue carcinoma and assessed for their prognostic value in site-specific manner. On comparison, only cyclin D1 showed significant difference in expression with higher accumulation in tongue tumors (r=+0.177, p=0.039). Moreover, univariate survival analysis showed that in buccal mucosa patients, loss of p16 and overexpression of H-ras were significant prognosticators for relapse-free survival (RFS) and overall survival (OS), respectively. However, in Cox multivariate analysis, they lost their significance after adjusting for significant clinicopathological parameters. On the other hand, in tongue cancer patients, Cox multivariate analysis showed that for RFS, Stat3 and c-myc, and for OS, Stat3, Bcl-2 and p53 were significant prognosticators after adjusting for significant confounding factors. Our findings indicated that buccal mucosa and tongue carcinoma exhibit different biological behavior which is reflected in prognosis. Therefore, this approach might be helpful to precisely identify patients for more effectively tailored treatment strategy.

Molecular alterations in oral carcinogenesis: significant risk predictors in malignant transformation and tumor progression.

In this study an attempt was made to establish the significance of a battery of molecular alterations and thereby identify risk predictors in oral carcinogenesis. For this purpose, EGFR, Stat3, H-ras, c-myc, p53, cyclin D1, p16, Rb, Ki-67 and Bcl-2 were localized immunohistochemically in normal mucosa (n=12), hyperplasia (n=35), dysplasia (n=25), early stage carcinoma (n=65) and advanced stage carcinoma (n=70). Deregulation occurred at an early stage and the number of alterations increased with disease progression. Using multivariate logistic regression analysis, the significant risk predictor for hyperplasia from normal mucosa was Ki-67 (OR=5.75, p=0.021); the significant risk predictors for dysplasia from hyperplasia were EGFR (OR=12.96, p=0.002), Stat3 (OR=17.16, p=0.0001), p16 (OR=5.50, p=0.039) and c-myc (OR=5.99, p=0.052); the significant risk predictors for early stage carcinoma from dysplasia were p53 (OR=6.63, p=0.0001) and Rb (OR=3.81, p=0.056); and the significant risk predictors for further progression were EGFR (OR=5.50, p=0.0001), Stat3 (OR=4.49, p=0.0001), H-ras (OR=4.05, p=0.001) and c-myc (OR=2.99, p=0.015). Cyclin D1 holds a key position linking upstream signaling pathways to cell cycle regulation. Gene products of the mitogenic signaling pathway play an equally significant role as cell cycle regulatory proteins in the hyperplasia-dysplasia-early-advanced-carcinoma sequence and together may provide a reference panel of markers for use in defining premalignant lesions and predicting the risk of malignant transformation and tumor progression.

Stat3 expression in oral squamous cell carcinoma: association with clinicopathological parameters and survival.

The present study sought to explore the occurrence of signal transducer and activator of transcription 3 (Stat3) in patients with oral squamous cell carcinoma (n=135) and its potential relationship with clinicopathological parameters and survival. Stat3 expression was studied by immunohistochemistry. Cytoplasmic or nuclear localization of Stat3 was observed in 62% of patients, whereas only nuclear Stat3 expression was found in 44%. Stat3 positivity in early-stage patients was 45% compared to 79% in advanced-stage patients. However, early-stage Stat3-positive patients showed a gradual increase in staining intensity, with intense staining seen in 52% of the tumors compared to 18% in Stat3-positive advanced-stage patients, where a gradual decrease in intensity expression was observed (p=0.001). Stat3 showed a significant positive correlation with disease stage (p=0.001), nodal status (p=0.033) and tumor size (p=0.001). Multivariate survival analysis using the Cox proportional hazard regression model showed that nuclear Stat3 was a significant independent prognosticator for both relapse-free survival (p=0.014) and overall survival (p=0.042) in early-stage patients. Our results indicated that Stat3 activation is an early event in oral squamous cell carcinoma and represents a potential risk factor for poor prognosis in early-stage patients.

p53 expression in leukoplakia and carcinoma of the tongue.

There is growing interest in assessing multistep carcinogenesis and predicting its course using different molecular markers. TP53 is a tumor suppressor gene and appears to be one of the molecular targets of tobacco-related carcinogens in oral cancer. The present study evaluated the role of p53 expression in patients with leukoplakia and carcinoma of the tongue. p53 expression was studied by immunohistochemistry. All patients with leukoplakia of the tongue were male tobacco users. Nuclear staining of p53 was observed in 79% of those patients. Fifty percent, 25% and 4% of the patients expressed 1+, 2+ and 3+ nuclear staining, respectively. When leukoplakia patients were graded according to histopathology, 67% had hyperplasia and 33% had dysplasia. Nuclear p53 accumulation was 88% in hyperplasia and 62% in dysplasia. In patients with tongue cancer, nuclear accumulation of p53 was seen in only 19% of the tumors, with a staining intensity of 1+ in 13%, 2+ in 2% and 3+ in 4% of the tumors. The prevalence of nuclear p53 positivity (79%) was significantly higher in patients with leukoplakia than in patients with tongue cancer (19%; chi2 = 34.32, r = -0.45, df = 1, p = 0.0001; odds ratio (OR) = 16.66, 95% CI, 5.25-52.86). Therefore, leukoplakia patients who show p53 expression have a higher risk of developing tongue cancer than those who do not show p53 expression. As the percentage of positivity of nuclear p53 was very low, none of the clinicopathological parameters or disease status showed any significant association with it. The interesting finding is that none of the female cancer patients showed nuclear p53 expression. Therefore, p53 accumulation is believed to be an early event in neoplastic progression of the tongue.

Telomerase activity in breast cancer in Western India (Gujarat).

The purpose of this study was to examine the association of telomerase activity with clinical and histopathological prognostic variables in primary breast cancer (n=64). Telomerase activity in breast cancer was also compared with that in benign (n=10) and non-malignant tissues (n=8; post-lumpectomy tissues histopathologically defined as containing no residual tumor). The parameter was assessed using the Telomerase PCR ELISA kit. Values above OD 0.120 were considered positive. Estrogen and progesterone receptors (ER and PgR) were assayed by the dextran-coated charcoal method and levels >10 fmol/mg cytosol protein were taken as positive. Telomerase activity was detected in 20% and 50% of the patients with benign lesions and primary breast cancer, respectively, and in 50% of post-lumpectomy breast tissues histopathologically defined as containing no residual tumor. Telomerase activity was present in all stages of breast carcinoma and showed a significant inverse correlation with lymph node status (p=0.006), lymphatic invasion (p=0.035) and necrosis (p=0.033). Moreover, when stage II patients were grouped according to nodal involvement, a trend towards significance was observed (p=0.055). No correlation was observed with ER and PgR. The results of our study suggest that telomerase activity might be associated with the presence of cancer cells. Furthermore, telomerase activation may occur early in breast cancer and may be periodically downregulated during subsequent tumor progression.

Molecular markers are predictors of recurrence and survival in patients with Dukes B and Dukes C colorectal adenocarcinoma.

PURPOSE: The goal was to investigate the prognostic value of various molecular markers like CEA, Cyclin D1, Bcl-2, c-Myc, p53, p21ras, Ki-67, CD44, Factor VIII-related antigen, cytokeratin-19, adenoma antigen, and prolactin in patients with Dukes B and Dukes C colorectal adenocarcinoma. METHODS: These molecular markers were localized immunohistochemically in nonmalignant (n = 36) and malignant (n = 98) diseases of the colorectum. Data were analyzed statistically using the SPSS software program. The patients with colorectal cancer were followed for a period of five years or their death within that period. RESULTS: The expression of carcinoembryonic antigen, Cyclin D1, Bcl-2, CD44, cytokeratin-19 and prolactin was significantly higher in malignant diseases (P < 0.05), whereas, p21ras was found to be significantly higher in nonmalignant diseases (P = 0.002) as compared with their respective counterparts. Besides Dukes stage, multivariate analysis indicated a significantly reduced relapse-free survival in patients expressing CD44 and cytokeratin-19 (P < 0.005). Similarly, besides Dukes stage, multivariate analysis indicated a significantly poor overall survival in patients expressing CD44, cytokeratin-19 and prolactin (P < 0.01). In patients with Dukes B disease, only cytokeratin-19 and CD44 expression attained statistical significance (P < 0.05), whereas in patients with Dukes C disease, CD44, p21ras- and c-Myc expression attained statistical significance (P < 0.018). Also, a multivariate analysis in relation to treatment given was performed using CD44 and cytokeratin-19. CONCLUSION: Besides Dukes stage, multivariate analysis of all the studied molecular markers showed that patients expressing CD44 and cytokeratin-19 had a significantly reduced relapse-free and poor overall survival. Moreover, patients expressing both these markers (CD44 and cytokeratin-19) had the lowest significant relative risk for developing recurrence than patients with both markers negative when treated with surgery followed by adjuvant chemotherapy as compared with patients treated with surgery alone. Thus, in patients with colorectal cancer, immunohistochemical localization of CD44 and cytokeratin-19 may be included as a part of routine pathologic evaluation along with conventional prognostic factors.

Ectopic production of prolactin by colorectal adenocarcinoma.

PURPOSE: This study assessed the role of prolactin in patients with Dukes B and C colorectal carcinoma. METHODS: Circulating prolactin and carcinoembryonic antigen were assayed using immunoradiometric assay and radioimmunoassay kits, respectively, in preoperative blood (n = 98) and tumor-draining venous blood (n = 34) samples of colorectal carcinoma patients. Immunohistochemical localization of prolactin (n = 98), carcinoembryonic antigen (n = 98), and prolactin receptors (n = 56) was performed. The expression of prolactin messenger ribonucleic acid (n = 50) and prolactin receptor messenger ribonucleic acid (n = 50) was studied by reverse transcriptase polymerase chain reaction. Further, prolactin amplimer was sequenced. RESULTS: Preoperative prolactin and carcinoembryonic antigen levels were significantly higher in patients with colorectal carcinoma than in controls (prolactin, P = 0.001; carcinoembryonic antigen, P = 0.0001). Univariate survival analysis showed that Dukes stage, histologic grade, and circulating prolactin were significant prognostic factors for determining overall survival (Dukes stage, P = 0.00001; histologic grade, P = 0.005; prolactin, P = 0.001). In multivariate analysis, besides Dukes stage, circulating prolactin emerged as the most significant independent prognostic factor influencing overall survival. Preoperative prolactin levels showed excellent significant correlation with response to therapy and progression of disease. A significant tenfold higher mean concentration of prolactin was observed in tumor-draining venous blood than in peripheral blood (P = 0.0001). Diffuse cytoplasmic staining for prolactin was seen in 51 percent (50/98) of the colorectal carcinomas. Prolactin messenger ribonucleic acid expression was seen in 88 percent (44/50) of the colorectal carcinomas. Sequence analysis of the 234-bp prolactin amplimer revealed that the sequence was homologous to exon 5 of pituitary prolactin messenger ribonucleic acid. CONCLUSION: These multiple approaches confirmed that prolactin is produced by colorectal carcinoma cells. Looking at its prognostic value and correlation with disease activity, it may provide new insights into treatment for patients with colorectal carcinoma.

Prolactin as a local growth promoter in patients with breast cancer: GCRI experience.

AIMS: The aim of this study was to evaluate the prognostic value of pre-operative prolactin (PRL) in conjunction with established prognosticators, and the risk of disease relapse in patients with early and advanced breast cancer. To confirm the hypothesis that PRL is produced by breast tumours molecular analysis of PRL, using immunohistochemistry, mRNA by RT-PCR and direct sequencing, was performed. Furthermore, presence of prolactin receptors (PRLR) was evaluated by immunohistochemical localization in these patients. METHODS: In 111 breast cancer patients, pre-operative PRL was determined by an immunoradiometric assay (IRMA) method. Immunohistochemical localization of PRL (IHL-PRL) and PRLR was performed on formalin-fixed, paraffin-embedded tissue sections. Expression of PRL mRNA was carried out by reverse transcriptase polymerase chain reaction (RT-PCR). RT-PCR PRL amplimer was sequenced and compared with human pituitary PRL amplimer. RESULTS: Fifty-eight per cent (64/111) of the patients had hyperprolactinaemia (PRL520.0 ng/ml). With increasing tumour size, a higher incidence of hyperprolactinaemia was noted which was statistically significant (r=0.34, P=0.0001). In stage III patients, and in node positive patients, the incidence of hyperprolactinaemia was significantly higher compared to their respective counterparts (stage II vs stage III, r=0.37, P=0.00006; node negative vs node positive, r=0.30, P=0.001). Hyperprolactinaemic patients had a significantly higher risk of developing recurrent/metastatic disease and a higher mortality risk as compared to patients with PRL <20.0 ng/ml. The multivariate survival analysis indicated that apart from disease stage, prognosis of patients with pre-operative hyperprolactinaemia was poorer than that of patients with PRL <20.0 ng/ml. Seventy-eight per cent (87/111) of the tumours showed positive immunoreactivity with PRL antibody indicating that PRL, or a similar molecule, is produced ectopically by breast tumours. PRL mRNA expression using RT-PCR confirmed the de novo synthesis of PRL. PRL mRNA expression was seen in 52% (33/63) of tumours. Sequence analysis of the 234 bp PRL amplimer revealed that the sequence was homologous to the sequence of exon 5 of human pituitary PRL mRNA. Furthermore, PRLR were present in 80% of tumours detected by immunohistochemical localization. A significant positive correlation was noted between IHL-PRL and PRLR (r=0.26, P=0.006). CONCLUSIONS: This multifaceted study of PRL suggests that breast cancer cells produce PRL and that this ectopically produced PRL may act as a major local growth promoter via autocrine and paracrine mechanisms. It may provide new insights into endocrine treatment of breast cancer.

Prognostic significance of immunohistochemically localized biomarkers in stage II and stage III breast cancer: a multivariate analysis.

BACKGROUND: The aim was to investigate the expression of a panel of biomarkers such as prolactin (PRL), p53, Bcl-2, c-erb B2, Ki-67, CD44, and factor VIII-related antigen (FVIII-RA) in primary tumors of stage II and stage III breast cancer and its correlation with disease prognostication. METHODS: The streptavidin-biotin peroxidase complex technique was used for the detection of these antigens. Cytoplasmic staining pattern was observed for PRL, Bcl-2, and Ki-67. Staining pattern for p53 was nuclear. Membranous and/or cytoplasmic staining was noted for c-erb B2 and CD44. Microvessel staining was noted for FVIII-RA. RESULTS: Of the 93 primary breast tumors analyzed, positivity for PRL was noted in 82%, for p53 in 56%, for Bcl-2 in 73%, for c-erb B2 in 68%, and for Ki-67 and CD44 in 78% each. The microvessel count (MVC) for FVIII-RA ranged from 0.0 to 29.0, with a median of 6.0, which was used as a cutoff. MVC > or = 6.0 was noted in 51% of breast tumors. With increasing tumor size, the higher frequency of positivity of MVC > or = 6.0 (P = .0001), CD44 (P = .001), PRL (P = .002), and c-erb B2 (P = .008), and higher frequency of Bcl-2 negativity (P = .033), was noted. In stage III patients, a higher positivity of the following biomarkers was noted, compared with stage II patients: MVC > or = 6.0 (P = .0004), PRL (P = .0002), c-erb B2 (P = .001), and CD44 (P = .005). Further, Bcl-2 positivity was significantly lower in patients with stage III disease compared with those with stage II disease (P = .024). In patients with nodal involvement, the frequency of c-erb B2 (P = .006), MVC > or = 6.0 (P = .011), and PRL (P = .032) was higher than in those without nodal involvement. Moreover, in these patients, with the increase in the number of involved lymph nodes, there was a significant increase in frequency of CD44+ (P = .0004) and PRL+ (P = .013) tumors. Abnormal expression of one biomarker was seen in 7% of tumors, of two biomarkers in 4%, of three in 15%, of four in 19%, of five in 28%, of six in 20%, and of all seven biomarkers in 7% of tumors. The frequency of an increasing number of biomarkers coexpressed was higher in stage III patients compared with stage II patients (P = .00003). In the total number of patients (n = 93), tumors with Bcl-2 negativity (P = .00001), MVC > or = 6.0 (P = .001), PRL positivity (P = .02), and CD44 positivity (P = .034) had a significantly poorer overall survival (OS) compared with their respective counterparts. In stage II patients (n = 40), only p53 expression was significantly associated with reduced relapse-free survival (P = .009) and OS (P = .040). In multivariate analysis, p53 expression was an independent prognostic factor that influenced relapse-free survival (P = .034) of stage II breast cancer patients. However, it failed to attain statistical significance for OS. In stage III patients (n = 53), tumors with Bcl-2 negativity (P = .0005) and MVC > or = 6.0 (P = .039) had a significantly poorer OS compared with their respective counterparts. In multivariate analysis of stage III patients, Bcl-2 was the only independent prognostic factor (P = .001) for predicting OS. There was a significant association between coexpression of the biomarkers and OS (P = .001). The OS rates decreased with the increase in number of abnormally expressed biomarkers. CONCLUSIONS: p53 expression in primary tumors was an independent prognostic factor that influenced relapse-free survival in patients with stage II disease. In stage III patients, lack of Bcl-2 expression was independently associated with a poor prognosis and, thus, may be an indicator of aggressive phenotype.

Overexpression of CD44: a useful independent predictor of prognosis in patients with colorectal carcinomas.

BACKGROUND: The goal was to investigate the potential correlation between overexpression of CD44, high microvessel count (MVC), and p21ras with length of relapse-free and overall survival in patients with colorectal adenocarcinomas. METHODS: CD44, factor VIII-related antigen (FVIII-RA), and p21ras were localized immunohistochemically in patients with colorectal adenomatous polyps (n = 8) and adenocarcinomas (n = 98). The correlation between the expression of CD44, MVC in the areas with highest density, and p21ras with relapse-free and overall survival time was investigated. Data were analyzed statistically using univariate and multivariate systems. RESULTS: In patients with adenomatous polyps, the positivity of CD44, FVIII-RA, and p21ras was 75%, 62%, and 88%, respectively. In patients with colorectal carcinomas the positivity of CD44 was 55%, and for p21ras it was 52%. The median of FVIII-RA was 4 MVC (range, 0.0 to 32.33). MVC was greater than 4 in 53% of the patients with colorectal carcinomas. In univariate analysis, a significantly longer relapse-free time (CD44: P = .0004; FVIII-RA: P = .0006) and overall survival time (CD44: P = .0001; FVIII-RA: P = .001) were observed for patients with CD44-negative tumors and MVC below 4 as compared to those with CD44-positive tumors and MVC greater than 4. Similar observations were noted in patients with Dukes B and C disease and the rectum as the site of tumor. In multivariate analysis, only CD44 correlated significantly with both relapse-free (P = .0003) and overall survival (P = .00001). CONCLUSION: Univariate analysis showed CD44 and MVC to be independent predictors of prognosis in colorectal carcinomas. Multivariate analysis showed that CD44 positivity was the most important indicator of an unfavorable prognosis for relapse-free and overall survival in patients with colorectal cancer. Thus, it can be deduced that whether CD44 is positive or negative in patients with colorectal cancer may have prognostic importance and in the future may be used as a factor in the pathologic evaluation of tumor specimens. This hypothesis needs to be tested prospectively in a larger number of patients.

Coexpression of Bcl-2, c-Myc, and p53 oncoproteins as prognostic discriminants in patients with colorectal carcinoma.

PURPOSE: This study was undertaken to evaluate the clinical use of Bcl-2, c-Myc, and p53 oncoproteins, either singly or in combination, as prognostic discriminants relative to recurrence and overall survival in patients with Dukes B or C colorectal carcinoma. METHODS: Analyses were made on archival pathology tissues of 48 patients with colorectal cancer. The oncoproteins were localized using commercially available monoclonal antibodies: clone 124 for Bcl-2, 9E11 for c-Myc, and DO-7 for p53. The avidin-biotin peroxidase complex method was used. Patients were followed up for a period of 2 to 36 months. RESULTS: Expression of Bcl-2 and c-Myc was cytoplasmic, whereas nuclear p53 immunoreactivity was localized in the tumor cells. Sixty percent (29/48), 65 percent (31/48), and 37 percent (18/48) of the tumors showed overexpression of Bcl-2, c-Myc, and p53 oncoproteins, respectively. Fifty-four percent (18/33) and 100 percent (9/9) of moderately and poorly differentiated tumors, respectively, were positive for Bcl-2 (P < 0.01). No such correlation was noted for c-Myc and p53 oncoproteins. Univariate analysis showed that patients with Bcl-2 and c-Myc overexpression were associated with poorer overall survival than patients with Bcl-2-negative (P < 0.0124) and c-Myc-negative (P < 0.036) tumors. In addition, when patients were subgrouped according to Dukes stage, a statistically significant poorer overall survival was observed in Dukes C patients with Bcl-2-positive tumors (P < 0.017). Furthermore, multivariate analysis revealed that coexpression of three oncoproteins was predictive of a worse prognosis than for those individuals expressing none of the oncoproteins (P < 0.031) and only one positive oncoprotein (P < 0.014). CONCLUSION: These findings suggest that oncoprotein coexpression possesses significant prognostic and potential therapeutic value; incorporation of molecular markers into future prospective randomized trials is advisable.