Modulation of proliferating cell nuclear antigen in the bronchial epithelium of smokers.

Clinical chemoprevention trials seek to intervene in the carcinogenic process to suppress, reverse, or delay the development of invasive cancer. Dysregulated cell growth is a hallmark of epithelial carcinogenesis, and proliferating cell nuclear antigen (PCNA) is a marker of dysregulated proliferation that is highly expressed in non-small cell lung cancers. Squamous metaplasia of the bronchial epithelium is found in chronic smokers and has been considered an early premalignant change. To evaluate the effect of 13-cis-retinoic acid (13-cRA) on PCNA modulation, we evaluated PCNA expression in a total of 706 bronchial biopsy specimens from histologically normal, hyperplastic, metaplastic, and dysplastic bronchial tissues obtained from 86 healthy smokers at baseline, of whom 69 subjects had completed 6 months of treatment on a randomized placebo-controlled chemoprevention trial of 13-cRA and had repeat bronchoscopic biopsies. PCNA expression was evaluated with respect to bronchial metaplasia and as an intermediate end point for response in the trial. In the bronchial biopsies obtained from six standardized pretreatment and posttreatment sites, high PCNA expression correlated significantly with more advanced histological grade (P < 0.001). Furthermore, smoking cessation during therapy correlated well with reduced PCNA expression (P = 0.006), although multivariate analysis indicated that this reduction in PCNA expression was associated with the reversal of squamous metaplasia. The level of PCNA expression appeared to correlate with the level of epidermal growth factor receptor expression both at baseline and at 6 months. In those patients who ceased smoking during the intervention, the 13-cRA also appeared to be more effective than placebo in reducing PCNA expression (P = 0.034 in all of the layers; P = 0.026 in basal layers). The efficacy of 13-cRA in the down-regulation of PCNA in quitters was independent of baseline PCNA expression levels. Our study demonstrated that increased PCNA expression was associated with histological progression from normal bronchial epithelium to squamous metaplasia and dysplasia. The modulation of PCNA by 13-cRA in patients who quit smoking suggests a potentially important role for regulating this proliferation marker in retinoid chemoprevention studies of former smokers.

A pilot clinical laboratory trial of paclitaxel and endobronchial brachytherapy in patients with non-small cell lung cancer.

Paclitaxel enhances microtubule assembly and causes a cell cycle arrest in mitosis, the most radiosensitive phase. We conducted this study to improve our understanding of paclitaxel effects in vivo and to determine the maximum tolerated dose of paclitaxel preceding endobronchial radiation therapy (brachytherapy). The treatment consisted of two cycles of paclitaxel infused over 24 hours followed by 192Ir brachytherapy; cycles were repeated every 3 weeks. Tumor samples were obtained at baseline, after each paclitaxel infusion, and 3 weeks after completion of therapy. Twenty-two non-small cell lung cancer patients with a documented endobronchial lesion were enrolled in the study and 20 patients received the therapy with different doses of paclitaxel, initially without and then later with granulocyte colony-stimulating factor (G-CSF) support (5 microg/kg subcutaneously on days 3 to 10). With the starting paclitaxel dose of 135 mg/m2, five of seven patients developed neutropenia and fever, which mandated a dose reduction to 120 mg/m2. At this dose level, three of three patients had neutropenic fever; thus, 120 mg/m2 of paclitaxel was considered above the maximum tolerated dose without G-CSF support. However, with G-CSF support the therapy was well-tolerated without dose-limiting toxicity and accrual is continuing at the paclitaxel 175-mg/m2 dose level. While no patient had achieved systemic tumor response, 11 patients achieved partial response of the endobronchial lesion, which represents 68.8% of 16 patients who received two courses of therapy and 91.8% of 12 patients who had full evaluation by bronchoscopy after completion of therapy. The in vivo paclitaxel effects were studied using the pre- and post-paclitaxel therapy tumor samples in eight patients. Four (50%) patients had a significant increase in mitotic cells after paclitaxel, as assessed by MPM-2 immunostaining that recognizes a large family of mitotic phosphoproteins. A substantial increase in the number of micronucleated apoptotic cells, another paclitaxel effect, was also found in six patients. These results clearly indicate that patients with endobronchial lesions from recurrent NSCLC could not tolerate this combined modality regimen without G-CSF support. However, this group of patients provided a unique opportunity to study in vivo paclitaxel effects in a clinical trial setting.

Comparison of p53 immunoreactivity in fresh-cut versus stored slides with and without microwave heating.

Because of technical problems in immunohistochemical staining of archival material, antigens can be masked or lost. A recent study reported diminished p53 immunoreactivity in slides that had been sectioned from parafinn-embedded tissue blocks and stored at room temperature. To investigate this issue, we performed immunohistochemical staining with use of a p53 monoclonal antibody (DO7) in 13 head and neck squamous cell carcinoma (HNSCC) and 13 non-small-cell lung carcinomas. The fresh-cut and stored slides were simultaneously stained with and without the use of a microwave heating (MWH) technique, and we compared the results of p53 immunostaining. The stored slides were sectioned from paraffin blocks 4 to 25 years old and kept at room temperature for 6 to 48 months. The slides were blindly evaluated for percentage of positivity and staining intensity. Twelve HNSCCs and six lung carcinomas showed p53 positivity. The stored slides showed a considerable decrease in staining intensity (P = 0.039), compared with fresh-cut slides. The difference in the percentage of positivity between the stored and fresh-cut slides was statistically significant, but the mean value of the difference was only 3.6%, which might not be meaningful for semiquantitation of immunostaining. MWH greatly enhanced staining intensity and percentage of positivity for both stored and fresh-cut slides. When MWH was applied, no significant difference in staining intensity (P = 0.063) was detected in fresh-cut versus stored slides, but the difference in the percentage of positivity was statistically significant (mean value, 3.1%). Individual cases showed a consistent p53 status regardless of the MWH treatment, storage duration, or age of the blocks. This study demonstrated a considerable decrease in p53 immunoreactivity in stored slides. Because the MWH successfully retrieved the p53 antigen without causing a change in p53 status, stored slides combined with an MWH antigen retrieval technique in a metal-containing solution should provide p53 immunostaining results similar to those from fresh-cut slides, as long as staining intensity is not a sole study parameter.

Expression of p53 oncoprotein in non-small-cell lung cancer: a favorable prognostic factor.

PURPOSE: Mutation of the p53 gene is one of the most common genetic abnormalities found in lung cancer. The purpose of this study was to evaluate the prognostic value of p53 oncoprotein expression in patients with non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: We studied 156 resected primary NSCLCs by the immunohistochemical staining technique, using the p53 antibody DO7. There were 81 adenocarcinomas, 16 large-cell carcinomas, and 59 squamous cell carcinomas; stages were I in 67, II in 30, and III in 59 cases. For each tumor, the percentage of p53 positivity was calculated by scoring a minimum of 1,000 cells on an arbitrary intensity scale of 0 to 3+. RESULTS: Overall, 103 (66%) tumors expressed p53 in more than 0.1% of cells, and squamous cell carcinomas tended to express more p53 than adenocarcinomas. Since 50% positivity marked the most distinct change in overall survival duration (P = .0008), we divided the cases into three groups, as follows: p53-negative (< or = 0.1%, n = 53), low p53 (0.1% to 50%, n = 54), and high p53 (> 50%, n = 49). Overall, patients in the high-p53 group survived longer than those in the low or negative groups, with respective median survival durations of more than 65, 26, and 33 months (P = .002). The survival difference among the three groups was statistically significant for non-squamous cell (P = .008), but not for squamous cell (P = .17) carcinomas. Among lymph node-negative patients, the survival difference between groups was not statistically significant. However, among lymph node-positive patients (n = 78), more than 65% of the high-p53 group survived for more than 70 months, while the median survival durations for the low and negative groups were 21 and 18 months, respectively (P = .001). A Cox regression analysis with multiple covariates showed that p53 positivity in more than 50% of tumor cells was an independent prognostic factor for better outcome. CONCLUSION: These results suggest that high expression of the p53 oncoprotein is a favorable prognostic factor in a subset of patients with NSCLC.