Abstract
Amplification of the CCDN1 gene encoding cyclin D1 was examined by Southern blotting and multiplex polymerase chain reaction (PCR) and occurred in 8 of 53 patients (15%) with primary resected non-small-cell lung cancer (NSCLC). These tumours and 17 additional tumours with a normal gene copy number showed overexpression of cyclin D1 (25/53, 47%), as assessed by immunostaining using a monoclonal antibody. In 22/25 cases, cyclin D1 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 D1 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 D1 overexpression (P = 0.01, Cox regression analysis). We conclude that genetic alteration of cyclin D1 is a key abnormality in lung carcinogenesis and may have diagnostic and prognostic importance in the treatment of resectable NSCLC.
Highlights
In order to examine the possibility whether the immunostaining result was due to fixation artifacts we analysed 33 nonsmall-cell lung cancer (NSCLC) tumour sections prepared in the Wythenshawe Hospital for cyclin Dl overexpression
In 8/53 (15%) of the NSCLC a 3- to 20-fold amplification of the CCNDI gene was identified (5/35 squamous cell, 2/11 adeno- and 1/2 large cell carcinoma, Figure 1). This result was confirmed by a polymerase chain reaction (PCR) multiplex comparing CCNDI amplification with the amplification of the adenosine deaminase gene, and numerical differences in chromosome 11 copy number were excluded by a PCR multiplex, comparing CCNDI with the progesterone receptor gene located on the same arm of chromosome 11 (Figure 1)
Five patients with amplification were informative for the cyclin DI gene polymorphism (Heighway, 1991); allelic imbalance was observed in each case supporting the amplification data obtained by Southern blotting and PCR multiplex
Summary
Tumour samples were obtained from 53 consecutive patients [49 men, four women, median age 64 years (45 -79)] who Correspondence: J Heighway Received 23 May 1995; revised 15 August 1995; accepted 22 August 1995 underwent resection of staged resectable NSCLC at the University Hospital of Berne, Switzerland. They had received no therapy before surgery [pneumonectomy (n = 15) or lobectomy (n= 38)]. Tumour size was measured by the pathologists on the fresh specimen. In order to examine the possibility whether the immunostaining result was due to fixation artifacts we analysed 33 NSCLC tumour sections prepared in the Wythenshawe Hospital for cyclin Dl overexpression
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