Role of circulating cytokeratin fragments and angiogenic factors in NSCLC patients stage IIIa-IIIb receiving curatively intended treatment.

Non-small cell lung cancer (NSCLC) is derived from epithelial cells and accounts for approximately 80% of all lung cancers. Cytokeratins are specific for epithelial cells and during malignant transformation the cytokeratin profile usually remains constant. Angiogenesis is the formation of new blood vessels out of the existing vascular bed. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are potent circulating angiogenic factors. The aim of the present study was to determine if increased levels of a new cytokeratin assay (MonoTotal, which in comparison with TPAcyk detects not only fragments of cytokeratins 8 and 18 but also of cytokeratin 19) is correlated with circulating angiogenic factors (VEGF and bFGF) and the secondary aim was to investigate if increased levels of these circulating markers are associated with survival. In the present study, a total of 45 NSCLC patients (26 patients stage IIIa and 19 patients stage IIIb) receiving only curatively intended treatment for advanced NSCLC were included. These patients donated a total of 291 serum samples during follow-up which was investigated for the presence of MonoTotal, VEGF and bFGF. MonoTotal was statistically significantly correlated with bFGF (R=0.26, p=0.00049) and VEGF (R=0.26, p=0.00007). From the time of histological diagnosis until time of death, MonoTotal increased by 603 U/l (p<0.0001). VEGF increased by 430 pg/ml (p=0.0004) whereas the corresponding value for bFGF was 5.93 pg/ml (p=0.018). MonoTotal, a newly developed commercial cytokeratin assay, seems to be a potentially very interesting serum marker that, in conjunction with other clinical data, might be used for monitoring of patients with NSCLC.

Evaluation of radiation-induced DNA damage and DNA repair in human lung cancer cell lines with different radiosensitivity using alkaline and neutral single cell gel electrophoresis.

Using the comet assay, radiation-induced DNA strand breaks were evaluated in human lung cancer cell lines with different radiosensitivity (U-1285, U-1906E, U-1752 and U-1810). Single strand breaks were more sensitive indicators of the radiation-induced damage than double strand breaks. However, there was no consistent pattern in the way the various cell lines responded to 1-5 Gy of gamma-irradiation and all cell lines showed a remarkably efficient DNA repair after 1 h. In a separate study of the repair kinetics of DNA double strand breaks, the radioresistant cell line U-1810 showed a more efficient initial strand rejoining than the radiosensitive cell line U-1285 after irradiation at 2 Gy. The latter finding suggests that the detection of early DNA repair may be useful when monitoring the intrinsic radiosensitivity of human lung cancer cells.

Serum VEGF and bFGF adds prognostic information in patients with normal platelet counts when sampled before, during and after treatment for locally advanced non-small cell lung cancer.

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have both been implicated to have roles in tumour angiogenesis. In the present retrospective study, serum VEGF and bFGF from patients with locally advanced non-small cell lung cancer (NSCLC) were analysed before, during and after treatment. Seventy-three patients and a total of 460 serum samples were analysed for VEGF and 443 serum samples were analysed for bFGF. Pre-treatment bFGF levels in patients with normal platelet counts, were correlated to poorer survival, P-value = 0.047. During chemotherapy, each rise of one unit bFGF corresponded to a hazard ratio of 4.06 (P=0.022). In patients with normal platelet counts, VEGF levels after radiotherapy significantly correlated to good prognosis (P=0.023), during radiotherapy VEGF levels indicated the same correlation (P=0.085). We conclude that serum VEGF and especially bFGF are of clinical interest as prognostic factors, especially in patients presenting with normal platelet counts.

Irradiation of brain metastases from lung cancer: a retrospective study.

A total of 94 patients with brain metastases from lung carcinomas were treated with irradiation of their brain metastases. Two fractionation schedules were applied, a non-conventional one (76 patients) mixing hypofractionation and accelerated hyperfractionation to a total dose of 47 Gy and a conventional one (18 patients), with 3 Gy once a day to a total dose of 30 or 36 Gy. No benefit was found for the non-conventional treatment schedule over the conventional one. A difference in survival was demonstrated between patients whose brain metastases originated from adenocarcinoma or squamous cell carcinoma of the lung with a median survival of 3.5 and 1.9 months, respectively (P = 0.006). Median survival of patients with brain metastases from small cell lung cancer (SCLC) was 2.8 months, and when compared with the squamous cell carcinoma group, there was no statistically improved survival (P = 0.12). There were indications of a better palliative effect in adenocarcinomas compared with squamous or large cell carcinomas. In a few patients (1/22 adenocarcinoma and 7/32 SCLC), the patients were free from malignant cells in the brain at autopsy, demonstrating that irradiation of brain metastases might be efficient in certain patients.

Different fraction schedules and combinations with chemotherapy in radiation treatment of non-small cell lung cancer.

BACKGROUND: In locally advanced non-small cell lung cancer (NSCLC), studies demonstrating advantages with hyperfractionated accelerated radiotherapy versus conventional radiotherapy have been published, so have studies demonstrating the value of chemotherapy concomitantly with radiotherapy. However, the value of non-conventional fractionation together with concomitant chemotherapy has not been investigated. MATERIALS AND METHODS: Consecutive patients from a single institution were studied in a retrospective non-randomised fashion. Inclusion criteria were stage III NSCLC, treatment with curative intent and a total dose above 50 Gy. RESULTS: Eighty-two patients were included and further divided into four different treatment groups. Multivariate analysis indicated a survival advantage with non-conventional radiotherapy (NCRT), especially if combined with concomitant chemotherapy. Toxicity was feasible, however there was a trend towards higher toxicity, mainly esophagitis, in patients given concomitant chemotherapy with NCRT. CONCLUSION: Our results suggest that accelerated hyperfractionated radiotherapy with concomitant chemotherapy could be an interesting test-arm in a future prospective study.

The frequency of micronuclei in lung cancer cell lines and their correlation to intrinsic radiation sensitivity.

BACKGROUND: Micronuclei arise from chromosomal, acentric, fragments or whole chromosomes that are not incorporated into the daughter nuclei at mitosis. The micronuclei assay is technically simple and requires only one mitosis in order to obtain information concerning the amount of micronuclei. This study was performed to investigate whether the formation of micronuclei could be used as a marker for intrinsic radiation sensitivity in lung cancer patients. MATERIALS AND METHODS: Two human lung cancer cell lines, a non-small cell lung cancer (NSCLC) cell line (U-1810) and a small cell lung cancer (SCLC) cell line (U-1906L), were used. Radiosensitivity data on the survival fraction at 2 Gy were obtained from the clonogenic assay. Radiation was delivered as one-fraction doses: 0, 1, 2, 4, 10 and 20 Gy. After irradiation, the cells were incubated for 0, 24, 48, 60, 72 and 96 hours before fixation and staining. RESULTS: The frequency of micronuclei in U-1906L was clearly elevated after 96 hours in the 20 Gy fraction. The frequency of micronuclei reached 5.5%. For U-1810 the micronuclei had a peak clearly different than the other settings after 48 hours in the 10 Gy fraction. The frequency of micronuclei was 1.2%. CONCLUSION: Counting micronuclei is not sensitive enough for estimation of radiosensitivity in clinical doses. However, our results demonstrated a distinct difference between NSCLC and SCLC cell lines at higher doses. This difference might be due to different repair fidelity, so future studies with this assay should aim to investigate this hypothesis.

Radiation responsiveness of human lung cancer cell lines measured with a short term semiautomatic assay.

BACKGROUND: Fluorometric microculture cytotoxicity assay (FMCA) is a short-term semi-automatic method, based on dye-inclusion of surviving cells. The assay was developed for investigations of drug resistance on tumour cells from biopsy material. In the present study, this short-term assay was evaluated, regarding usefulness in determining radio-sensitivity. MATERIALS AND METHODS: Eight human lung cancer cell lines were used. There were five small cell lung cancer (SCLC and three non-small cell lung cancer (NSCLC cell lines. Results were compared with the corresponding data derived from the clonogenic assay and/or the extrapolation method. RESULTS: The surviving fraction (SF) after 2, 5 and 10 Gy compared with data from the clonogenic assay were not in accordance for 5 of the 8 cell lines. The FMCA assay overestimated SF- values for the SCLC cell lines. CONCLUSION: The FMCA assay is not useful as a quick screening method for the radioresponsiveness in vitro of human tumour cell lines.

Serum p53 autoantibodies as prognostic marker in patients with oesophageal carcinoma.

BACKGROUND: Oesophageal carcinoma is one of the more aggressive cancers and the patients usually seek medical attention only when the disease is already advanced. Therefore it is important to have a tool, which is simple and fast, to measure and to predict the prognosis for these patients. Mutations in the p53 gene are among the most common genetic abnormalities in oesophageal carcinoma. The present study is the first study, to our knowledge, in which the relationship between the presence of p53 autoantibodies in the serum and survival has been investigated in patients with oesophageal carcinoma. PATIENTS AND METHODS: Serum from patients with oesophagal carcinoma was collected between 1996 and 1999 at the Department of Oncology, Uppsala University Hospital, Sweden. The serum samples were analysed for the presence of p53 autoantibodies using a sandwich ELISA. RESULTS: In a multivariate analysis, the presence of p53 autoantibodies was associated with decreased survival (p=0.047). Patients with extensive disease had a poor prognosis and time to death was decreased in these patients (p=0.000022). The one-year survival was 0% for these patients if they had p53 autoantibodies compared to 36% for patients with no p53 autoantibodies and extensive disease. CONCLUSION: We conclude that the presence of serum p53 autoantibodies is associated with decreased survival for patients with oesophageal carcinoma.

P53 auto-antibodies in non-small cell lung cancer patients can predict increased life expectancy after radiotherapy.

BACKGROUND: In this study we investigated whether the presence of p53 antibodies in sera before of during/after radiation therapy can predict increased survival in patients with non-small cell lung cancer. PATIENTS AND MATERIALS: Sera from 67 patients with a histopathologically confirmed diagnosis of nonsmall cell lung cancer have been investigated using sandwich ELISA (Dianova, Hamburg, Germany). Sera was collected before or during/after radiation therapy. RESULTS: Antibodies were detected in 18 (27%) patients. 46/67 (69%) of the sera had been taken before start of radiation therapy and the presence of p53 antibodies was a statistically significantly good prognostic factor in terms of increased survival (p = 0.025). CONCLUSION: p53 antibodies in sera, before the start of radiation therapy, can predict increased survival after radiation treatment in patients with non-small cell lung cancer.

Basic fibroblast growth factor and vascular endothelial growth factor in sera from non-small cell lung cancer patients.

BACKGROUND: The formation of new microvessels from the existing vascular bed is known as angiogenesis and is normally under the tight regulatory control of angiogenic factors. This control is lost in malignant tumours. Previous studies have correlated increased microvessel density with poor prognosis in patients with primary lung cancer. MATERIALS AND METHODS: Our group measured levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in sera from 68 patients with non-small cell lung cancer (NSCLC) and compared elevated levels of VEGF and bFGF with clinical outcome. Serum basic FGF and VEGF were measured using commercially available enzyme- linked immunosorbent assays (R & D Systems Inc., Minneapolis, MN USA). RESULTS: In 26/68 (38%) patients we found that elevated circulating levels of bFGF and in 27/68 (39%) serum samples levels of VEGF were elevated. Elevated bFGF values in sera was a statistically significant good prognostic factor, p- value = 0.048, when adjusted to stage and there was a trend in that patients with elevated levels of bFGF had a higher fraction of adenocarcinomas compared with squamous epithelial carcinomas (chi 2 = 2.0). No significant correlations could be demonstrated when elevated levels of VEGF in serum was present. Elevated levels of both VEGF and bFGF was present in 45% of the patients. CONCLUSIONS: We found that elevated levels of bFGF is a good prognostic factor when measured in sera from NSCLC patients. As this result disagrees with earlier studies on other malignancies the results from our study needs to be further investigated in a prospective study.

Cytokeratin 8 and 18 fragments measured in serum and their relation to survival in patients with non-small cell lung cancer.

BACKGROUND: In this study we investigated if the newly developed monoclonal antibodies against Cytokeratin 8 and 18 fragments (Cyk 8/18) have prognostic information in patients with non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Serum from 69 patients with NSCLC was investigated using a sandwich ELISA, Cyk 8/18, provided by IDL Biotech, Sollentuna Sweden. RESULTS: Cyk 8/18 levels varied between 0.34-14.2 ng/mL, compared with a cut-off value of 1.0 ng/mL for healthy individuals (95% specificity). Using that cut-off value, 80% of NSCLC patients had elevated levels. A statistically significant diminished survival was found for Cyk 8/18 values of 8.0 ng/mL or higher (p = 0.0001). When survival data and Cyk 8/18 levels were analysed according to continuous Cox regression analysis, increased levels of Cyk 8/18 were significantly related to decreased survival (p = 0.016). CONCLUSIONS: The Cyk 8/18 monoclonal antibody had in this study prognostic information regarding survival in patients with NSCLC.

Complete sequence of p53 gene in 20 patients with lung cancer: comparison with chemosensitivity and immunohistochemistry.

In this study the entire p53 complementary DNA has been sequenced in 20 non-small cell lung carcinomas (NSCLC) and the results correlated with chemosensitivity, immunohistochemistry and clinical data. Ten patients had mutations in p53, 8 missense mutations and 2 nonsense mutations. The method discovered two mutations never described previously and two other mutations that have never been described before in connection with NSCLC tumours. Chemosensitivity data, according to a short-term assay (FMCA), indicated that tumours with p53 mutation were more resistant to cisplatin and cyclophosphamide. Immunohistochemical studied demonstrated a 70% concordance between over-expression of p53 protein and mutation in p53. No conclusions or trends could be drawn from the immunohistochemical studies of Bcl-2 and Bax.

HER-2 overexpression (3+) in patients with squamous cell esophageal carcinoma correlates with poorer survival.

The incidence of esophageal carcinoma is increasing worldwide. In Sweden, approximately 400 patients are diagnosed each year. The present study retrospectively investigates survival in 97 patients with esophageal carcinoma in regard to their HER-2 status as examined by immunohistochemistry (IHC) and chromogen in situ hybridization (CISH). Sixty-eight patients had localised disease and 29 patients had advanced disease. Seventy patients had squamous cell carcinoma, and nine of these patients (13%) had HER-2 overexpression (3+). Eight (30%) of 27 adenocarcinoma patients overexpressed (3+) HER-2. In patients overexpressing (3+) HER-2 a statistical trend towards poorer survival was observed (P = 0.057). In squamous cell carcinoma patients, HER-2 overexpression (3+) correlated with poorer survival (P = 0.035), whereas in adenocarcinoma patients, HER-2 status (3+) did not. HER-2 amplification according to CISH was present in five (two squamous cell carcinomas and three adenocarcinomas) out of 17 HER-2 overexpressing (3+) tumours. In conclusion, HER-2 overexpression (3+) seems to be associated with poorer survival in esophageal carcinomas, especially in patients with squamous cell esophageal carcinoma.

Genes associated with telomerase activity levels in esophageal carcinoma cell lines.

Telomerase activity levels have been shown to correlate with tumor progression in several malignancies. However, the genetic regulation of telomerase activity levels is not fully understood. The aim of the present study has been to identify a gene expression profile, predicting correlation with the telomerase-activity test. Ten human esophageal carcinoma cell lines were investigated using the telomerase activity assay (TRAPeze) Telomerase Detection Kit), followed by further characterization using the GeneChip Human Genome U133A 2.0 Array (Affymetrics Inc., USA), including 14 500 human genes. Telomerase activity levels were detected in all cell lines with a broad range of activity levels. Using a high correlation coefficient, r > 0.90, the following genes were found to be positively correlated with telomerase activity levels: N-myristoyltransferase 2; ribosomal protein L3; retinoblastoma-like 2 (pRb2/p130); and cyclin G2. Only one gene was negatively correlated with telomerase activity levels, zinc finger protein 207. In conclusion, the present microarray data provide primary validation data indicating possible candidates for prognostic and prediction factors in esophageal cancer in relation to telomerase activity.

Newly developed assay measuring cytokeratins 8, 18 and 19 in serum is correlated to survival and tumor volume in patients with esophageal carcinoma.

Esophageal carcinoma is the seventh most common cause of cancer-related death in the Western world. In Sweden, approximately 400 new esophageal carcinomas are diagnosed yearly. Cytokeratins (CK) are specific for epithelial cells and the expression profile usually remains unchanged even when the epithelium undergoes malignant transformation. In the present study, MonoTotal, a newly developed RIA-assay detecting circulating CK 8, 18 and 19 fragments, was investigated in sera from patients with esophageal carcinoma. Serum samples from 40 patients with esophageal carcinoma were collected. The median value of circulating CK 8, 18 and 19 measured with MonoTotal was 378 U/L (range 53-6843) and with regard to the defined cut-off (< 75 U/L), 39/40 (98%) patients were shown to have elevated levels of circulating CK 8, 18 and 19. Patients with localized disease had a median value of circulating CK 8, 18 and 19 of 305 U/L (mean: 500 U/L), whereas the corresponding value for metastatic disease was 771 U/L (mean: 1506 U/L). This difference was statistically significant (P = 0.016). Circulating CK 8, 18 and 19, according to cut-off, were not associated with survival in univariate analysis (P = 0.34). However, continuous values of circulating levels of CK 8, 18 and 19 were associated with survival (P = 0.000083) in univariate as well as in the multivariate analysis (P = 0.03). In conclusion, circulating CK 8, 18 and 19 correlates with increased tumor burden and might, in conjunction with other clinical parameters, aid the clinician in estimating the prognosis of the individual patient.