Protective immunity of single and multi-antigen DNA vaccines against schistosomiasis.

We evaluated the usefulness of the combination of three plasmids encoding tegumental (pECL and pSM14) and muscular (pIRV5) antigens of the Schistosoma mansoni on improving the protective immunity over the use of a single antigen as DNA vaccines. Female BALB/c mice were inoculated twice with 25 micro g DNA plasmid within two weeks interval. The challenge was performed with 80 cercarias of a regional isolate of S. mansoni (SLM) one week after the last immunization. Six weeks after challenge, all mice were perfused for worm load determination. The following groups were analyzed: saline; empty vector; monovalent formulations of pECL; pSM14 and pIRV5 and also double combinations of pECL/pIRV5 and pIRV5/pSM14 and a triple combination of pECL/pIRV5/pSM14. The protection was expressed as a percentage of worm loads in each group compared with the saline group. The results obtained were 41% (p < 0.05); 52% (p < 0.05); 51% (p < 0.05); 48% (p < 0.05); 55% (p < 0.05); 45% (p < 0.05); 65% (p < 0.05) for each group respectively.

Protective immunity of single and multi-antigen DNA vaccines against schistosomiasis

We evaluated the usefulness of the combination of three plasmids encoding tegumental (pECL and pSM14) and muscular (pIRV5) antigens of the Schistosoma mansoni on improving the protective immunity over the use of a single antigen as DNA vaccines. Female BALB/c mice were inoculated twice with 25 æg DNA plasmid within two weeks interval. The challenge was performed with 80 cercarias of a regional isolate of S. mansoni (SLM) one week after the last immunization. Six weeks after challenge, all mice were perfused for worm load determination. The following groups were analyzed: saline; empty vector; monovalent formulations of pECL; pSM14 and pIRV5 and also double combinations of pECL/pIRV5 and pIRV5/pSM14 and a triple combination of pECL/pIRV5/pSM14. The protection was expressed as a percentage of worm loads in each group compared with the saline group. The results obtained were 41 percent (p < 0.05); 52 percent (p < 0.05); 51 percent (p < 0.05); 48 percent (p < 0.05); 55 percent (p < 0.05); 45 percent (p < 0.05); 65 percent (p < 0.05) for each group respectively

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.

Prolactin as a local growth promoter in patients with locally advanced tongue cancer: GCRI experience.

BACKGROUND: The purpose of this study was to assess the role of prolactin (PRL) in men with locally advanced tongue cancer. METHODS: Circulating PRL was assayed immunoradiometrically in pretherapeutic and sequential blood samples of 99 patients with locally advanced tongue cancer. Patients were followed for 3 years or until their death within a stipulated time. Immunohistochemical localization of PRL was performed on formalin-fixed paraffin-embedded tissue sections. Tumoral prolactin receptors (PRLR) were estimated by ligand binding assay; the expression of PRL mRNA and PRLR mRNA were carried out by reverse transcriptase polymerase chain reaction (RT-PCR). Furthermore, PRL amplimer was sequenced and compared with human pituitary PRL amplimer. RESULTS: Pretherapeutic PRL levels were significantly higher in patients with locally advanced tongue cancer compared with controls (p =.01). Thirty-four percent (34 of 99) of the patients had hyperprolactinemia (PRL >/=15.0 ng/mL). Univariate survival analysis showed that patients with pretherapeutic hyperprolactinemia had a significantly shorter overall survival than patients with pretherapeutic PRL <15.0 ng/mL serum (p =.0009). In multivariate analysis, PRL emerged as the most significant independent prognostic factor influencing overall survival. Furthermore, changes in serial PRL levels showed excellent correlation with response to therapy and progression of disease. Forty-four percent (24 of 54) of the tumors showed positive immunoreactivity with PRL antibody, indicating that PRL or a molecule similar to it is produced by tongue tumors. PRL mRNA expression was seen in 85% (43 of 50) of the tumors and confirmed the de novo synthesis of PRL. Sequence analysis of the 234 bp PRL amplimer revealed that the sequence was homologous to exon 5 of pituitary PRL mRNA. The action of PRL is mediated by PRLR, and it was observed that the PRLR positivity by ligand binding assay was 33%. The expression of PRLR mRNA by RT-PCR showed two forms of PRLR mRNA (ie, intermediate form [500-600 bp] seen in 82% (41 of 50 ) of the tumors and the long form [800-900 bp] seen in 36% (18 of 50) of the tumors. In 82% (41 of 50) of the tumors, either the intermediate or long form was seen. CONCLUSIONS: This multifaceted study of PRL suggests that tongue cancer cells produce PRL, and this ectopically produced PRL might be acting as a major local growth promoter by means of autocrine and paracrine mechanisms. Looking at its prognostic value and correlation with disease activity, it may provide new insights into treatment of tongue cancer.

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.

Clinical significance of p53, nm23, and bcl-2 in T3-4N1M0 oesophageal carcinoma: an immunohistochemical approach.

BACKGROUND AND OBJECTIVE: The purpose of this retrospective study was to test whether the expression of p53, nm23, and bcl-2 in T3-4N1M0 oesophageal carcinoma is associated with patient survival. METHODS: Immunohistochemical localisation of p53, nm23, and bcl-2 was performed on formalin-fixed, paraffin-embedded tissue sections (N = 46). The observed range of follow-up period was 0.2-24.0 months with a median of 11.0 months. A total of 85% (39/46) of the patients died within 24.0 months, which could be due to advanced disease at presentation. The immunohistochemical signal was expressed as the proportion of positive cells. The immunostaining for p53 was nuclear, whereas that for nm23 and bcl-2 was cytoplasmic in the neoplastic cells. RESULTS: p53 was expressed in 70% (32/46) of cases; nm23 in 29% (13/45), and bcl-2 in 67% (29/43) of tumours. The univariate analysis showed that the expression of two markers, i.e., expression of p53 and absence of nm23 were independently associated with unfavourable overall survival time. Despite a small number of patients treated with adjuvant therapy, we observed that tumours positive for p53 had an unfavourable prognosis when compared with tumours negative for p53. CONCLUSIONS: Our preliminary findings suggest: expression of p53 and nm23 negativity may be related to an unfavourable prognosis in patients with advanced oesophageal carcinoma.

Node negative breast carcinoma: hyperprolactinemia and/or overexpression of p53 as an independent predictor of poor prognosis compared to newer and established prognosticators.

The purpose of this study was to investigate a prognostic indicator that can differentiate node negative breast cancer patients (N = 39, T2N0M0) with high risk and low risk for the development of recurrence or metastases. Preoperative plasma prolactin (PRL) was estimated by radioimmunoassay. The expression of PRL, p53, nm23, and c-erbB2 was investigated by immunohistochemical (IHC) localization; cathepsin D (CD, Enzyme Linked Sorbant Assay) and estrogen- and progesterone-receptors (ER and PR, Dextran coated charcoal method) were estimated in the tumor cytosols. The follow-up period was 2-6 years. Statistical comparisons were made between each marker for relapse-free survival (RFS) and overall survival (OS). Of the 39 patients, 18 had hyperprolactinemia (PRL > 20.0 ng/ml plasma), whereas overexpression of p53 was observed in 55% (17/31) tumors. These were independently and in combination associated with a reduced RFS and OS. The rest of the investigated markers did not show promising results. Hyperprolactinemia and/or overexpression of p53 were associated with aggressiveness of the tumor, early disease relapse or metastases, and poor OS in patients with node negative breast cancer. These two markers may enhance our ability to identify node negative breast cancer patients with aggressive tumors, for whom the use of adjuvant chemo and/or endocrine therapy is unequivocally justified.

Interrelationship of prolactin and its receptor in carcinoma of colon and rectum: a preliminary report.

The prolactin receptors (PRLR) were correlated with circulating prolactin and various clinicopathologic parameters to investigate its prognostic value in patients with colorectal cancer. The prolactin (by radioimmunoassay) and its receptors (by radioligand method) were estimated in a total of 71 male patients with colorectal cancer enrolled at the Gujarat Cancer and Research Institute, Ahmedabad. The patients were followed for a period of 3 years. We have observed that 51% colorectal tumors were PRLR+. Significant correlation was not observed between presence/absence of PRLR and clinicopathologic variables. Dukes' D patients were lost to follow-up after 2-3 months; therefore, the results of prognostic significance were analysed only in patients with Dukes' A, B, and C (N = 64). Statistically significant difference in overall survival was not observed when the patients were subgrouped according to the presence/absence of PRLR and according to the cutoff level (i.e., 2%). PRLR+ hyperprolactinemic (Prolactin > 20.0 ng/ml plasma) patients had better overall survival than that of patients with PRLR- hyperprolactinemia, although the difference was statistically nonsignificant. However, PRLR- hyperprolactinemia patients had a more unfavourable prognosis than that of their counterparts. A similar trend was observed in patients with Dukes' B and C disease. Our preliminary study suggests an unequivocal finding, that PRLR- with concomitant hyperprolactinemia probably characterises a subgroup of patients with aggressive colorectal cancer.

Levels of circulating peptide and steroid hormones in men with lung cancer.

Levels of circulating peptide (FSH, LH, prolactin, ACTH, calcitonin, gastrin and insulin-like growth factor-1 [IGF-1]) and steroid (estradiol, progesterone, DHEA-S and testosterone) hormones were estimated by radioimmunoassay (RIA) and immunoradiometric assay (IRMA) in male patients with lung cancer (n = 37) pre-therapeutically and compared with 25 age matched healthy controls. In this retrospective study, FSH, LH, prolactin, ACTH, calcitonin, gastrin and IGF-1 were significantly higher with concomitant lower levels of DHEA-S and testosterone, while the difference was statistically non-significant for estradiol and progesterone in patients with lung cancer when compared with controls. Early stage patients (Stage II) exhibited higher levels of gastrin as compared to advanced stage patients (Stages III and IV). It is suggested that hormonal imbalance might play an important role in the development and progression in male patients with lung cancer.