Diagnostic Accuracy Of Vascular Endothelial Growth Factor And Human Telomerase Reverse Transcriptase In Malignant Pleural Effusions

Background: While the early diagnosis of cancer has been fully respected, it is still however often difficult for clinicians to confirm malignant pleural effusions (PE), which essentially indicate the end-stage cancer. It has now been demonstrated that vascular endothelial growth factor (VEGF) is a pivotal angiogenesis factor and associated with tumor growth and metastasis. The aim of this study was then to assess the diagnostic performance of VEGF in malignant PE.Methods: In this controlled and blinded prospective study, 113 consecutive patients with PE were recruited. For each recruited patient, during the course of routine diagnostic procedures and upon the patient's informed consent, 10 mL PF and 2 mL serum would be saved simultaneously for VEGF test while the first thoracentesis was performed if possible. Given the patient's informed consent or qualified specimens unavailable, this case would then be excluded. As did if the clinically definite etiological diagnosis of PE or the laboratory conclusive result of index test was absent finally. For each eligible case, the VEGF levels of pleural fluid (PF) and serum were examined simultaneously using enzyme immunoassay. The reference standard for malignant PE was clinical evaluation and PF cytology with pleural biopsy, other examination and follow-up added as needed. The laboratory VEGF test was completely independent of clinical diagnosis and treatment. The readers of the index tests and reference standard were blind to the results of the other test. Only after completion of all the experimental tests of VEGF and clinical data collection, we analyzed the results of VEGF test in accordance with those of reference standard. For each VEGF test strategy, the results in malignant and benign groups were firstly described and compared. If there was a statistical difference, the sensitivity and specificity of this test for malignant PE, including rates of sample and 95％confidence intervals, were reached by counting. For measurement data, the diagnostic cutoff point was primarily calculated via receiver operating characteristic curve. Followed by statistical analysis, different test strategies regarding VEGF in diagnosing malignant PE were assessed and compared.Results: According to the final diagnoses, 81 qualified cases were grouped as malignant (n=32) and benign (n=49) PE. For PF VEGF level, the mean in malignant group was higher than that in benign group (1358±1493 pg/mL vs. 422±317 pg/mL, P=0.001). As did for serum VEGF level (650±533 pg/mL vs. 137±189 pg/mL, P＜0.001). Using receiver operating characteristic analysis, the determined diagnostic cutoff points of VEGF levels of PF and serum for malignant PE were 959.25 pg/mL and 212.36 pg/mL, with sensitivities of 47％, 69％and specificities of 96％, 88％, respectively. For cascade connection and parallel operation of PF VEGF and serum VEGF, the sensitivities were 34％, 81％at specificities of 98％, 86％, respectively.Conclusions: These findings suggest that VEGF could be used in diagnosing malignant PE as a useful adjunct of conventional algorithm. Different VEGF test strategies, including test on PF, serum and both, may be selected according to practical needs. Background: Detection of human telomerase reverse transcriptase (hTERT) mRNA by in situ hybridization (ISH) may be valuable in the diagnosis of cancer. We assessed the diagnostic performance of hTERT mRNA in cells from pleural fluid in malignant pleural effusions (PE).Methods: We used a 2-step ISH with digoxin-labelled oligonucleotide probes to detect hTERT mRNA in a blinded prospective study of cells from 103 unselected pleural fluid specimens. For detection of hTERT mRNA in cells from pleural fluid by a 2-step ISH, the chief procedures of manipulation were as follows: preparation and preservation of cell smears; treatment before hybridization, such as deactivation of endogenous peroxidase, revelation of mRNA in cells on the slides, prehybridization and blockage of non-specific IgG; the 2-step hybridization in situ: treatment after hybridization, such as blockage of endogenous biotin, addition of mouse biotinylated anti-digoxin antibody and subsequent incubation, addition of high sensitive peroxydase complex and subsequent incubation and coloration; observation, record and analysis of experimental results. To primarily observe the specificity and sensitivity of experimental results respectively and compare the experimental results with theoretical expectation, negative control and positive control were established in this test of hTERT mRNA ISH. Based on the results of preliminary experiments, under the light microscope, tumor cells showed dark-staining cytoplasm, and the nucleus was nearly colorless except for a few stained spots. Other cells on the slices of positive control were semitransparent, almost without color. The background of slices almost had no color, or merely with superficial pigmentation. On the negative control sections, almost all cells were semitransparent, and without coloration, and the background of slices almost had no color. In the absence of probes or after treatment of cells with RNase, specimens that served as positive control showed cells with abnormal morphology could be seen indistinctly. So, the criteria to judge positive and negative results of experiment were established as follows: if cells with dark-staining cytoplasm and the nucleus almost without coloration except for a few stained spots, the result would be judged as positive regardless of the cytomorphological findings; other result would be called negative. All ISH slides were assessed and recorded independently by two observers. Discrepancies were resolved by discussion including other experienced workers. The reference standard for malignant PE was clinical evaluation and pleural fluid cytology, combined with pleural biopsy, other examination and follow-up as needed. After completion of hTERT mRNA testing and clinical data collection, we compared the results of detection of hTERT mRNA in cells from PF by two-step ISH with the relevant clinical data, such as the results of cytological examination and the outcome of over one year of follow-up.Results: According to the final diagnoses, there were 41 malignant PE, 55 benign PE and 7 cases with uncertain etiology. When the 7 cryptogenic cases were excluded, the sensitivity and specificity of detectable hTERT mRNA for malignancy were 80% and 95%, respectively. When detection of hTERT mRNA was combined with clinical repeated pleural fluid cytology, the sensitivity and specificity were 90% and 95%, respectively. Conclusions: Detection of hTERT mRNA in cells from pleural fluid by ISH could potentially be used in diagnosing malignant PE as an aid. Further investigations with stricter controls and cross-validation tests will be warranted.