Summary Amplification of the CCDNI gene encoding cyclin DI was examined by Southern blotting and multiplex polymerase chain reaction (PCR) and occurred in 8 of 53 patients (15%) with primary resected nonsmall-cell lung cancer (NSCLC). These tumours and 17 additional tumours with a normal gene copy number showed overexpression of cyclin Dl (25/53, 47%), as assessed by immunostaining using a monoclonal antibody. In 22/25 cases, cyclin DI was localised in the cytoplasm, but some (7/25) had simultaneous nuclear staining. This result is in marked contrast to that reported in breast, hepatocellular and colorectal carcinoma studies where immunostaining was invariably nuclear. Examination of a restriction fragment length polymorphic (RFLP) site within the 3'untranslated region of the cDNA following reverse transcriptase (RT)-PCR (29/53 informative cases) showed a strong association between cytoplasmic staining and imbalance in allele-specific message levels. Cyclin Dl overexpression was associated with a poorly differentiated histology (P=0.04), less lymphocytic infiltration of the tumour (P=0.02) and a reduction in local relapse rate (P =0.01). The relative risk of local relapse was 9.1 in tumours without cyclin Dl overexpression (P = 0.01, Cox regression analysis). We conclude that genetic alteration of cyclin Dl is a key abnormality in lung carcinogenesis and may have diagnostic and prognostic importance in the treatment of resectable NSCLC.

Quantitative reverse transcriptase real-time PCR (QRT-PCR) is a robust method to quantitate RNA abundance. The procedure is highly sensitive and reproducible as long as the initial RNA is intact. However, breaks in the RNA due to chemical or enzymatic cleavage may reduce the number of RNA molecules that contain intact amplicons. As a consequence, the number of molecules available for amplification decreases. We determined the relation between RNA fragmentation and threshold values (Ct values) in subsequent QRT-PCR for four genes in an experimental model of intact and partially hydrolyzed RNA derived from a cell line and we describe the relation between RNA integrity, amplicon size and Ct values in this biologically homogenous system. We demonstrate that degradation-related shifts of Ct values can be compensated by calculating delta Ct values between test genes and the mean values of several control genes. These delta Ct values are less sensitive to fragmentation of the RNA and are unaffected by varying amounts of input RNA. The feasibility of the procedure was demonstrated by comparing Ct values from a larger panel of genes in intact and in partially degraded RNA. We compared Ct values from intact RNA derived from well-preserved tumor material and from fragmented RNA derived from formalin-fixed, paraffin-embedded (FFPE) samples of the same tumors. We demonstrate that the relative abundance of gene expression can be based on FFPE material even when the amount of RNA in the sample and the extent of fragmentation are not known.

Summary Four patients underwent intraoperative photodynamic therapy after surgery with meso-tetra-(hydroxyphenyl)-chlorin (mTHPC-PDT) for diffuse malignant mesothelioma. Preliminary procedures were performed in two patients in order to establish the efficacy of mTHPC-PDT and to optimise its tumoricidal effect. The (Faber, 1988). Improved local control does require additional measures, but the disease responds poorly to radio-and chemotherapy (Lerner et al., 1983). As photodynamic therapy (PDT) has been reported to be effective in human mesothelioma xenografts (Feins et al., 1990), it might allow for an appropriate 'clean-up' of the thoracic cavity after surgery. For clinical purposes, the currently used sensitisers for PDT are haematoporphyrin derivatives (HpD) and dihaematoporphyrin ether (DHE) . However, PDT with meso-tetra-(hydroxyphenyl)-chlorin (mTHPC) was superior to DHE-PDT with respect to antitumour activity and tissue selectivity in rodents without causing significant toxicity (Berenbaum, 1989). mTHPC might therefore be better fitted to large surface PDT as required for diffuse malignant mesothelioma treatment. A pilot study was done to evaluate mTHPC-PDT for diffuse malignant mesothelioma with respect to its antitumour activity and the feasibility of a combined modality approach under clinical conditions. Patients and methodsFour patients underwent mTHPC-PDT for diffuse malignant mesothelioma. Each patient was informed in detail about the experimental nature of the procedure and consent was obtained from each patient and from the local Human Investigations Committee of our institution.The four men were aged 46 (patient 1), 48 (patient 2), 65 (patient 3) and 50 years (patient 4), all having had possible occupation related exposure to asbestos. The main symptoms at admission were dyspnoea due to pleural effusion, chest pain and loss of weight. There was no evidence of disease in the peritoneal and contralateral chest cavity on CT-scans at admission. The right side was involved in patients 1, 2 and 4 and the left in patient 3. Previous biopsies revealed an epithelial (Figure la), a biphasic (Figure 2a), a sarcomatous and a mixed type of mesothelioma in the four patients and was confirmed in every case by immunohistologic examinations.Preliminary PDT To establish the efficacy of mTHPC-PDT and to optimise its tumouricidal effect, preliminary PDT was performed in patients 1 and 2 prior to its definitive application. Modulations of mTHPC dose, light dose and of the time interval between mTHPC application and activation were tested. mTHPC (Scotia Pharmaceuticals Ltd, Guildford, UK) was dissolved in 20% ethanol, 30% polyethylene glycol 400 and 50% H20 and administered over 15 min i.v. through a bacterial filter under sterile conditions within 60 min of preparation. Argon-pumped dye laser light of 650 nm (Coherent Innova 200 and Dye CR 599, GMP SA, Lausanne, Switzerland) was delivered through a sterilised optical fibre on tumour areas of 3 cm diameter. The power at the end of the optical fibre was measured wi...

No abstract

No abstract