Genes of the polycomb group function by silencing homeotic selector genes that regulate embryogenesis. In mice, downregulation of one of the polycomb genes, bmi-1, leads to neurological alterations and severe proliferative defects in lymphoid cells, whilst bmi-1 overexpression, together with upregulation of myc-1, induces lymphoma. An oncogenic function has been further supported in primary fibroblast studies where bmi-1 overexpression induces immortalization due to repression of p16/p19ARF, and where together with H-ras, it readily transforms MEFs. It was the aim of this study to assess the expression of bmi-1 in resectable non-small cell lung cancer (NSCLC) in association with p16 and p14ARF (=human p19ARF). Tumours (48 resectable NSCLC (32 squamous, 9 adeno-, 2 large cell, 4 undifferentiated carcinomas and 1 carcinoid); stage I, 29, II, 7, III, 12; T1, 18, T2, 30; differentiation: G1 12, G2 19, G3 17) were studied by immunohistochemistry for protein expression and by comparative multiplex PCR for gene amplification analysis. In tumour-free, normal lung tissue from patients, weak – moderate bmi-1 staining was seen in some epithelial cells, lymphocytes, glandular cells and in fibroblasts, whereas blood, endothelial, chondrocytes, muscle cells and adipocytes did not exhibit any bmi-1 expression. In tumours, malignant cells were negative/weakly, moderately and strongly positive in 20, 22 and 6 cases, respectively. As assessed by multiplex PCR, bmi-1 gene amplification was not the reason for high-level bmi-1 expression. Tumours with moderate or strong bmi-1 expression were more likely to have low levels of p16 and p14ARF ( P = 0.02). Similarly, tumours negative for both, p16 and p14ARF, exhibit moderate–strong bmi-1 staining. 58% of resectable NSCLC exhibit moderate–high levels of bmi-1 protein. The inverse correlation of bmi-1 and the INK4 locus proteins expression (p16/p14ARF) supports a possible role for bmi-1 misregulation in lung carcinogenesis. © 2001 Cancer Research Campaign www.bjcancer.com

The CDKN2 locus expresses two di erent mRNA transcripts, designated a and b. The protein product of the a transcript is the cell cycle inhibitor and tumour suppressor p16INK4a. The b transcript is translated in an alternate reading frame (ARF) and in humans encodes a 15 kDa protein (p19ARF). Immunohistochemical and Western analysis of p16INK4a has shown that the protein is downregulated in a signi®cant number of tumours, but less is known on the expression of the p19ARF. We have examined the expression of p16INK4a and p19ARF in resectable non-small cell lung cancer (NSCLC) by immunostaining (n=49) and multiplex RT ± PCR (n=28). In order to investigate the mechanism responsible for p16INK4a downregulation, exon 1a methylation was analysed in a PCR-based assay. Of 49 tumours examined by immunostaining, 24 and 20 tumours expressed p16INK4a and p19ARF at nil to low levels, respectively. p19ARF was localized primarily to the nuclei of tumour cells, but was also seen to varying degrees in nuclei of lymphocytes, chondrocytes, ®broblasts, and epithelial cells. No tumour with normal p16INK4a had decreased p19ARF expression. Among 16 tumours with nil to low p16INK4a expression, 11 tumours exhibited full methylation of at least one site within exon 1a and these tumours showed normal p19ARF expression. In contrast, no methylation of exon 1a was observed in ®ve tumours which also lacked p19ARF. In normal lung, p16INK4a and p19ARF were not expressed at detectable levels, the multiplex RT ± PCR results were balanced, and sites within exon 1a were strongly methylated. In tumours, imbalanced multiplex RT ± PCR data (p16INK4a5p19ARF) predicted methylation of exon 1a (P=0.0006) as well as downregulation of p16INK4a. p19ARF downregulation was inversely correlated with p53 overexpression (P=0.025), whilst negative immunostaining for p16INK4a was inversely correlated with pRb downregulation (P=0.003) and directly correlated with p53 overexpression as assessed by immunostaining (P=0.015). Our results show that: (1) p16INK4a and p19ARF expression are altered in almost half of resectable NSCLC; (2) methylation within exon 1a is a frequent, but not the only mechanism of p16INK4a downregulation; and that (3) the inverse association of p19ARF and p53 alteration is consistent with a linked pathway.

Summary Proteins of the Bcl-2 family as well as p53 are important regulators of apoptosis. Alterations in the expression of these proteins can contribute to the formation of cancer, as well as influence tumour response to chemo-and radiotherapy. We used antibodies specific for the human Bcl-2, Mcl-1, Bax, Bak and p53 proteins to examine the expression of these apoptosis-regulating genes in 49 archival specimens of patients with radically resected non-small-cell lung cancer (NSCLC). Tumour cells containing immunostaining for the antiapoptotic proteins Bcl-2 and Mcl-1 were present in 31% and 58% of the cases evaluated, respectively, whereas immunopositivity for the proapoptotic proteins Bax and Bak was found in 47% and 58% of the samples. p53 immunopositivity was detected in 61% of the samples. The expression of Bcl-2 and p53 and the expression of Mcl-1 and Bax showed a positive association (P = 0.02 and P = 0.06 respectively), whereas the expression of Bax was inversely related to p53 (P = 0.008). The expression of Bcl-2 had a negative influence on relapse-free survival in this population of primary resected NSCLC patients (P = 0.02). The expression of p53 and Bcl-2 was significantly associated with metastasis-free survival (P < 0.01). Only patients with p53-positive tumours developed metastases during the follow-up period. Our results establish the frequent expression of the Bcl-2 family proteins Bcl-2, Mcl-1, Bax and Bak in NSCLC. It can be expected that Bcl-2 family members have no straightforward impact on clinical outcome in this disease because their interactions in the regulation of apoptosis are complex.Keywords: non-small-cell lung cancer; p53; Bcl-2; Mcl-1; Bax; Bak 952British Journal of Cancer (1999) 79(5/6), 952-958 © 1999 Cancer Research Campaign Article no. bjoc.1998 Received 19 Lung cancer is the leading cause of cancer death in Europe and the USA (Boring et al, 1994). More than 150 000 new cases are diagnosed in Europe every year, but less than 10% of these patients can be cured. Eighty per cent of malignant lung tumours are non-smallcell lung cancer (NSCLC). Despite wide surgical excision, metastases and local relapse are frequent after resection of early stage NSCLC. This makes the availability of prognostic factors for these events highly desirable to guide the use of (neo)adjuvant treatment modalities and to aid the decision about the extent of the operative procedure. The present study examined the differential expression pattern and the prognostic significance of the Bcl-2 family members Bcl-2, Mcl-1, Bax and Bak and of p53 in primary resected NSCLC. PATIENTS AND METHODS Patient featuresTumour samples were obtained from 49 consecutive patients (three women, 46 men) who underwent complete surgical resection for NSCLC at the Division of Thoracic Surgery of the University Hospital of Bern, Switzerland. ImmunohistochemistryParaffin-embedded tumour specimens that had been fixed in neutralbuffered formalin were sectioned (4 µm) and immunostained using anti-peptide polyclonal antisera specific f...

Progression through the mammalian cell cycle is controlled by a series of cyclins, cyclin‐dependent kinases (cdks) and cdk inhibitors. Cyclin D1, cdk4 and the tumour suppressors p16 and retinoblastoma protein (pRb) are thought to comprise a linked system governing cell passage through the G1 phase of the cell cycle. Extending an earlier study on cyclin D1 expression, a series of resectable non‐small cell lung carcinomas (NSCLCs) was examined for defects in other elements of this control system. Forty‐six of fifty‐one NSCLC specimens exhibited at least one alteration of these cell‐cycle regulators. Immunohistochemical analysis revealed that 33% and 47% of the tumours failed to express pRb and p16, respectively. Failure to detect pRb did not correlate with loss of heterozygosity at the RB1 locus. Eleven of 12 tumours showing positive (normal) pRb staining over‐expressed nuclear localised cyclin D1, including 8 with amplification of the cyclin D1 gene (CCND1). However, in a number of lesions (n = 5) where cyclin D1 was over‐expressed but localised to the cytoplasm, pRb expression was undetectable. Sequencing of exons 1 and 2 of the p16 gene (CDKN2) revealed 3/51 tumours with somatic mutations (in addition to 1 case with a germ‐line alteration). All of these lesions were positive for p16 protein. No clear homozygous deletions of CDKN2 were observed by multiplex PCR. As assessed by immunostaining using a p16 monoclonal antibody, there was an inverse correlation of pRb and p16 down‐regulation. Whilst patients with tumours over‐expressing cyclin D1 had a significantly lower incidence of local relapse, the group whose tumours failed to express pRb had a significantly greater risk of local relapse and tended to have shortened event‐free survival. Our data show that alteration of at least one cell cycle–regulator gene occurs in the majority of resectable NSCLCs. Int. J. Cancer 74:556–562, 1997. © 1997 Wiley‐Liss, Inc.