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The objective in much of the proteomics literature today is to establish the difference between healthy and disease states at the protein level using blood plasma. A critical component in this endeavor is to establish what is normal. The focus of the work reported here was to do this with oxidized proteins that might relate to oxidative stress and oxidative stress-related diseases. Oxidative stress is known to increase markedly in cancer, diabetes, heart disease, and neurodegenerative diseases. Since proteins are one of the targets of ROS, generated by oxidative stress, oxidized proteins are excellent biomarker candidates for these diseases. But first it is necessary to identify oxidized proteins that occur in the healthy state. Healthy rat plasma was used in this study as a source for the identification of naturally oxidized proteins. Freshly drawn blood was treated with biotin hydrazide to selectively derivatize carbonyl groups in oxidized proteins. Oxidized proteins thus biotinylated were separated from the other plasma proteins using avidin affinity chromatography. Affinity selected proteins were further fractionated on a C(8) RP column and fractions collected. The collected fractions were then tryptic digested and the peptides identified using a combination of LC/MS/MS and database searches. One hundred forty-six proteins were identified using 700 signature peptides from the tryptic digested chromatographic fractions. The most frequently encountered proteins in the samples were keratins. Brain and liver were among the organs contributing the most oxidized proteins to plasma followed by heart and kidney.

Pre-treatment serum squamous cell carcinoma antigen [SCC] levels are elevated in 28-88% of patients with squamous cell cervical cancer, and are related to tumour stage, tumour size, depth of stromal invasion, lymph-vascular space status, parametrial involvement and lymph node status. The clinical relevance of pre-treatment serum SCC assay is still debated. Some authors reported that it has no prognostic value, some others found that it is related to survival at univariate analysis, and some others detected that is an independent prognostic variable for survival. Serial SCC measurements reflect both the tumour response to treatment and the clinical outcome of patients. Increasing SCC levels can precede the clinical diagnosis of recurrent disease in 46-92% of the cases, with a mean lead time ranging from 2 to 8 months. According to some authors serum SCC assay during the follow-up does not improve the cure rate of patients who will ultimately develop a recurrence. However, it has been recently reported that the performance of a positron emission tomography [PET] in patients with asymptomatic SCC elevation can sometimes allow an earlier diagnosis of relapse with a survival benefit. SCC is a more sensitive serum tumour marker than CYFRA 21-1 for squamous cell cervical cancer in most series. Pre-treatment CA 125 levels are raised in 20-75% of patients with cervical adenocarcinoma, and reflect tumour stage, tumour size, histological grade, cervical stromal invasion, lymph-vascular space status and lymph node status. Elevated serum CA 125 has been also detected in patients with squamous cell cervical cancer, but with a positivity rate lower than that found in patients with cervical adenocarcinoma. Pre-treatment CA 125 levels appear to have a prognostic value, and rising serum CA 125 during follow-up may precede or be coincident with the clinical diagnosis of recurrent cervical adenocarcinoma. Serum levels of vascular endothelial growth factor [VEGF] are often elevated in patients with cervical cancer, and decrease significantly after successful treatment. However, the clinical relevance of serum VEGF assay is still investigational.

The patient was a 63-year-old man who consulted our hospital with complaints of a cough and breathing difficulties. His chest CT revealed a 25-mm mass in his right S1 hilar area with spiculation, disseminated nodule in right lung, and pericardial effusions. Also, bronchoscope and TBLB revealed squamous cell carcinoma. This patient was diagnosed as lung cancer (cT4N3M1, stage IV), and chemotherapy was initiated. The chemotherapy was given in the order of CBDCA (AUC3) +GEM (1,000 mg/m(2)), DOC (60 mg/m(2)), and VNR (25 mg/m(2)), and the tumor response was PD. S- 1 (120 mg/body/day, continuous administration for 2 weeks followed by 1 week of rest) was chosen as fourth-line treatment, and a breast CT detected tumor size reduction following completion of the first course. However, after completion of three courses, the breast CT found tumor-enlargement again. Then the chemotherapy was changed to amrubicin (35 mg/m(2)), but the treatment was discontinued due to skin rash. We once experienced a size reduction with S-1, so S-1 (100 mg/body/day, day 1-14) plus CPT-11 (60 mg/m(2), day 1, 7, 14) combination chemotherapy was conducted at 4-week intervals. After two courses were completed, tumor size reduction was observed by breast XP and CT. The response rate was 40.0%. Currently, seven courses were completed, and we will continue this treatment due to the tumor response of SD. The S-1 single treatment and S-1+CPT-11 combination chemotherapy showed efficacy for this difficult case of NSCLC with refractoriness to multiple cancer drug chemotherapy. This combination treatment should be investigated further for its therapeutic benefit.

Background: A high serum cytokeratin 19 fragment (CYFRA21-1) concentration in patients with various cancers is associated with poor prognosis. This study aimed to establish the clinical significance of preoperative serum CYFRA21-1 in patients with intrahepatic cholangiocarcinoma., Methods: CYFRA21-1, carcinoembryonic antigen (CEA), and carbohydrate antigen (CA) 19-9 concentrations were measured in sera from 71 patients with intrahepatic cholangiocarcinoma. The prognostic significance of serum CYFRA21-1 levels was assessed by univariate and multivariate analyses., Results: Analysis of the areas under the receiver operator characteristic (ROC) curves clearly showed better discrimination between intrahepatic cholangiocarcinoma and benign liver diseases for CYFRA 21-1 than for CEA or CA 19-9. Based on the maximization of the Youden's index, the optimal cut-off value was 2.7 ng ml(-1) for CYFRA 21-1 (sensitivity, 74.7%; specificity, 92.2%). The serum CYFRA21-1 concentration was related to tumor stage, since the CYFRA21-1 concentrations varied according to tumor size, vascular invasion, and number of tumors. The 3-year recurrence-free survival rates for patients with high and low concentrations of CYFRA21-1 were 25.0% and 76.2%, respectively (log-rank test, p < 0.01). The 3-year overall survival rates for patients with high and low concentrations of CYFRA21-1 were 39.4% and 63.6%, respectively (p = 0.01). On multivariate analysis, a high concentration of CYFRA21-1, nodal metastases, and a microscopic resection margin involvement were independent prognostic factors associated with both tumor recurrence and postoperative death., Conclusions: A high serum CYFRA21-1 concentration is associated with tumor progression and poor postoperative outcomes in patients with intrahepatic cholangiocarcinoma.

Prognostic implication of serum cytokeratin 19 fragments (CYFRA 21-1) was explored in 60 advanced NSCLC patients, whereas in 45 patients assessable for serological response a >or=35% CYFRA 21-1 decline after two chemotherapy cycles was strongly associated with non-progression (NP), defined as a sum of objective response (OR)+stable disease (P<0.0001) and survival (P=0.0002). Association of OR with survival was not significant. In multivariate survival analysis, >or=35% marker decline and radiological NP status were found as major determinants of prolonged survival with RR: 0.37 (P=0.01) and 0.63 (P=0.01), respectively. In advanced NSCLC patients, NP reflects therapeutic efficacy better than traditional OR. CYFRA 21-1 >or=35% decline seems to be a reliable surrogate marker of treatment efficacy in terms of survival.

Background: The role of tumour markers such as carbohydrate antigen (CA) 125, CA 15-3, CA 19-9 and CYFRA 21-1 (a fragment of cytokeratin 19) in differentiating malignant pleural effusions (MPE) from benign effusions is not yet clear., Methods: After a systematic review of English language studies, sensitivity, specificity and other measures of accuracy of pleural concentrations of CA 125, CA 15-3, CA 19-9 and CYFRA 21-1 or their combinations in the diagnosis of MPE were pooled using random effects models. Summary receiver operating characteristic curves were used to summarise overall test performance., Results: Twenty-nine studies met the inclusion criteria for the analysis. The summary estimates of the sensitivity and specificity of these tumour markers were as follows: CA 125, 0.48/0.85; CA 15-3, 0.51/0.96; CA 19-9, 0.25/0.96; CYFRA 21-1, 0.55/0.91 for diagnosing MPE. The estimated summary receiver operating characteristic curves showed that the performance of pleural CA 125 and CA 19-9 measurement in the diagnosis of MPE was limited, whereas that of CA 15-3 and CYFRA 21-1 was better. When two or more of the above four tumour markers were combined, or combined with carcinoembryonic antigen, the sensitivity and specificity were all increased to different extents., Conclusions: The current evidence does not recommend using one tumour marker alone for the diagnosis of MPE, but the combination of two or more tumour markers seems to be more sensitive. The results of tumour marker assays should be interpreted in parallel with clinical findings and the results of conventional tests.

Cytokeratin 19 (CK19) is an acidic protein of 40 kDa that is part of the cytoskeleton of epithelial cells and is highly expressed by differentiated thyroid carcinomas, mainly of the papillary subtype. The soluble fragments of CK19 (Cyfra 21.1) can be measured by immunometric assays employing specific monoclonal antibodies. The present study was planned to assess the serum expression of Cyfra 21.1 in patients with benign thyroid nodules and thyroid malignancies. We enrolled 135 patients with histologically proven benign thyroid nodules (n=79) and thyroid carcinomas (n=56). No differences were found in serum Cyfra 21.1 levels between patients with benign nodules and patients with carcinomas. When thyroid malignancies were subdivided according to tumor histology, serum Cyfra 21.1 increased significantly from classical differentiated thyroid carcinomas (papillary or follicular) to less differentiated or undifferentiated carcinomas (poorly differentiated or anaplastic). CK19 release into the bloodstream is strongly related to the apoptotic pathway, and particularly to hyperproliferation-related apoptosis. These pathways characterized anaplastic and poorly differentiated thyroid carcinoma but not classical forms of differentiated thyroid carcinoma. Consequently, Cyfra 21.1 may be regarded as a circulating marker of poorly differentiated and anaplastic thyroid carcinoma. Additionally, a role of Cyfra 21.1 as a dedifferentiation marker in patients with classical differentiated thyroid carcinomas may be postulated and should be explored by further focused studies.

We aimed to investigate whether pretreatment serum levels of squamous cell carcinoma (SCC) antigen (SCC-Ag), cytokeratin 19 (CYFRA 21-1) and two mucins (CA 15-3 and CA 125) identify patients with occult disease in early-stage SCC of the cervix. Therefore, pretreatment serum samples were obtained from 78 patients with SCC of the cervix (52 IB, 9 IIA and 18 IIB), and tumor markers were measured with commercial immunoassays. SCC-Ag, CYFRA 21-1 and CA 15-3 (analyzed as continuous variables) were significantly associated with overall (OS) and disease-free survival (DFS) in univariate analysis (p < 0.001 in all cases). Multivariate analysis identified lymph node status as the strongest predictor for OS and DFS (p < 0.001 and p = 0.001, respectively), followed by CYFRA 21-1 (p = 0.060 and p = 0.027, respectively) and CA 15-3 (p = 0.082 and p = 0.017, respectively). Clinical cutoff values for each marker were defined by maximizing the log-rank statistics for OS in the total population: 1.1 microg/l for SCC-Ag (n = 47, 60.3%), 1.4 microg/l for CYFRA 21-1 (n = 47, 60.3%), 40 U/ml for CA 15-3 (n = 11, 14.1%) and 30 U/ml for CA 125 (n = 10, 12.8%). Stage IB patients with positive SCC-Ag and CYFRA 21-1 had significantly lower OS (mean 8.3 years, 95% confidence interval, CI, 5.8-10.7 years) and DFS (mean 7.3 years, 95% CI 4.6-10 years) than all other stage IB patients (OS, mean 14.5 years, 95% CI 13.5-15.5 years; DFS, mean 13.9 years, 95% CI 12.5-15.4 years). Stage IB patients with tumors <4 cm or with negative lymph nodes and positive SCC-Ag and CYFRA 21-1 had significantly poorer OS and DFS compared to all other patients in the same group. Elevated levels of both CA 125 and CA 15-3 (3 patients) were associated with an extremely poor prognosis. In conclusion, a combination of SCC-Ag and CYFRA 21-1 may help to identify early-stage cervical cancer patients with occult disease requiring adjuvant therapy., ((c) 2008 S. Karger AG, Basel)

Squamous cell carcinoma antigen (SCC) is still a widely used tumor marker for monitoring non-small cell lung cancer (NSCLC), although recent reports discourage its routine use because of low sensitivity. This is a study evaluating the efficacy of SCC and CYFRA21-1 in diagnosing NSCLC. A chart review was performed in a university hospital in Japan, covering a period of 10 years, up to October 2004. During the study period, 142 (35.5%) among 400 NSCLC patients diagnosed, received serum assays of both SCC and CYFRA21-1. Elevated SCC and CYFRA21-1 levels were found in 29.6% and 59.2% of patients, respectively. SCC sensitivity was only 13.0% but CYFRA21-1 sensitivity rose to 73.9% in metastatic patients. The adjunct of SCC increased the CYFRA21-1 sensitivity by 6.3% in the overall population and by only 2.2% for patients with metastases. SCC determination should be considered an inefficient method as a potential diagnosing tool for NSCLC patients, and it provides no additional value when used in combination with CYFRA21-1.

Objective: To validate the prognostic value of preoperative levels of CYFRA 21-1, CEA and the corresponding tumor marker index (TMI) in patients with stage I non-small cell lung cancer (NSCLC)., Methods: Two hundred forty stage I NSCLC patients (80 in pT1 and 160 in pT2; 100 squamous cell carcinomas, 91 adenocarcinomas, 32 large-cell carcinomas, 17 with other histologies; 171 males and 69 females) who had complete resection (R0) between 1986 and 2004 were included in the analysis. CYFRA 21-1 and CEA were measured using the Elecsys system (Roche) and AxSym-System (Abbott), respectively. Univariate analysis was performed using the Kaplan-Meier method to identify potential associations between survival and age, gender, CYFRA 21-1, CEA and TMI., Results: Overall 3- and 5-year survival rates were 74 and 64%, respectively. Male gender (p = 0.0009) and age >70 years (p = 0.0041) were associated with a worse prognosis; there were no differences between pT1 and pT2 nor between histological subtypes. Three-year survival was 72% for CYFRA 21-1 levels >3.3 ng/ml versus 75% for levels 6.7 ng/ml versus 75% for CEA 0.05). Corresponding 5-year survival rates were near 64% both for patients with CYFRA 21-1 values above and below the cutoff (3.3 ng/ml), and 49 and 66% for patients with values above and below the CEA cutoff (6.7 ng/ml), respectively (both p values >0.05). Overall survival did not vary in the different TMI risk groups (p = 0.73)., Conclusions: In this cohort of early-stage NSCLC patients, male gender and age >70 years were associated with a worse outcome, but elevated levels of CEA and CYFRA 21-1, and TMI risk were not., ((c) 2008 S. Karger AG, Basel.)

Background & Objective: Breast cancer, a whole body disease, can metastasize at early stage. This study was to explore the correlation of peripheral blood cancer cell (PBCC) content to distant metastasis of breast cancer., Methods: The PBCC content of 65 breast cancer patients and 8 healthy donors was detected by multi-parameter flow cytometry (FCM) with CD45 and cytokeratin staining., Results: Cancer cells were detected in peripheral blood samples from 57 of the 65 patients; the positive rate was 87.7%. No cancer cell was found in peripheral blood samples from healthy donors. The positive rate of PBCCs was correlated to T stage (r=0.271,P=0.017) and N stage (r=0.393, P=0.002). The patients were followed for 5 years; 2 were lost. Distant metastasis was found in 25 patients with PBCCs. In contrast, no metastasis was found in 8 patients without PBCCs (P<0.05)., Conclusion: Preoperative PBCC content is closely related to distant metastasis of breast cancer. The detection of PBCCs might be useful for individual treatment decision for breast cancer.

This study aimed to establish the clinical significance of preoperative serum cytokeratin 19 fragment (CYFRA21-1) and Sialyl Lewis(x) (SLX) in patients with stage I non-small cell lung cancer (NSCLC). The study involved 137 patients (87 male, 50 female; median age 69 years) with completely resected stage I NSCLC. SLX, carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), and CYFRA21-1 were examined. Receiver operator characteristic (ROC) curves were constructed to determine prognostic cut-off values. Among the 137 patients, we identified 30 with recurrence within 3 years. The 5-year survival rates in patients with (n=30) and without (n=107) recurrence were 14% and 81%, respectively. The serum concentrations of SLX, CEA, and CYFRA21-1 in the recurrence group were significantly higher than those in the non-recurrence group. The areas under the ROC curve (AUC) were 0.72, 0.65, 0.53, and 0.64 for SLX, CEA, SCC, and CYFRA21-1, respectively. The prognostic cut-off values were 36U/ml, 7.8ng/ml, 1.5ng/ml, and 3.2ng/ml for SLX, CEA, SCC, and CYFRA21-1, respectively. A log-rank test revealed that age, performance status, T factor, lymphatic invasion, vascular invasion, SLX, CEA, SCC, and CYFRA21-1 were all significantly associated with survival. By multivariate analysis, age, performance status, lymphatic invasion, SLX (risk ratio, 4.11) and CYFRA21-1 (risk ratio, 3.47) were independent prognostic factors. For patients positive for both CYFRA21-1 and SLX, the relative risk was 5.32 compared with patients who were negative for both markers. The 5-year survival rates were 80% in the group negative for both markers (n=86); 52% in the group positive for one of the markers (n=43); and 13% for the group positive for both markers (n=8) (p<0.001). We concluded that serum SLX and CYFRA21-1 were prognostic markers for stage I NSCLC. Their combination should contribute to the classification of stage I NSCLC patients. There is a need to consider adjuvant and neoadjuvant therapies to improve prognosis in patients positive for both tumor markers.

Background: The aims of the present study were to compare the diagnostic and clinical value of seven commercially available assays for second-generation anti-cyclic citrullinated peptide (CCP2) antibodies, anti-keratin antibodies (AKA) and rheumatoid factor (RF) determination in patients with rheumatoid arthritis (RA), and to check the potential advantages of a third-generation anti-CCP (CCP3) antibodies assay., Methods: Serum samples from 120 RA patients and from 170 controls were used to determine the sensitivity, the specificity, the positive (PPV) and negative predictive values (NPV) of CCP2 and CCP3 antibodies, AKA and RF assays. The respective performances of these tests were compared using a receiver-operating characteristics (ROC) curves methodology., Results: We found no significant differences in sensitivity and specificity between the tested anti-CCP assays. The CCP3 antibodies assay we evaluated was not significantly more sensitive than those of the second generation. Compared with RF technique, all anti-CCP assays showed better specificity, but lower sensitivity. In contrast, while of equivalent specificity, they proved to be more sensitive than AKA in the discrimination of RA from other rheumatic diseases., Conclusions: Anti-CCP antibodies determination proved to be a powerful diagnostic tool, especially in RA patients with negative AKA or RF. The investigated CCP3 antibodies assay had no better diagnostic performances than the second-generation assays.

Background: The analysis of tumour markers is based on the evaluation of data in relation to defined cut-off values. Changes in the method of determination or reference study group have led to different results. Cut-off-independent diagnostic evaluation of laboratory parameters can avoid laboratory-based and method-derived systematic errors. The decision guarantee (DG) is an appropriate parameter that can be determined using a defined reference population and its respective receiver operating characteristic (ROC) curve. The influence of ROC differences on the determination of DG is examined., Patients and Methods: A group of 281 consecutive patients with newly diagnosed, histologically confirmed lung cancer and a control group of 231 patients were examined. Histological classification of the tumour cases defined in 59 small-cell carcinoma, 102 squamous cell carcinomas, 66 adenocarcinomas and 54 large-cell carcinomas or mixed bronchial carcinomas without classification. The control group without tumours consisted of 23 healthy subjects, 125 patients with silicosis or asbestosis, 27 with chronic obstructive pulmonary diseases (COPD) and 56 suffering from inflammatory lung diseases., Results: Cytokeratin-19 fragments (CYFRA 21-1) was the most sensitive marker with a sensitivity of 57.3% and a specificity of 94.9%. Sensitivity and specificity influence each other. Related to the ROC curve, the method described here ensured the diagnosis of lung cancer on the basis of the data collected in comparison with a reference population. Thus, it was possible to determine with statistical certainty whether the evaluation of the sample data would lead to a diagnosis of lung cancer., Conclusion: The DG provides the basis for a laboratory-and method-independent support for a diagnosis including fairer information about the reference population in the data analysis.

Objective: The clinical importance of preoperative CYFRA 21-1 measurement in early-stage non-small cell lung cancer (NSCLC) is still unclear. The aim of this study is to clarify the prognostic value of preoperative CYFRA 21-1 levels in clinical stage (c-stage) I NSCLC., Methods: The records of 101 c-stage I NSCLC patients who had undergone complete resection were analyzed to correlate preoperative CYFRA 21-1 levels to both the pathologic factors of resected specimens and postoperative outcomes. The cut-off value was set at 3.5 ng/ml., Results: Six cases (5.9%) showed high CYFRA 21-1 (> or =3.5 ng/ml). The 5-year survival of normal and high CYFRA 21-1 groups was 83.3% and 50.0%, respectively. Patients with high CYFRA 21-1 had significantly poor outcomes (P=0.006). In univariate analysis, preoperative serum CYFRA 21-1 level, pT, pN, and p-stage were significantly associated with prognosis. Multivariate analysis showed that only CYFRA 21-1 level was retained as an independent prognostic factor (relative risk=9.79, P=0.002)., Conclusions: CYFRA 21-1 is an independent predictor of poor outcome for c-stage I NSCLC. Elevated preoperative CYFRA 21-1 levels in early-stage NSCLC may indicate a subgroup at high risk of early death, which has the potential for better survival with additional systemic chemotherapy.

No validated tumor marker is currently available for the management of patients with cervical cancer. However, some tumor-associated antigens have been measured in the sera from patients with this malignancy and have been related to the clinical course of disease. Squamous cell carcinoma antigen (SCC) is more sensitive than CYFRA 21-1 for squamous cell cervical cancer. Serum SCC levels are elevated in 28-88% of patients with this malignancy, and correlate with tumor stage, tumor size, cervical stromal invasion, lymph-vascular space involvement, and lymph node status. Some authors reported that pre-treatment serum SCC has no prognostic value, whereas others found that it is related to disease-free survival and/or overall survival at univariate analysis or at multivariate analysis. Serial SCC measurements correlate with tumor response to radiotherapy and/or chemotherapy and the clinical outcome of patients. Increasing serum SCC can precede the clinical diagnosis of relapse in 46-92% of cases, with a median lead time ranging from 2 to 8 months. According to some authors serum SCC assay during the follow-up does not improve the cure rate of patients who will ultimately develop a recurrence. However, it has been recently reported that the performance of a PET in asymptomatic patients with serum SCC elevation can sometimes allow an earlier diagnosis of relapse with a survival benefit. Serum CA 125 levels are raised in 20-75% of patients with cervical adenocarcinoma, and reflect tumor stage, tumor size, histological grade, cervical stromal invasion, lymph-vascular space involvement, and lymph node status. Pre-treatment CA 125 levels appear to have a prognostic value, and rising serum CA 125 may precede or be coincident with the clinical diagnosis of recurrent cervical adenocarcinoma.

IMeasurement of tumor markers level in human body fluids, mainly in serum may be useful for diagnosis, therapy monitoring and early recurrence detection. SCC-Ag, Cyfra 21-1, CEA, CA 125 and TPS are of a clinical value in uterine cervical cancer despite their diverse significance. Other biological markers that could be measured in serum as well as in tumor tissue are cytokines: VEGF, IL-6, IL-8, IL-4, IL-10, leptin, stromal drive factor (SDF- 1), although assessment of their importance needs further examination. Elevation of uterine cervical cancer hypoxia markers such as HIF 1a, CA 9, GLUT-1 is combined with poorer clinical outcome prognosis.

Background: The aim of this retrospective study was to assess the prognostic value of serum tumor markers (carcinoembryonic antigen (CEA) and CYFRA21-1) in patients with pathologic (p-) stage I non-small cell lung cancer (NSCLC) undergoing complete resection., Methods: Two hundred and seventy-five patients (163 males, 112 females, mean age 67.1 years) with p-stage I NSCLC who underwent complete resection at our institution between April 1999 and October 2004 were examined. Patients who had received preoperative chemotherapy or radiotherapy were excluded, as were patients who had multiple malignancies including multiple lung cancer. The serum levels of tumor markers were measured using commercially available immunoassays within 1 month before surgical resection. Serum levels of CEA and CYFRA21-1 higher than 5.0 and 2.8 ng/ml, respectively, were considered as positive according to the manufacture's instructions., Results: The histological classification was adenocarcinoma in 193 patients, squamous cell carcinoma in 71, large cell carcinoma in 5, and other histological type in 6. One hundred and fifty-seven patients had T1 disease and 118 patients had T2 disease. The positive ratio of CEA and CYFRA21-1 was 25.7% and 13.7%, respectively, and in relation to histological type was 27.8% and 7.8% in adenocarcinoma, and 20.6% and 28.4% in squamous cell carcinoma. The overall 5-year survival rate was 79.3%. With a median follow-up of 35.5 month for surviving patients, those with initial CYFRA21-1 serum levels higher than 2.8 ng/ml had a significantly worse prognosis (p=0.0041). Patients with an elevated preoperative CEA level exceeding 5.0 ng/ml had a shorter disease-free survival period (p=0.0003). In patients with adenocarcinoma, a CEA level above 5.0 ng/ml was associated with shorter survival and early recurrence, whereas CYFRA21-1 showed no such association. In patients with squamous cell carcinoma, elevated preoperative CEA was not related to survival and recurrence. In these patients, preoperative CYFRA21-1 level exceeding 2.8 ng/ml was associated with a poorer outcome, whereas preoperative CYFRA21-1 level was not associated with cancer recurrence., Conclusion: The patients with p-stage I adenocarcinoma whose preoperative CEA level was high might be considered as good candidates for adjuvant chemotherapy. The prognostic value of CYFRA21-1 could not be confirmed for stage I NSCLC, and preoperative CYFRA21-1 level was not useful in selecting the candidates for adjuvant chemotherapy.

A large part of the cell cytoplasm of the cells consists of components forming cytoskeleton. The cytoskeleton is represented by actin-containing microfilaments, tubulin-containing microtubules and filaments of intermediate size. The last ones have diameters between 7-11 nm. Based on morphological, immunological and biochemical criteria the filaments of intermediate size are divided into: a) nuclear lamins; b) neurofilaments which are typical for neuronal cells; c) vimentin and vimentin-like filaments present especially in mesenchyma derived cells and d) filaments containing keratin-like proteins--cytokeratins. Cytokeratin filaments are a family of many different polypeptides and are normal components of epithelial cell cytoskeleton. Cytokeratins are expressed in various types of epithelia in different combinations. This is why antibodies against various cytokeratins have been used to characterize a wide variety of epithelial tumors. For example immunohistochemical detection of cytokeratin can identify micrometastases, not detected by conventional hematoxylin and eosin staining. Also serum cytokeratins levels are widely used as the markers of tumors of epithelial origin. In this review clinical applications of cytokeratins detection are presented.

In recent years, there has been an increasing awareness among physicians of the value of therapeutic interventions in patients suffering from lung cancer and mesothelioma. A search for an optimal approach using surgery, irradiation and chemotherapy in different settings of the tumour disease, including curatively aimed adjuvant chemotherapy after locoregional surgery or radiotherapy, has resulted in gradually improved survival rates. Still, early detection is crucial if there is to be a possibility of curing patients or prolonging life in cases of relapsed disease. Several studies have been initiated in which surrogate markers are evaluated in comparison to chest X-rays and computer tomography. The present review focuses on the predictive and prognostic value of using serological cytokeratins as tumour markers for patients suffering from thoracic malignancies.

Background: The analysis of tumour markers is based on the evaluation of data in relation to defined cut-off values. Changes in the method of determination or reference study group have led to different results. Generally, fuzzy logic-based classifiers for the evaluation of tumour-marker profiles have increased sensitivity and higher specificity. Drawing from experiences with cut-off-oriented evaluation, the suggestion that diagnosis be made using a non-cut-off-based approach is discussed., Patients and Methods: Two hundred and eighty-one consecutive patients with newly diagnosed, histologically confirmed lung cancer and a control group of 231 patients were examined. Histological classification of the tumour cases yielded 59 small cell carcinomas, 102 squamous cell carcinomas, 66 adenocarcinomas and 54 large cell carcinomas or mixed bronchial carcinomas without classification. The control group without tumours consisted of 23 healthy subjects, 125 patients with silicosis or asbestosis, 27 patients with chronic obstructive pulmonary diseases (COPD) and 56 persons suffered from inflammatory lung diseases., Results: CYFRA 21-1 was the most sensitive marker with a sensitivity of 57.3% and a specificity of 94.9%. The sensitivity increased when various tumour markers were evaluated. The loss of specificity through this type of multiparametric analysis could be compensated for by using a multiparametric evaluation such as a fuzzy logic classifier. A determination of the sensitivity-adapted confidence in diagnosis should be made. The decision guarantee and the cut-off-based evaluation are qualitatively equal because the decision threshold of 0.5 corresponds by definition to the cut-off value at 95% specificity. Every cut-off-based system of classification could be restated by using the decision guarantee instead of the measured values., Conclusion: The evaluation based on decision guarantee has proven to be unaffected by changes in laboratory systems. The multiparametric system of classification based on decision guarantee of individual markers is self-adjusting and differences in measurement levels are eliminated if the ROC curves of the individual markers are in agreement.

The aim of this study was to evaluate the individual diagnostic utility of tumour and inflammatory markers in patients with different pulmonary diseases. The usefulness of neuron-specific enolase (NSE), carcino-embryonic antigen (CEA), serum pro-gastrin releasing peptide (ProGRP) and CYFRA 21-1, as tumour markers, and C-reactive protein (CRP) and tumour necrosis factor-alpha (TNFalpha) as inflammatory markers for diagnosis, treatment and monitoring of patients with different pulmonary afflictions was investigated. Eighty healthy individuals were also included. Serum samples were also obtained from 20 patients suffering from bronchitis, 20 with lung fibrosis and 30 with sarcoidosis. Moreover, serum marker levels were analyzed in 139 patients with different pulmonary malignancies: 29 patients with adenocarcinoma, 30 patients with squamous cell carcinoma, 80 patients with small cell lung cancer (SCLC). All tumour markers showed significantly elevated values in malignant diseases. The levels of ProGRP in patients with benign diseases were significantly higher than those in the healthy group (35.4 +/- 6.6 compared with 21.3 +/- 9.2 pg/ml respectively). The serum ProGRP levels were elevated in SCLC patients (1673.9 +/- 706 pg/ml). The elevation was significantly higher than that of the benign reference group. The acute phase response had a wide range in patients with malignant tumours. Serum CRP levels were significantly higher in patients with SCLC (38.5 +/- 7.6 mg/dl) than in the benign reference group. In conclusion, when serum tumour markers are abnormally elevated in patients with lung cancer, CEA, CYFRA 21-1, NSE and ProGRP are useful clinical markers, good indicators of disease extent and may have important prognostic value. In particular, NSE and ProGRP have a very high sensitivity for SCLC detection.

Background: The aim of this study was to evaluate the diagnostic power of a fuzzy classifier and a marker panel (CYFRA 21-1, NSE, CRP) for the detection of lung cancers in comparison to asbestosis patients at high-risk of developing lung cancer., Patients and Methods: A panel of four tumour markers, i.e. CEA, CYFRA 21-1, NSE, SCC and CRP, was measured in newly diagnosed lung cancer patients of different histological types and stages in comparison to asbestosis patients. In this prospective study, a fuzzy classifier was generated with the data of 216 primary lung cancer patients and 76 patients suffering from asbestosis. The patients and controls were recruited in the clinics of the University in Giessen., Results: At 95%-specificity, it was possible with this tool to detect non-small cell lung cancers in 70% at stage I (n = 30), in 95% at stage II (n = 22), in 98% at stage III (n = 56), in 92% at stage IV (n = 50) and small cell lung cancers with limited disease status (n = 21) in 90.7% and with extensive disease status (n = 37) in 97.3%. In contrast, single markers had a detection rate significantly far below these. The application of the classifier was examined on an independent collective of 38 non-small cell lung cancers and 76 asbestosis patients. The latter underwent stationary rehabilitation in the clinics for occupational diseases in Bad Reichenhall or Falkenstein. The fuzzy classifier showed correct negative classification in 75 out of the 76 cancer-free asbestosis patients, which confirmed a specificity of 97.4%. The overall sensitivity for lung cancer detection in high risk populations was 73.6%. All large cell carcinomas were detected. The positive predictive value was 77.7%. The negative predictive value reached 94.8%., Conclusion: With the fuzzy classifier and a marker panel, a reliable diagnostic tool for the detection of lung cancers in a high risk population is available.

Objective: Reverse transcriptase-polymerase chain reaction assay results have indicated that tumor cells sometimes appear during surgery for primary non-small-cell lung cancer. In this study, we attempted to determine whether cancer cells can be detected during and after surgery using an immunocytology method., Methods: Nine patients undergoing a lobectomy for non-small-cell lung cancer were studied. The presence of circulating tumor cells was determined by the detection of magnified EpCAM antibodies. The criteria used to identify circulating tumor cells were a round-to-oval morphology with a visible nucleus (4'-6'-diamidino-2-phenylindole (DAPI)-positive), which were positive for cytokeratin and negative for CD45., Results: One patient showed evidence of circulating tumor cells at thoracotomy, and 3 patients did so after surgery. Ten days after the operation, the circulating tumor cells had disappeared in all these cases. The median follow-up period was 14 months, and there was no cancer recurrence in any of the patients., Conclusion: Using this technique, tumor cells were detected in the peripheral blood of patients before and after lobectomy procedures. It could be argued that this method can provide useful information about patients undergoing lung cancer treatment.

Aim: The aim of this study was to examine the clinical usefulness of cytokeratin 19 fragments (CYFRA 21-1) compared with squamous cell carcinoma (SCC) antigen in patients with esophageal cancer treated with radiation therapy., Methods: Fifty-one patients with stage I-IV esophageal cancer were evaluated. CYFRA 21-1 and SCC antigen serum levels were measured at the start and the end of radiation therapy., Results: CYFRA 21-1 (> 3.5 ng/mL) and SCC antigen (> 1.5 ng/mL) before radiation therapy were elevated in 63% and 53% of the patients, respectively. The CYFRA 21-1 levels were significantly correlated with TNM stages, tumor depth and lymph node metastasis (P = 0.0003, P = 0.019 and P = 0.019, respectively), whereas no correlation was observed between SCC antigen and these factors. The values of CYFRA 21-1 in all patients who survived without recurrence were under the cutoff level at the end of treatment, but the values in all patients with locoregional recurrence were above the level. However, there was no significant correlation between SCC antigen level at the end of treatment and any clinical outcome., Conclusions: The results suggest that the evaluation of CYFRA 21-1 would be useful not only for assessment before radiation therapy but also for monitoring after radiation therapy in the treatment for esophageal cancer.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumour in the oral and maxillofacial region, and has a poor prognosis. Cyfra 21-1 is a useful tumour marker for squamous cell carcinoma, but the clinical value of Cyfra 21-1 in OSCC has not been confirmed. In order to investigate the diagnostic and prognostic value of serum Cyfra 21-1 in primary OSCC patients, the preoperative serum Cyfra 21-1 concentration of 100 OSCC patients and 56 healthy subjects was detected by enzyme-linked immunosorbent assay (ELISA). The cut-off value was calculated with a receiver operating characteristic (ROC) curve, and prognostic analysis was performed using the Kaplan-Meier method and Cox regression models. The preoperative serum Cyfra 21-1 concentration in OSCC patients (1.18+/-1.20 microg/L) was significantly higher (t=6.585, P<0.001) than that in healthy subjects (0.40+/-0.16 microg/L). With a cut-off value of 0.65 microg/L, the diagnostic sensitivity and specificity was 0.570 and 0.964, respectively. There was significant correlation with tumour recurrence and survival rate: the higher the serum Cyfra 21-1; the higher the tumour recurrence rate and lower the survival rate. Serum Cyfra 21-1 was an independent prognostic factor for OSCC using univariate and multivariate Cox models.

Objective: To determine the levels of the tumor markers carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA21-1) and carbohydrate antigen 15-3 (CA 15-3) in the blood and pleural fluid of patients with benign or malignant pleural effusion, evaluating the sensitivity of each marker in these fluids., Methods: We prospectively evaluated 85 patients with pleural effusion. The study of the pleural fluid observed the criteria established in the literature. Levels of the markers were determined using electrochemiluminescence. The sensitivity was determined on the condition that the specificity was > or = 90%., Results: Of the 85 cases, 36 (42.4%) were malignant, 30 (35.3%) were benign, and the results were inconclusive in 19 (22.3%). In the malignant cases, the CEA and CYFRA21-1 levels were higher in the pleural fluid than in the blood, which was not observed for CA 15-3. In the benign cases, the CYFRA21-1 levels were higher in the pleural fluid than in the blood, whereas the opposite was found for CEA and CA 15-3. There were significant differences between malignant and benign cases for all markers, in pleural fluid and blood. In the pleural fluid, the sensitivity of CEA, CYFRA21-1 and CA 15-3 was 69.4, 69.4 and 66.7%, respectively, and the combined sensitivity was 80.6%. In the blood, the sensitivity was 57.1%, 71.4% and 48.6% for CEA, CYFRA21-1 and CA 15-3, respectively, and the combined sensitivity was 77%., Conclusion: The results suggest that these markers might be useful in the differentiation between malignant and benign pleural effusion.

Cytokeratins have been extensively used as serum tumour markers for monitoring of disease progression in cancer patients. The source of cytokeratins in the circulation as well as the mechanisms of release from cells have long been unclear. Recent evidence suggests that cytokeratins present in the circulation of cancer patients are released from apoptotic or necrotic tumour cells. CK18 is cleaved by caspases during apoptosis and a monoclonal antibody (M30) specific to caspase-cleaved forms is available. The molecular form of CK18 released from cells (caspase-cleaved or not) can conveniently be determined by immunoassays (M30-Apoptosense and M65 ELISA assays; Peviva AB, Bromma, Sweden) to determine cell death mode--apoptosis or necrosis. Recent studies where these assays were used to evaluate the response to cytotoxic anticancer drugs using cancer patient serum have been encouraging. CK18 is attracting considerable interest as a response biomarker during clinical trials of anticancer drugs. Properties such as excellent antigen stability and the epithelial specificity of cytokeratins contribute to make this biomarker attractive.

Background: Sertoli-Leydig cell tumours of the ovary account for only 0.2% of malignant ovarian tumours. Two-thirds of all patients become apparent due to the tumour's hormone production., Methods: A 41-year-old patient (gravida 4, para 4) presented with dyspnoea, enlarged abdominal girth and melaena. Diagnostic imaging was suspicious for an ovarian cancer. The standard tumour marker for ovarian cancer (CA 125) was elevated to 984 U/mL., Results: Surgical exploration of the abdomen revealed a mouldering tumour of both adnexes extending to the level of the navel. Frozen sections showed an undifferentiated carcinoma of unknown origin. Radical surgery was performed. The final histological report described a malignant sex-cord stroma tumour, a Sertoli-Leydig cell tumour, emanating from both ovaries. Analysis of preoperative blood serum showed elevated levels of CYFRA 21-1 (10.4 ng/mL), neuron-specific enolase (36.2 ng/mL), oestradiol (485 pg/mL) and CA-125 (984 U/mL). Adjuvant chemotherapy and regional hyperthermia were performed due to the malignant potential and incomplete resection of the tumour., Conclusions: Undifferentiated Sertoli-Leydig cell tumours show a poor clinical course. As only two-thirds of patients with this rare disease present with elevated hormone levels, new markers deserve further investigation to offer more specific, individualised tumour monitoring.

The usefulness of selected serum biomarkers (KL-6, SP-A, SP-D, IL-8, MCP-1, CYFRA-21) in diagnosis, monitoring and prognosis prediction of idiopathic pulmonary fibrosis is discussed in this paper.

Lung cancer is the most common cancer worldwide with 900,000 new cases each year in men and 330,000 in women, being also the major cause of death from cancer. In Greece about 4,000 persons die every year due to lung carcinoma. One of the major problems in the follow up of these patients is the difficulty of early detection of recurrent disease. Tumor markers are of particular interest in this respect. Cytokeratines, especially fragment 19, are specified epithelial tissue-proteins that show increased levels in patients with carcinomas. CYFRA 21-1 assays determine the serum cytokeratin 19 fragment. The aim of our study was to evaluate the importance of serum CYFRA 21-1 studied by immunoradiometric assay in patients with various types of lung cancer after surgery or chemotherapy. Ninety-six consecutive patients were studied during a two years period. Forty-five of them had small cell lung cancer (SCLC) and 51 had non-small cell lung cancer (NSCLC). Moreover, 52 healthy individuals were studied to estimate the cut off value of CYFRA 21-1. Increased serum levels of the marker were found in patients with lung cancer compared to controls (P<0.001). The cut off value was estimated as 3.3 ng/ml with 96% specificity. Before the treatment there was no difference in the sensitivity of CYFRA 21-1 for patients with SCLC (21/45 patients had increased CYFRA 21-1 levels, 47%) and for patients with NSCLC (27/51 had increased levels, 52%). Also, before treatment there was a higher sensitivity in NSCLC than in SCLC and especially in SCC among other histotypes of NSCLC when different stages of the disease were compared. Patients with extended disease, metastatic or recurrent disease had also more increased levels of the marker (P<0.001). One month after surgical ablation of the primary lung lesion, 28/58 patients showed a drop in the levels of the marker as an indication of the tumor ablation. From the 58 operated patients 35 relapsed and 31/35 showed an increase in CYFRA-21-1 levels with a sensitivity of 92% and specificity of 95%. From the 38 patients that underwent chemotherapy treatment, 24 had a depravation of the disease and 21/24 had a great increase of serum CYFRA 21-1 with a sensitivity of 89% and specificity of 94%. In conclusion, CYFRA 21-1 is a useful tumor marker before and after surgical treatment in lung cancer.

Unlabelled: OBJECTIVE. Tumour markers are widely used for monitoring cancer patients and for screening certain tumours. It has recently been shown that the concentrations of some tumour markers are higher in patients with chronic kidney disease (CKD) than in healthy subjects. We analysed the influence of renal function and hemodialysis treatment on the serum levels of CA19-9, CA125, alpha fetoprotein (AFP), CA15.3, CA72.4, CYFRA 21-1, neuron-specific enolase (NSE) and squamous cell carcinoma antigen (SCC-Ag)., Material and Methods: 232 non-dialysis patients with CKD and 37 uraemic patients treated with maintenance hemodialysis were enrolled in this study. The nondialysis patients were divided into three groups depending on their creatinine clearance (Ccr) levels: group 1 = Ccr < or =25 mL/min; group 2 = 25.1-49.9 mL/min; group 3 = Ccr > or =50 mL/min. For comparison, we chose 37 non-dialysis patients with similar Ccr, age and same gender as controls., Results: The serum concentrations of CA19-9, CA125 (male), CYFRA 21-1, NSE and SCC-Ag correlated negatively with Ccr, while there were no significant differences in the concentrations of CA125 (female), AFP, CA15.3, CA72.4. The serum levels of CA19-9, CA125, AFP, CA15.3, CA72.4, CYFRA 21-1, NSE and SCC-Ag showed no differences between hemodialysis patients and non-dialysis controls (p > 0.017)., Conclusions: The increase in the serum levels of CA19-9, CA125 (in males), CYFRA 21-1, NSE and SCC-Ag in patients with CKD affects the specificity of these markers in the diagnosis of cancer. Hemodialysis does not affect the serum levels of CA19-9, CA125, AFP, CA15.3, CA72.4, CYFRA 21-1, NSE and SCC-Ag.

Background: This study aimed to establish the clinical significance of preoperative serum cytokeratin 19 fragment (CYFRA21-1) and sialyl-Lewis x (SLex) as prognostic markers., Methods: The study involved 272 patients (181 male, 91 female; median age 69 years; range, 32 to 92) with non-small cell lung cancer (NSCLC) who underwent pulmonary resection with mediastinal lymph node dissection. Tumor markers carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), CYFRA21-1, and SLex were examined., Results: A log-rank test revealed that age, gender, performance status, CEA, SCC, CYFRA21-1, and SLex were associated with the survival rate. By multivariate analysis, age, gender, performance status, CYFRA21-1 (risk ratio, 2.42) and SLex (risk ratio, 6.18) were independent prognostic factors. For patients positive for both markers, the relative risk was 6.10 compared with patients negative for both markers. The patients were divided into three groups: negative for both CYFRA21-1 and SLex (n = 97); positive for either marker (n = 136); and positive for both markers (n = 39). The 1-, 3-, and 5-year survival rates were the following: 98%, 82%, and 75% in the first group; 90%, 63%, and 49% in the second group; and 62%, 31%, and 25% in the third group (p < 0.001). Sixty-four percent of patients positive for both markers were histologic stage III/IV, and 68% of patients negative for both markers were stage I., Conclusions: Serum CYFRA21-1 and SLex were prognostic markers for NSCLC. Their combination should contribute to the classification of NSCLC patients. Preoperative staging should be carefully performed in patients positive for both tumor markers.

Background: The authors assessed the predictive and prognostic role of decline in the serum levels of carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA 21-1) during chemotherapy in patients with advanced nonsmall cell lung cancer (NSCLC)., Methods: Changes in serum levels of CEA and CYFRA 21-1 during first-line, conventional chemotherapy were studied prospectively with an immunometric assay at baseline and every 2 courses in 117 patients with advanced NSCLC. Data were correlated with radiologic objective response (OR) and survival., Results: One hundred seven patients were evaluable for radiologic and serologic response assessment after 2 chemotherapy courses. The radiologic OR rate was 44% overall. The CEA and CYFRA 21-1 responses (> or =20% reduction over baseline level; assessed after the second course of chemotherapy) were 38% and 61%, respectively. Statistically significant correlations were observed between CEA and CYFRA 21-1 responses and OR (P = .01 and P = .004, respectively). The median survival from response assessment (landmark analysis) was 9 months. In a univariate analysis, disease stage, performance status, baseline lactate dehydrogenase level (LDH), OR, CEA response, and CYFRA 21-1 response were correlated significantly with survival. In particular, the median survival was 13 months for patients who had a CEA response and 11 months for patients who had a CYFRA 21-1 response compared with 8 months and 6 months for patients who did not respond, respectively. In a multivariate analysis, performance status (P = .005), baseline LDH level (P = .02), CEA response (P = .03) and CYFRA 21-1 response (P = .01) were confirmed as independent prognostic factors for survival., Conclusions: CEA and CYFRA 21-1 responses appeared to be reliable surrogate markers of chemotherapy efficacy in patients with advanced NSCLC., (Copyright 2006 American Cancer Society.)

Background: The efficacy of screening for colorectal cancer using a simple blood-based assay for the detection of tumor cells disseminated in the circulation at an early stage of the disease is gaining positive feedback from several lines of research. This method seems able to reduce colorectal cancer mortality and may replace colonoscopy as the most effective means of detecting colonic lesions., Methods: In this work, we present a new microarray-based high-throughput screening method to identifying candidate marker mRNAs for the early detection of epithelial cells diluted in peripheral blood cells. This method includes 1. direct comparison of different samples of colonic mucosa and of blood cells to identify consistent epithelial-specific mRNAs from among 20,000 cDNA assayed by microarray slides; 2. identification of candidate marker mRNAs by data analysis, which allowed selection of only 10 putative differentially expressed genes; 3. Selection of some of the most suitable mRNAs (TMEM69, RANBP3 and PRSS22) that were assayed in blood samples from normal subjects and patients with colon cancer as possible markers for the presence of epithelial cells in the blood, using reverse transcription-polymerase chain reaction (RT-PCR)., Results: Our present results seem to provide an indication, for the first time obtained by genome-scale screening, that a suitable and consistent colon epithelium mRNA marker may be difficult to identify., Conclusion: The design of new approaches to identify such markers is warranted.

Objective: To investigate the changes and clinical value of circulating endothelial cells (CEC) in the peripheral blood of advanced NSCLC patient., Methods: Sixty-seven advanced NSCLC patients were randomly divided into either the treatment group with NP plus endostatin or control group with NP alone. Level of CEC and cytokeratin (CK) in the peripheral blood were measured by flow cytometry., Results: The response rate and benefit rate was 44.4%, 80.0% in the treatment group, and 27.3%, 50.0% in the control group, respectively (P = 0.176 and P = 0.012). Time to tumor progression (TTP) was 146.7 days in the treatment group and 91.1 days in the control group (P = 0.061). However, when the cut-off of TTP was defined as > 170 days, there was a significant difference between two groups (cut-off = 170, P = 0.034; cut-off = 180, P = 0.009). The number of CEC decreased by 0.29 +/- 0.47 in the treatment group and by 0.01 +/- 0.43 in the control group (P = 0.033). The correlation between CEC and CK was found to be positive either before (r = 0.381, P = 0.013) or after the treatment (r = 0.450, P = 0.004)., Conclusion: Chemotherapy combined with endostatin is superior to chemotherapy alone in the treatment of NSCLC. CEC, as a biomarker, may be useful in predicting the efficacy of the combined treatment.

Purpose: This study was designed to examine whether disseminated tumor cells in peripheral blood of locally advanced rectal cancer patients undergoing preoperative chemoradiation have the potential to serve as a marker for therapy response. Studies suggest that patients with advanced rectal cancer who respond to preoperative chemoradiation most likely benefit from this treatment., Methods: From advanced rectal cancer patients undergoing preoperative chemoradiation, peripheral blood was obtained at defined times: before, during, and after chemoradiation and during surgery. Patients were divided into histopathologic responders (ypT0-T2) and nonresponders (ypT3-T4). Cytokeratin 20 and carcinoembryonic antigen reverse transcriptase-polymerase chain reaction were performed to detect disseminated tumor cells. A blood sample was deemed positive for disseminated tumor cells if both carcinoembryonic antigen and cytokeratin 20 were detected., Results: The overall population (n = 26) showed a positivity rate of 32 percent for disseminated tumor cells before initiation of chemoradiation. Of the responders (n = 8), 63 percent were positive for disseminated tumor cells before chemoradiation, whereas only 18 percent of nonresponders (n = 18) were positive (P = 0.026). From initiation of chemoradiation to the end of surgery, a significant decrease was seen in tumor cell positivity in the blood of responders (P = 0.042). Moreover, the responders represented a trend toward a decrease in tumor cell positivity during chemoradiation (P = 0.079). In contrast, there were no noticeable alterations within the treatment course in nonresponders., Conclusions: This prospective proof of principle study demonstrates that locally advanced rectal cancer with preoperative chemoradiation shows different biologic behavior in terms of tumor cell dissemination in peripheral blood when therapy responders compared with nonresponders.

The clinical value of prostate-specific antigen (PSA)-positive circulating tumor cells (CTCs) is still a matter of debate and it is also still unclear if these CTCs actually represent the primary tumor. Therefore, we isolated PSA-positive CTCs from the peripheral blood of patients suffering from multifocal cancers and did genetic profiling of each cancer focus by a multiplex PCR-based microsatellite analysis (D7S522, D8S522, NEFL, D10S541, D13S153, D16S400, D16S402, D16S422, and D17S855). In 17 of 20 prostate cancer cases, the loss of heterozygosity (LOH) pattern of the CTCs was identical with only one focus of the primary tumor. Moreover, in six cases, the LOH pattern suggested that smaller foci, down to 0.2 cm3, might deliver CTCs. Interestingly, the highest number of LOHs was observed at the marker D10S541 (85%), the PTEN gene, which was observed much less frequently in unifocal prostate cancer (48%). Furthermore, the infrequently occurring LOH in the BRCA1 gene (38%) was found in four of the five cases where a biochemical recurrence was seen within 3 years after prostatectomy. Therefore, the data might support the assumption that CTCs in prostate cancer are derived from distinct foci of a primary tumor. The size of the tumor focus is not related to the delivery of cells. Although the number of cases that were investigated in this study was small, it might be suggested that the LOH at distinct markers such as D10S541 and D17S855 represent the genes PTEN and BRCA1, which might be associated with the occurrence of CTCs in the peripheral blood of patients as well as an early biochemical recurrence.

Objective: To evaluate the performance of 18F-FDG three-head tomography with coincidence imaging and serum tumor marker assays in identifying lung lesions in 104 patients with abnormal findings on chest X-ray or computer tomography., Methods: A prospective evaluation of 18F-FDG coincidence imaging and the measurement of 3 serum markers for lung cancer ( carcinoembryonic antigen, CYFRA21-1 and neuron specific enolase) were performed within one week in 104 inpatients with suspected lung malignancy. All images were analyzed visually. It was considered positive for malignancy if the 18F-FDG uptake was increased relative to that in the adjacent lung tissue, and was focal. The serum tumor marker test was considered positive for malignancy if the serum level of at least one marker was elevated., Results: 66 patients were proven to have lung cancer by pathology, and 38 patients had benign lung diseases. The sensitivity, specificity, accuracy of 18F-FDG coincidence imaging and serum tumor markers in assessing lung cancers were 80. 0% , 77. 2% , 77. 9% and 56. 0% , 60. 9%, 64. 4% , respectively. 18F-FDG coincidence images in assessing lung lesions showed significantly higher sensitivity, specificity and accuracy than serum tumor markers. Four patients with lung cancer had negative findings on 18F-FDG coincidence images but showed positive serum markers., Conclusion: 18F-FDG coincidence imaging is a powerful tool for evaluating patients with lung lesions suggestive of malignancy. Although the determination of serum marker levels is less accurate than 18F-FDG coincidence imaging, the combination of a positive 18F-FDG coincidence result and positive tumor markers may be helpful in improving the diagnosis of lung cancers.

Purpose: To evaluate the predictive and prognostic value of peripheral blood cytokeratin-19 (CK-19) mRNA-positive cells in axillary lymph node-negative breast cancer patients., Patients and Methods: Peripheral blood was obtained from 167 node-negative breast cancer patients before the initiation of any systemic adjuvant therapy, and was analyzed for the presence of CK-19 mRNA-positive cells using a real time polymerase chain reaction assay. The association with known prognostic factors and the effect of CK-19 mRNA-positive cells on patients' prognosis was investigated., Results: CK-19 mRNA-positive cells were detected in the blood of 36 (21.6%) of the 167 patients. There was no correlation between the detection of CK-19 mRNA-positive cells in the peripheral blood and the various known pathologic and clinical prognostic factors; only overexpression of HER2 receptor (score 2+/3+) on the primary tumor was associated with a higher incidence of CK-19 mRNA-positive cell detection (P = .033). Multivariate analysis revealed that detection of peripheral blood CK-19 mRNA-positive cells was associated with early clinical relapse (P < .00001) and disease-related death (P = .008)., Conclusion: Detection of peripheral-blood CK-19 mRNA-positive cells is an independent predictive and prognostic factor for reduced disease-free interval and overall survival, respectively, in node-negative breast cancer patients.

Our previous proteomics study on human hepatocellular carcinoma (HCC) cell strains revealed that cytokeratin 19 (CK19) was expressed in cells with high metastasis potential; we further studied serum CK19 fragment CYFRA 21-1 level in HCC patients and nude mice model of HCC metastasis. HCC cell line HCCLM3 was injected subcutaneously into 30 nude mice which were then randomized into 6 groups of 5 mice each. The murine serum CYFRA 21-1 and pulmonary metastases were determined 2, 3, 4, 5, 6, and 7 weeks after injection. Serum CYFRA 21-1 levels of 101 normal controls and 108 HCC patients were also determined. In nude mice model, CYFRA 21-1 level increased significantly when pulmonary metastases occurred. Among 108 HCC patients, 24 (22.2%) had increased serum CYFRA 21-1 level. The presence of portal vein tumor emboli was significantly higher in CYFRA 21-1 increased cases (33.3%, 6/24) than in CYFRA 21-1 normal cases (6.0%, 5/84) (x2=7.403, P < 0.01). In addition, the percentage of TNM stage III/IV tumor was significantly higher in CYFRA 21-1 increased patients (54.2%, 13/24) than in CYFRA 21-1 normal cases (21.4%, 18/84) (x2=9.776, P < 0.005). These results suggest that CK19 may play an important role in HCC metastasis.

Circulating proteinic biomarkers are secreted by tumor cells or by their environmental cells and they have a variable specificity. In case of breast cancer, carcino-embryonic antigen (CEA) was for a long time the only circulating biomarker used. Nowadays, the most useful biomarkers measure circulating levels of fragments of MUC1-polymorphic epithelial mucin (MUC1-PEM): cancer antigen (CA) 15.3, mucin-like carcinoma-associated antigen (MCA), CA 27-29, CA 549... They are useful for general disease follow-up. Other circulating markers belonging to keratins (tissue polypeptide antigen, TPA, TPS or Cyfra 21.1) are correlated with proliferative activity of breast tumors. More recently, the measure of the c-erb B2 circulating part (extra cellular domain, ECD) was proposed as a prognostic biomarker for breast tumors with c-erb B2 overexpression. Moreover, the determination of urinary level of trefoil factor1 (PS2-TFF1) might be useful for the follow-up of hormonodependent breast cancers. The present review describes the clinical interest of these different circulating biomarkers in case of breast cancer, emphasizing their biological characteristics.

In patients with nonalcoholic fatty liver disease (NAFLD), a liver biopsy remains the only reliable way to differentiate simple steatosis from nonalcoholic steatohepatitis (NASH). Noninvasive methods are urgently needed. Increasing evidence suggests hepatocyte apoptosis is a key mediator of liver injury in NAFLD. The aim of this study was to quantify hepatocyte apoptosis in plasma from patients with NAFLD and correlate it with histological severity. Plasma was obtained from 44 consecutive patients with suspected NAFLD at the time of liver biopsy. Histology was assessed blindly. Caspase-3-generated cytokeratin-18 fragments were measured in situ via immunohistochemistry and in vivo using a novel enzyme-linked immunosorbent assay. Plasma cytokeratin-18 fragments were markedly increased in patients with NASH compared with patients with simple steatosis or normal biopsies (median [interquartile range]: 765.7 U/L [479.6-991.1], 202.4 U/L [160.4-258.2], 215.5 U/L [150.2-296.2], respectively; P < .001). Cytokeratin-18 fragment levels independently predicted NASH (OR 1.95; 95% CI 1.18-3.22; P = .009 for every 50 U/L increase). A cutoff value of 395 U/L calculated using the receiver operating characteristic curve approach showed a specificity of 99.9%, a sensitivity of 85.7%, and positive and negative predictive values of 99.9% and 85.7%, respectively, for the diagnosis of NASH. In conclusion, these findings strongly suggest that determination of hepatocyte caspase activation in the blood is a strong and independent predictor of NASH in human subjects. These data highlight the potential usefulness of this test as a noninvasive diagnostic means of determining histological disease severity in patients with NAFLD.

Aim: To evaluate the efficacy of postoperative serial assay of carcinoembryonic antigen (CEA) and cytokeratins for the detection of recurrent disease in patients with colorectal adenocarcinoma after radical surgery., Methods: Between 1993 and 2000, 120 patients with colorectal adenocarcinoma underwent radical surgery in the Department of Surgical Gastroenterology, Federal University of Sao Paulo-Escola Paulista de Medicina, Sao Paulo, Brazil. Periodic postoperative evaluation was performed by assaying markers in peripheral serum, colonoscopy and imaging examination. Presence of CEA was detected using the Delfia method with 5 microg/L threshold, and cytokeratins using the LIA-mat TPA-M Prolifigen method with 72 U/L threshold., Results: In the first postoperative year, patients without recurrent disease had normal levels of CEA (1.5 +/- 0.9 microg/L) and monoclonal tissue polypeptide antigen-M (TPA-M, 64.4 +/- 47.8 U/L), while patients with recurrences had high levels of CEA (6.9 +/- 9.8 microg/L, P < 0.01) and TPA-M (192.2 +/- 328.8 U/L, P < 0.05). During the second postoperative year, patients without tumor recurrence had normal levels of CEA (2.0 +/- 1.8 microg/L) and TPA-M (50.8 +/- 38.4 U/L), while patients with recurrence had high levels of CEA (66.3 +/- 130.8 microg/L, P < 0.01) and TPA-M (442.7 +/- 652.8 U/L, P < 0.05). The mean follow-up time was 22.3 mo. There was recurrence in 23 cases. Five reoperations were performed without achieving radical excision. Rises in tumor marker levels preceded identification of recurrences: CEA in seven (30%) and TPA-M in eleven individuals (48%)., Conclusion: Intensive follow-up by serial assay of CEA and cytokeratins allows early detection of colorectal neoplasm recurrence.

Caspase-cleaved proteins are released from disintegrated apoptotic cells and can be detected in the circulation. We here addressed whether caspase-cleaved cytokeratin 18 (CK18-Asp396) can be used as a serum biomarker for assessment of the clinical efficiency of chemotherapy in hormone-refractory prostate cancer (HRPC). A total of 82 patients with HRPC were evaluated during 751 treatment cycles, either with estramustine (EMP)/vinorelbine or with EMP/docetaxel. The levels of CK18-Asp396 and of total CK18 were measured in patient serum before and during therapy by ELISA. Docetaxel induced significant increases in serum CK18-Asp396 (P<0.0001) and total CK18 (P<0.0002), suggesting induction of apoptosis. Similarly, vinorelbine induced increases in both CK18-Asp396 and CK18 (P<0.001 and 0.011). In contrast, EMP induced increases in total serum CK18 (P<0.0001), but not in CK18-Asp396 (P=0.13). The amplitudes of docetaxel-induced increases were associated with baseline prostate-specific antigen (PSA) and CK18 serum levels in these patients, consistent with tumoral origin of caspase-cleaved fragments. Docetaxel induced significant increases in CK18-Asp396 during second-, third- and fourth-line therapy and induced increased levels of CK18-Asp396 during treatment cycles 1-8. In contrast, vinorelbine induced significant increases only during cycles 1-3. In a subgroup of 32 patients that received EMP/vinorelbine in second line followed by EMP/docetaxel in third line, docetaxel induced stronger increases than vinorelbine (P=0.008). These results show that the CK18-Asp396 serum marker can be used to assess tumour apoptosis in vivo and suggest that the clinical efficiency of docetaxel in HRPC is due to induction of apoptosis during multiple treatment cycles.

This study is aimed at establishing a sensitive approach to detect disseminated tumor cells in peripheral blood and evaluate its clinical significance. A total of 198 blood samples including 168 from colorectal carcinoma (CRC) patients and 30 from healthy volunteers were examined by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to evaluate the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and cytokeratin 19 (CK19) mRNA. CEA mRNA was detected in 35.8% of patients and 3.3% of controls, CK20 mRNA in 28.3% of patients and 6.7% of controls, and CK19 mRNA in 41.9% of patients and 3.3% of controls. CEA and CK20 mRNA positive ratio increased with the advancing Dukes stages, but there was no significant difference in positive ratio between any two stages (P>0.05). Also, relatively high positive ratio of CEA, CK20 and CK19 mRNA expression was observed in some CRC patients with earlier Dukes stages. A higher positive ratio was obtained when two or three detection markers were combined compared to a single marker. Our study indicates that quantitative real-time RT-PCR detection for CEA, CK20 and CK19 mRNA in peripheral blood is a valuable tool for monitoring early stage dissemination of CRC cells in blood circulation.