Mario D'Costa

p53 protein is absent from the serum of patients with lung cancer

p53 protein, which accumulates intracellularly in over half of all human tumours, has also been reported to be present in the sera of patients with various malignancies, including lung cancer. Using a quantitative immunoassay, we measured p53 protein concentrations in 216 sera from 114 lung cancer patients of whom 75 provided matched lung tumour tissues, which were also assayed for p53 protein. p53 protein levels above the detection limit of 0.04 ng ml-1 were detected in only two sera from lung cancer patients (0.14 ng ml-1 and 0.27 ng ml-1), but not in any of 13 sera from non-malignant lung disease patients or in 100 sera from normal non-diseased individuals. The presence of these apparent traces of serum p53 protein concentrations could not be related either to the p53 protein expression status of the primary lung tumours or to the tumour stage, grade or histological type. By pretreating these two sera with anti-p53 antibody linked to solid phase, and by the addition of mouse serum to neutralise possible heterophilic antibodies, the signals arising from these sera were shown to be non-specific and possibly caused by heterophilic antibodies. We conclude that our data do not support previous reports of p53 protein in the sera of lung cancer patients. Since immunoassays are subject to numerous sources of interference in serum, including heterophilic antibodies, we suggest that the results of p53 protein analysis of serum specimens should be interpreted with caution.

Lecithin/sphingomyelin ratios in tracheal aspirates from newborn infants.

ABSTRACT. A sensitive and rapid high-performance liquid chromatography method for determining lecithin/sphingomyelin (L/S) ratios in small volume tracheal aspirates has been evaluated in 115 infants sampled within 3 h of delivery. Respiratory distress syndrome was present in 41 infants and all had L/S ratios of ≥15 (sensitivity 100%). Respiratory distress syndrome was absent in 74 infants, 67 of whom had L/S ratios of >15 (specificity 91%). Serial tracheal aspirates (n = 68) in 27 infants showed little change in nine respiratory distress syndrome infants with L/S ratio ≥15 over 3 h while 18 nonrespiratory distress syndrome infants with L/S >15 were more variable. In two infants initial immature ratios rose to maturity rapidly after birth. Comparison of amniotic fluid L/S ratios obtained within 2 h of delivery correlated with the corresponding tracheal aspirate L/S in 24 infants (r = 0.81, p < 0.001) although the latter were three times higher. This method may have potential routine application in the assessment of surfactant replacement and mechanical ventilator therapies.

Quantitative analysis of p53 protein in non-small cell lung cancer and its prognostic value

Accumulation of mutant p53 protein occurs frequently in human malignancies, including 40–60% of non‐small cell lung carcinomas. The implications of such p53 over‐expression, usually assessed by immunohistochemical techniques, for the prognosis of lung cancer patients remain undetermined. In this study, we used a time‐resolved immunofluorometric assay to measure p53 protein concentrations in extracts prepared from 86 primary non‐small cell lung tumours and examined the associations between p53 protein levels (corrected for total protein) and other clinico‐pathologic variables, including post‐surgical disease‐free and overall survival. Contingency tables analysed by χ2 tests revealed no significant relationships between p53 status, defined by a median cut‐off point, and patient gender, age, disease stage, histologic grade and type, lymph node extension, smoking history and administration of adjuvant chemotherapy or radiation. However, multivariate Cox proportional hazard regression analysis demonstrated a dose‐response relationship between p53 concentration, expressed as a 4‐level, quartile‐divided variable, and increased risk of relapse (p = 0.010) and death (p = 0.016). Patients whose tumours contained p53 concentrations exceeding the median value had over 3‐fold higher risk of relapse (p= 0.002) and death (p = 0.007) than those whose tumours had lower p53 concentrations. We also provide evidence suggesting that the impact of p53 on survival is greater in patients with squamous cell carcinoma than in those with adenocarcinoma. Although the latter finding needs confirmation, our results suggest that application of an immunoassay of p53 protein on non‐small cell lung tumour extracts may identify patients at increased risk of unfavourable outcome. Int. J. Cancer (Pred. Oncol.) 79:494–501, 1998.© 1998 Wiley‐Liss, Inc.

Selective determination of urinary free cortisol by liquid chromatography after solid-state extraction

We have developed a selective and precise high-performance liquid chromatographic method for urinary free cortisol with an improved and efficient sample clean-up using C18 Sep-Pak cartridges. The urine sample (2 ml), with 11-deoxycortisol as internal standard, is applied to the Sep-Pak, which is then sequentially washed with acetone-water (1:4, v/v), water and hexane. Cortisol is eluted with diethyl ether, evaporated to dryness and redissolved in 2 ml of water. The wash cycle is repeated once using the same Sep-Pak cartridge. This double extraction greatly improves sample clean-up and allows modification of the mobile phase (tetrahydrofuran-methanol-water) so that cortisol is rapidly eluted as a single well resolved peak at 13 min. Chromatography is performed isocratically on a reversed-phase column with detection at 254 nm. Detection limits for urinary free cortisol by this procedure were two or three times lower than those obtained with two commercial radioimmunoassay kits. The chromatographic method was used successfully in the diagnosis of patients with hypercortisolism and Cushings syndrome.

Validation of a simple rapid high performance liquid chromatographic method for amniotic fluid lecithin/sphingomyelin ratios

A simple, rapid and sensitive HPLC method for the determination of L/S ratios in amniotic fluids is described. The method is based on isocratic separation in the normal phase with UV detection. The procedure has good precision and was validated clinically and by comparison with a routine TLC method. Although L/S ratios differed from those obtained by TLC, the clinical correlation between these methods was good. In single and serial samples from 39 mothers (42 babies) the HPLC method predicted respiratory distress syndrome (RDS) in all 9 babies with L/S ratios less than 7. No babies with ratios above 7 developed RDS. This method has potential clinical usefulness for the assessment of fetal lung maturity.

p21WAF1 protein expression determined by quantitative immunoassay in relation to non-small-cell lung cancer aggressiveness

Purpose: p21WAF1, a cyclin-dependent kinase inhibitor, is an important mediator of the cell-cycle arrest and tumor suppression induced by the protein p53. Although alterations of the p53 gene and its overexpression are frequent in most malignancies, including non-small-cell lung cancer (NSCLC), and may be associated with poor patient prognosis, the clinical utility of p21WAF1 expression in NSCLC has not been established. Methods: We have used a commercial enzyme-linked immunosorbent assay (ELISA) kit for p21WAF1 to test soluble extracts of 54 NSCLC specimens with known clinicopathological properties. Results: There was no correlation between p21WAF1 and p53 concentrations, the latter being determined by a time-resolved immunofluorometric assay developed in-house. Furthermore, p21WAF1 levels were not associated with patient age, tumor/node/metastasis (TNM) stage, lymph node metastasis, histological grade or type, or smoking history, in Mann-Whitney analysis. χ2-tests, based on cutoffs equal to the 25th, 50th, or 75th percentiles of the p21WAF1 distribution, similarly did not reveal any statistically significant associations between p21WAF1 and other clinicopathological variables. Because of the small number of patients and the median follow-up of only 18 months, a meaningful survival analysis could not be performed. Conclusion: In summary, this preliminary study suggests that ELISA-quantified p21WAF1 levels in NSCLC extracts are weaker than p53 in terms of prognostic value and do not contribute to the further subclassification of patients.