Higher importance of interleukin 6 than classic tumor markers (carcinoembryonic antigen and squamous cell cancer antigen) in the diagnosis of esophageal cancer patients.

It has been suggested that interleukin 6 (IL-6) plays a potential role in the growth and progression of tumors, including esophageal cancer (EC). The aim of the study was to compare clinical significance of serum IL-6 with classic tumor markers - carcinoembryonic antigen (CEA) and squamous cell cancer antigen (SCC-Ag) - in EC patients in relation to its histological types - squamous cell carcinoma of esophagus (ESCC) and adenocarcinoma (AD) of esophagus. The study included 53 EC patients and 90 healthy subjects. Serum IL-6 and CEA levels were determined using immunoenzyme assays, while SCC-Ag - chemiluminescent assay. The diagnostic criteria and prognostic values for markers were defined. The levels of all proteins tested in EC, ESCC, and AD were higher than in healthy subjects. The percentage of elevated results was substantially higher for IL-6 (86%) than for CEA (30%) and SCC-Ag (24%) in EC, similarly as in ESCC (87%, 23%, and 33%) and AD (87%, 39%, and 13%, respectively) patients. Concentrations of IL-6 depended on distant metastases and patients' survival in EC and were significantly higher in ESCC patients with more advanced tumor stage and nodal metastases. The IL-6 area under receiver operating characteristic curve (0.92) was larger than for CEA (0.84) and SCC-Ag (0.62) in EC, likewise in ESCC (0.92, 0.87, 0.77) and AD (0.91, 0.79, 0.57, respectively). Our findings indicate better usefulness of IL-6 than classic tumor markers in the diagnosis of EC, especially in patients with ESCC.

Vascular endothelial growth factor C and D expression correlates with lymph node metastasis and poor prognosis in patients with resected esophageal cancer.

Vascular endothelial growth factors C (VEGF-C) and D (VEGF-D) are important lymphangiogenic factors in human cancers. We studied the expression of VEGF-C and VEGF-D using immunohistochemistry in 73 resected esophageal cancer specimens, and correlated the results with patient clinicopathologic features and survival. High expression of VEGF-C was identified in 40 (54.7%) patients, and it correlated positively with histological grade (p=0.038), tumor stage (p=0.01), depth of tumor invasion (p=0.036) and lymph node metastasis (p=0.001). In 48 of 73 (65.7%) tumors, the VEGF-D protein was also expressed at high levels. VEGF-D immunoreactivity significantly correlated with tumor location (p=0.027), size of tumor (p=0.015), histological grade (p=0.02), depth of invasion (p=0.001) and lymph node metastasis (p=0.018). In logistic multivariate analysis, high expression of VEGF-C (OR 1.941, 95% CI 1.263-7.289, p=0.024) was associated with lymph node metastasis. Calculating the prognostic relevance revealed that both VEGF-C and VEGF-D correlated with decreased overall survival (p=0.01, p=0.003), disease free survival (p=0.02, p=0.006), and cancer-specific survival (p=0.03, p=0.005). In conclusion, our results suggest that high levels of both VEGF-C and VEGF-D proteins are associated with lymph node involvement, and that VEGF-C expression is an independent predictor of risk for lymph node metastasis in esophageal cancer. In locally advanced disease, overexpression of VEGF-C and VEGF-D may be useful in identifying patients who are more likely to have a poor prognosis even after curative resection.

Auditory thalamus responses to guinea-pig vocalizations: a comparison between rat and guinea-pig.

Although neuronal responses to species-specific vocalizations have long been described, very few between-species comparisons have been made. In a previous study, a differential representation of species-specific vocalizations was found in the auditory cortex (ACx): marmoset ACx neurons responded more, and more selectively, to marmoset calls than did cat ACx neurons [Wang, X., Kadia, S.C., 2001. Differential representation of species-specific primate vocalizations in the auditory cortices of marmoset and cat. J. Neurophysiol. 86, 2616-2620]. The present study analyzed responses of guinea-pig and rat auditory thalamus neurons to four well-defined guinea-pig vocalizations. Neurons of guinea-pigs (n = 96) and rats (n = 87) displayed similar response strength to guinea-pig vocalizations, and did not exhibit a preference for the natural over the time-reversed version of the calls in both species. This difference with the study by Wang and Kadia might suggest that, in mammals, the selectivity for the natural version of species-specific vocalizations is prominent only at the cortical level.

The preoperative study of mediastinal lymph nodes metastasis in lung cancer by endoscopic ultrasonography (EUS) and helical computed tomography (CT).

OBJECTIVE: Accurate staging of mediastinal lymph nodes in patients with lung cancer is fundamental for their treatment and prognosis. The aim of this study was to compare the value of EUS and CT staging in patients with non-small-cell lung cancer (NSCLC) with postsurgical stage. METHODS: Ninety two patients with NSCLC underwent EUS and CT for preoperative detection of metastases to the mediastinal lymph nodes. EUS examinations were done with the ultrasonic linear array scanning echoendoscope (FG 32 UA, Hitachi/Pentax), CT-Toshiba Exvision GX scanner, with 24-s spiral acquisition, pitch 1:1 (7 mm collimation, 4 mm reconstruction index), during i.v. administration of non-ionic iodinated contrast media. RESULTS: The frequency of mediastinal involvement was 22.7%. The regions most accessible by EUS evaluation were subaortic, subcarinal and paraoesophageal lymph nodes. On a per-patient basis, EUS and CT results were: sensitivity 70.0 and 60.0%, specificity 80.6 and 72.6%, accuracy 77.2 and 68.5%. On a per-sites basis, the sensitivity of EUS evaluation was 78.8%, specificity 89.9%, accuracy 87.7%, comparing with CT-63.6, 84.0, 79.9%, respectively. When the EUS and CT images were analysed in combination, the sensitivity increased to 86.4%. CONCLUSION: We believe that EUS and CT should be used together for preoperative non-invasive staging of mediastinal lymph nodes in patients with NSCLC.

Prognostic molecular markers in non-small cell lung cancer.

Although TNM stage is the most significant prognostic parameter in lung cancer, additional parameters are required for explaining variability of survival. Hence molecular alterations in lung cancer have been extensively studied. Most prominent among them are alterations in the p53-p21 pathway, controlling the G1/S transition. They are the most commonly observed aberrations in non-small cell lung cancer (NSCLC). The results of p53 mutations on an individual patient's changes for survival are rather controversial. In a recent study however, after analyzing p53 abnormalities both by direct sequencing and immunohistochemistry together with evaluation of bcl-2 protein expression, we have found that p53 alterations were significantly associated with poor overall survival. Recently, a more sensitive yeast functional assay for altered p53 protein has been developed, with about 70% positivity in NSCLC patients and a correlation with shortened survival. The clinical significance of p21WAF1, the protein encoded by the target gene of p53 transcription, is still controversial; however expression has been associated with favorable prognosis in squamous cell carcinoma type. The 'Rb pathway' involving two oncogenes (cyclins D and E) and two tumor suppressor genes (Rb and p16) represents another major source of molecular alterations in lung cancer. Loss of Rb does not seem to significantly influence prognosis, white loss of p16 has been show repeatedly to be a factor for poor survival. Hypermethylation of the promoter region has been proposed as an alternative mechanism for inactivation of the p16 gene. The relation between cyclin D and E expression and prognosis, still is matter of controversy. Ras mutations are reported especially in adenocarcinoma; considered alone they bear no clear relation with prognosis, in opposition when considering them together with other molecular alterations. As a conclusion, a variety of molecular markers have been implicated in the prognosis of NSCLC. However, conflicting results were reported in the literature. Thus further investigations will be required, especially the use of newer molecular assays and the development of appropriate markers or panels of molecular markers.

Prognostic value of serum p53 antibodies in patients with resected non-small cell lung cancer.

BACKGROUND: Serum antibodies against p53 protein (p53-Abs) have been detected in some cancer patients. The significance and use of p53-Abs as a marker of the clinical behavior of lung cancer is currently under investigation. PURPOSE: In this study, we measured the serum p53-Abs in 84 patients with operable non-small cell lung cancer (NSCLC) and evaluated potential association between the presence of these antibodies and prognosis. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to detect p53-Abs in serum. Survival and disease-free survival curves related to initial p53-Abs status were estimated using the Kaplan-Meier method. RESULTS: At the time of diagnosis 19 (22.6%) of 84 analyzed patients had positive result from the serum p53 antibodies (p53-Abs) test. No association was found between p53-Abs, histological types of tumors and clinical stage of disease. We found that patients with a positive result from the p53-Abs test had lower probability of overall and disease-free survival. The unfavorable effect was significant both in the univariate analysis, as well as in the multivariate analysis (after adjustment for sex, histopathological type of tumor. TNM stage). CONCLUSION: The results of the present study indicate that serum p53 antibodies may be an independent prognostic factor in NSCLC, especially in the squamous cell carcinoma (SqCC) patients and may be useful in identifying resected lung cancer patients at high risk for treatment failure.

Prognostic value of pretreatment CEA, SCC-Ag and CA 19-9 levels in sera of patients with non-small cell lung cancer.

The levels of carcinoembryonic antigen (CEA), squamous cell associated antigen (SCC-Ag) and carbohydrate antigenic determinant 19-9 (CA 19-9) were assessed in 70 patients with non-small cell lung cancer (NSCLC) and in 20 patients with non-malignant lung diseases. Increased levels of CEA and CA 19-9 were observed in 55.7 and 44.2%, respectively, mostly in patients with adenocarcinoma (adeno C; 69.5 and 56.5%). Increased levels of SCC-Ag were observed in 45.7%, first in patients with squamous cell carcinoma (68.6%). Serum CEA, CA 19-9 and SCC-Ag levels were correlated with the postoperative, pathological stage of disease. Positive CEA levels in patients with adeno C were present in 50% of stage 1, 66.6% of stage 2 and 88.8% of stage 3; positive CA 19-9 levels in patients with adeno C were present in 30% of stage 1, 66.6% of stage 2 and 80% of stage 3; positive SCC-Ag levels were present in patients with squamous LC in 50% of stage 1, 83.3% of stage 2 and 73.7% of stage 3. The study proved that preoperative CEA, SCC-Ag and CA 19-9 determination have been shown to be of prognostic value in patients with NSCLC. A high preoperative antigen value suggests a worse prognosis than a lower value.

Evaluation of CYFRA 21-1 as a new marker for non-small cell lung cancer.

The levels of the new tumour marker CYFRA 21-1 were assessed in 115 patients with non-small cell lung cancer (NSCLC) and in 45 patients with non-malignant lung disease. Increased levels of CYFRA 21-1 were observed in 47.8%, mostly in patients with squamous cell carcinoma (SCC; 69.1%). Serum CYFRA 21-1 levels were correlated with the stage of SCC type. Positive CYFRA 21-1 levels in patients with SCC were present in 40% of stage I, 61.1% of stage II, and 85.2% of stage III. In addition, SCC patients who presented mediastinal lymph nodes (N2) demonstrated higher serum CYFRA 21-1 levels, compared with patients without mediastinal lymph nodes metastases (N0 or N1). With regard to tumour size, significant difference was observed between T1, T2 and T3. The study also showed that the percentage of patients who survived 18 months with normal preoperative level of CYFRA 21-1 was higher compared with those patients with elevated preoperative levels of this marker, but the differences were not statistically significant.

p53 gene mutation and protein expression in operable non-small cell lung cancer in Poland.

We investigated the association of p53 abnormalities (gene mutations by DNA sequencing and protein over-expression by immunostaining) with clinical data and prognosis in 74 patients with resected non-small cell lung cancer (NSCLC). DNA analysis of exons 5-8 of the p53 gene showed 34 mutations in 74 resected primary NSCLC (45.9%). Immunohistochemical study of the p53 protein revealed that 41 of 74 (55.4%) samples had positive staining. We found strong agreement between the results of the p53 protein expression test (p53-PE) and the p53 gene mutation test (p53-M) (Cohen's kappa = 0.65, 95% CI 0.48-0.82). Joint distribution of the results (analysed using the bivariate Dale model) was mainly influenced by, histological type of tumour. A positive result for the p53-PE test significantly increased (estimated odds ratio 84.5; 95% CI 8.89-803.03) the odds of observing a positive result in the p53-M test. In the univariate analysis (log rank test), positive results in the p53-M test and the p53-PE test were significantly associated with overall survival (P < 0.001 and P = 0.005, respectively). In the multivariate analysis (Cox's proportional hazard model), a positive result for the p53-M test significantly increased relative risk for overall survival (RR 9.56; 95% CI 2.62-34.87; P < 0.001). When the result of the p53-M test was accounted for, a positive result for the p53-PE test did not offer any additional prognostic information due to the strong dependence of results of the tests. However, when the result of the p53-M test was removed from the model, a positive result for the p53-PE test became a significant unfavourable prognostic factor (P = 0.009). We conclude that p53 gene mutation and protein expression analyses are in a strong agreement. Joint distribution of the results depends mainly on histological type of tumour. When considered separately, both tests are unfavourable prognostic factors in NSCLC. When the result of the p53-M test is taken into account, the p53-PE test does not offer any additional prognostic information.

A correlative study of bronchial cytology, bronchoalveolar lavage (BAL) and serum tumor markers in the diagnosis of lung carcinoma.

In order to evaluate the diagnostic value of lavage CEA and SCC-Ag in combination with other parameters such as serum CEA and SCC-Ag, sputum and bronchial brushing cytology, we studied 30 patients with lung carcinoma and 12 with nonmalignant lung disease. All four tests combined, resulted in an increase in the predictive accuracy for diagnostic lung carcinoma to 87 per cent, a value which is much above the approximately 50 per cent level achieved by each test alone. Hence lavage CEA and SCC-Ag appears to be a useful accessory test in the diagnosis of lung carcinoma, particularly when combined with other diagnostic procedures currently in use.

Detection of p16 abnormalities in early-stage non-small cell lung cancer.

We investigated the clinical significance of p16 abnormalities (mutations by sequencing and hypermethylation by methylation-specific PCR) in 52 radically resected stage I-II non-small cell lung cancers (NSCLCs). P16 abnormalities were detected in 20 (38%) patents (point mutations in 4 (8%) and promoter hypermethylation in 16 (31%) cases. No correlation was found between p16 abnormalities and various clinicopathologic factors, including sex, histological type of tumor and TNM (I vs. II) stage. The multivariate analysis of survival was performed using the Cox proportional hazard model. When the types of 16 inactivation were analyzed, p16 hypermethylation rather than point mutation was associated with poor Prognosis. The presented results prompt the conclusion that hypermethylation of p16 is the major mechanism for p16 gene inactivation in early stage NSCLC and could be a useful molecular marker for the prognosis.

Detection of P53 abnormalities in non-small cell lung cancer by yeast functional assay.

We assessed the status of P53 in 32 surgically treated non-small cell lung cancers (NSCLC) by using yeast functional assay. For functional assay, total RNA extracted from fresh-frozen specimens was reverse transcribed and P53 cDNAs were PCR-amplified using Pfu DNA polymerase (Stratagene). The transcriptional competence of the P53 cDNA was then tested in a yeast reporter strain. 20 of the 32 (69%) NSCLC patients contained mutant P53 in the yeast functional assay with the higher frequency in squamous cell carcinoma (14/17, 82%) than in adenocarcinoma (5/10, 50%) and large cell carcinoma (3/5, 60%) (p<0.01, chi2 test). No significant difference was observed with respect to the TNM. Preliminary survival analysis showed that patients scoring positive for the yeast test had shorter disease-free survival (median = 10 months) than those that scored negative (median > 21 months). Our results suggest that yeast functional assay is not only an improved method to examine the status of P53, but might hopefully improve understanding of the role of mutant P53 in the clinical evaluation of NSCLC.

Vascular endothelial growth factor and basic fibroblast growth factor in patients with squamous cell oesophageal cancer.

It is suggested that vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) play an important role in tumor-induced angiogenesis. The purpose of this study was to estimate the correlation between VEGF and bFGF levels and tumor pathological status according to pTNM classification in patients with squamous cell oesophageal cancer. A group of 25 healthy controls and 32 consecutive patients with oesophageal cancer were included in this study. Serum VEGF and bFGF levels were determined by enzyme-linked immunosorbent assay (Quantikine R&D Systems). Serum VEGF and bFGF levels were significantly elevated in the patient groups (VEGF: 146.0 pg/ml, 79.0-386.3 pg/ml vs. 38.0 pg/ml, 6.5-135.1 pg/ml, p<0.005, and bFGF: 5.2 pg/ml, 1.2-10.6 pg/ml vs. 2.06 pg/ml, 0.07-4.0 pg/ml, p<0.02 Fisher test). The highest correlation between serum VEGF and bFGF levels were found in patients with advanced cancers, especially with: T4, N1, and M1 factors. The VEGF and bFGF levels were significantly higher in patients with pT4 (p<0.01). Patients with N1 lymph node invasion, compared with N0 factor, have higher levels of angiogenetic factors (p<0.04). Also in patients with advanced cancers with liver metastases the serum levels VEGF and bFGF were significantly higher (M1 vs. M0, VEGF p<0.001 and bFGF p<0.05). Consecutive monitoring of VEGF and bFGF serum levels may be a useful prognostic marker for patients with squamous cell oesophageal cancer.

Strong association between P53 protein accumulation, serum antibodies and gene mutation in non-small cell lung cancer.

DNA sequencing is the gold-standard method, but it is not feasible on a routine clinical basis. Immunohistochemistry is the most widely used method for assessing P53 alterations in human cancers. It can be performed during routine analysis, but some mutations may not lead to P53 protein accumulation, or P53 overexpression may be detected in the absence of mutations of the P53 gene. Recent studies have demonstrated the appearance of P53 antibodies in the serum of patients with lung cancer, probably due to the accumulation of mutant P53 protein in tumor cells. Using a logistic regression model applied to data for 102 non-small cell lung cancer (NSCLC) patients, we show a strong association between results of P53 mutation (P53-M) test, TNM stage and results of anti-P53 antibody in serum (P53-Abs) and P53 protein expression (P53-PE) tests. According to that model, we propose a procedure allowing for prediction of result of P53-M test. The percentage of correct predictions for independent data of 15 NSCLC patients was 80%. We conclude that in the absence of P53-M test result, a reasonably precise prediction of the test can be obtained using TNM stage and results of P53-Abs and P53-PE tests. The prediction can in turn be used to evaluate prognosis of NSCLC patients.

Circulating anti-p53 antibodies in esophageal cancer patients.

Circulating anti-p53 protein antibodies (p53-Abs) have been detected in some cancer patients. The aim of the study was to determine the presence of circulating anti-p53 protein antibodies and their clinical significance in patients with esophageal carcinoma. Serum specimens from 75 consecutive patients with squamous cell carcinomas and 10 healthy subjects were studied. Enzyme linked immunosorbent assay (ELISA--Pharma Cell) was used to detect p53-Abs. At the time of diagnosis 20 (26.6%) of 75 analyzed patients had positive result in the p53-Abs test, but not any of the healthy subjects. The positive rate was 25% (1/4) cases in stage I, 41% (10/24) cases in stage IIA, 0% (0/8) cases in stage IIB, 28% (8/28) cases in stage III and 9% (1/11) cases in stage IV. In respect of tumour differentiation, cases graded as G1, G2 and G3 were positive in 28.5% (4/14), 25.9% (7/27) and 26.4% (9/34), respectively. There was no correlation between presence of p53-Abs and stage, rumour differentiation, lymph nodes metastases, tumour size, patient age and sex. In conclusion, the results of the present study indicate that serum p53-Abs did not correlate with cliniocopathologic feature of esophageal carcinoma.

G1 phase of the cell cycle control and lung cancer: biological and clinical implications. Minireview.

Neoplastic diseases, including lung cancer are characterized by an uncoordinated cell growth. Cellular proliferation follows an orderly progression through the cell cycle, which is governed by protein complexes composed of cyclins and cyclin-dependent kinases. These complexes exert their regulatory function by phosphorylation of key proteins involved in cell cycle transitions. Abnormalities of cyclins and cyclin-dependent kinases have been reported and proposed to be oncogenic events. It appears that the molecular networking of these proteins and complexes exerts an impact on two fundamental cell cycle regulators; p53 and pRb. Interactions between these two nuclear proteins are being delineated, implying potential links between p53 and Rb in cell cycle control, apoptosis, and tumor progression. Furthermore, the detection of alterations in p53 and Rb pathways appears to be of clinical significance.

Evaluation of squamous cell carcinoma antigen (SCC-Ag) in the diagnosis and follow-up of patients with non-small cell lung carcinoma.

The usefulness of squamous cell carcinoma (SCC) antigen as a tumor marker was investigated in 72 patients with histologically verified non-small cell lung carcinoma (NSCLC). Increased level of SCC-Ag was observed in 41%, mostly in patients with squamous cell carcinoma (69%). Positive serum SCC-Ag was correlated with lymph node metastases and with the stage of disease. The positive rate of SCC-Ag observed in patients without and with nodal metastases was 52.9% and 84.2%, respectively. Positive SCC-Ag level was observed in 50% of Stage I, 71.4% of Stage II and 78.9% of Stage III patients with squamous cell carcinoma of the lung. The study proved that preoperative SCC-Ag determination in patients with squamous cell carcinoma of the lung and the course of levels of this marker during postoperative follow-up was of importance. A high preoperative and postoperative SCC-Ag value suggested a worse prognosis.

Carcinoembryonic antigen, neuron-specific enolase and creatine kinase-BB as tumor markers for carcinoma of the lung.

Carcinoembryonic antigen (CEA), neuron-specific enolase (NSE) and creatine kinase-BB (CK-BB) were estimated in blood serum of 75 patients with primary lung carcinoma and of 20 patients with nonmalignant lung diseases. CEA and NSE were determined by immunoenzymatic method using monoclonal antibodies (Abbott CEA-EIA and Roche NSE-EIA) and CK-BB was assayed using kits supplied by Boehringer-Mannheim (Monotest CK-NAC aktiviert). Enhanced levels of CEA were observed in 64% of patients with lung carcinoma, mainly with adenocarcinoma. Increased activities of NSE and CK-BB were obtained in 47% and 39% of patients, respectively, principally of those with small cell carcinoma. The CEA level was dependent on the stage of advanced NSCLC carcinoma and of NSE and CK-BB on the stage of advanced SCLC carcinoma. The complex analysis of the three markers has given 100% specificity of test.

Evaluation of carcinoembryonic antigen (CEA) and brain-type creatine kinase (CK-BB) in serum from patients with carcinoma of the lung.

The levels of carcinoembryonic antigen (CEA) and the activities of creatine kinase isoenzyme BB (CK-BB) were assessed in 84 patients with primary lung carcinoma and in 20 patients with nonmalignant lung diseases. The level of CEA was measured by the immunoenzymatic method using monoclonal antibodies (Abbott). The activity of CK-BB was assayed using a commercial kit (Boehringer Manheim, Monotest CK-NAC aktiviert). Increased levels of CEA were observed in 62% of patients, mostly in patients with nonsmall cell lung carcinoma (NSCLC), while enhanced activities of CK-BB were found in 39%, first of all in patients with small cell lung carcinoma (SCLC). A relationship was found between enhanced levels of CEA or CK-BB and the degree of carcinoma advance. The increased values of studied markers seem to indicate the limited possibility of surgical treatment and they are also important in prognosis after the resection of lung tissue.

Usefulness of a multiple biomarker assay in bronchoalveolar lavage (BAL) and serum for the diagnosis of small cell lung cancer.

To evaluate the diagnostic usefulness of simultaneous determinations of carcinoembryonic antigen (CEA), neuron specific enolase (NSE), chorionic gonadotrophin (HCG) and carbohydrate antigenic determinant 19-9 (CA 19-9), we studied 48 patients with small cell lung carcinoma (SCLC) and 15 with nonmalignant lung disease. The combination of CEA-BAL, NSE-BAL, and NSE-serum taken together with results of bronchoscopy (histologic and cytologic) showed the highest discriminating power between malignant (SCLC) and nonmalignant lung disease. The sensitivity of bronchoscopy alone was 35%. However, when bronchoscopy results were combined with 3 positive markers, the sensitivity increased to 71%, with at least 2 positive markers to 94%, and with at least 1 positive marker to 100%. When both bronchoscopy and all 3 markers were negative, the results showed a negative predictive value of 100%.