Origin and prognostic value of circulating KRAS mutations in lung cancer patients.

Because of the current controversy on the origin and clinical value of circulating KRAS codon 12 mutations in lung cancer, we screened 180 patients using a combined restriction fragment-length polymorphism and polymerase chain reaction (RFLP-PCR) assay. We detected KRAS mutations in 9% plasma samples and 0% matched lymphocytes. Plasma KRAS mutations correlated significantly with poor prognosis. We validated the positive results in a second laboratory by DNA sequencing and found matching codon 12 sequences in blood and tumor in 78% evaluable cases. These results support the notion that circulating KRAS mutations originate from tumors and are prognostically relevant in lung cancer.

An expectation-maximization algorithm for the analysis of allelic expression imbalance.

A significant proportion of the variation between individuals in gene expression levels is genetic, and it is likely that these differences correlate with phenotypic differences or with risk of disease. Cis-acting polymorphisms are important in determining interindividual differences in gene expression that lead to allelic expression imbalance, which is the unequal expression of homologous alleles in individuals heterozygous for such a polymorphism. This expression imbalance can be detected using a transcribed polymorphism, and, once it is established, the next step is to identify the polymorphisms that are responsible for or predictive of allelic expression levels. We present an expectation-maximization algorithm for such analyses, providing a formal statistical framework to test whether a candidate polymorphism is associated with allelic expression differences.

Overexpression of aurora B kinase (AURKB) in primary non-small cell lung carcinoma is frequent, generally driven from one allele, and correlates with the level of genetic instability.

Aurora kinases are key regulators of chromosome segregation during mitosis. We have previously shown by microarray analysis of primary lung carcinomas and matched normal tissue that AURKB (22 out of 37) and AURKA (15 out of 37) transcripts are frequently over-represented in these tumours. We now confirm these observations in a second series of 44 carcinomas and also show that aurora B kinase protein levels are raised in the tumours compared to normal tissue. Elevated levels of expression in tumours are not a consequence of high-level amplification of the AURKB gene. Using a coding sequence polymorphism we show that in most cases (seven out of nine) tumour expression is predominantly driven from one AURKB allele. Given the function of aurora B kinase, we examined whether there was an association between expression levels and genetic instability. We defined two groups of high and low AURKB expression. Using a panel of 10 microsatellite markers, we found that the group showing the higher level of expression had a higher frequency of allelic imbalance (P=0.0012). Analysis of a number of other genes that are strongly and specifically expressed in tumour over normal lung, including SERPINB5, TERT and PRAME, showed marked allelic expression imbalances in the tumour tissue in the context of balanced or only marginally imbalanced relative allelic copy numbers. Our data support a model of early carcinogenesis wherein defects in the process of inactivation of lung stem-cell associated genes during differentiation, contributes to the development of carcinogenesis.

The use of allelic expression differences to ascertain functional polymorphisms acting in cis: analysis of MMP1 transcripts in normal lung tissue.

Summary Aberrant expression of matrix metalloproteinase 1 (MMP1) has been implicated in a number of pathological conditions of the lung. In vitro results and analysis of tumours and cell lines suggest that an insertion/deletion polymorphism at position -1607 in the promoter of the gene can influence expression levels. However, whether this polymorphism is associated with differences in expression in normal lung tissue remains to be established. Polymorphisms affecting expression in cis will lead to alleles with different expression levels and will result in unequal expression of both alleles in heterozygous individuals (allelic expression imbalance, AEI). This can be detected using a transcribed marker. Here we follow a new approach and use AEI to ascertain that the -1607 polymorphism is associated with allelic expression differences of MMP1 in normal lung tissue. This approach could be used to map the sites associated with inter-individual expression differences in other genes. This is of particular interest since such sites allow prediction of expression levels, and can be used to test whether genetically determined differences in expression influence inter-individual differences of a phenotype of interest, such as disease predisposition.

S100A2 is strongly expressed in airway basal cells, preneoplastic bronchial lesions and primary non-small cell lung carcinomas.

S100A2 gene products were shown to be frequently and dramatically over-represented in non-small cell lung cancer (NSCLC) lesions over normal tissue by microarray analysis. We have now analysed an independent series of NSCLC tumours and multiple matched normal bronchial epithelial sites by RT-PCR and immunohistochemistry to investigate: whether this expression pattern can be confirmed and whether elevated expression is associated with tumour histology, clinical outcome or preneoplasia. In this second series, S100A2 was strongly expressed in 76% (35 out of 46) of tumours, more frequently in squamous cell than adenocarcinomas (P<0.002). This strong expression was not related to high-level gene amplification, but was associated in one of five informative cases with an allele-specific imbalance in transcript levels. Most tumours strongly expressed the DeltaNp63 transcript, the product of which is a putative regulator of S100A2 transcription and while all but one of the tumours positive for DeltaNp63 expressed S100A2, others negative for this regulator also expressed the gene. Contrary to the hypothesis that S100A2 is a tumour suppressor, no somatic mutations were identified in the coding sequence in 44 tumours. Furthermore, an examination of multiple tumour-free epithelial sites from 20 patients showed that strong expression was often associated with increasing levels of disorder in preinvasive bronchial lesions (P<0.0001).

METH-2 silencing and promoter hypermethylation in NSCLC.

The antiangiogenic factor METH-2 (ADAMTS-8) was identified in a previous dual-channel cDNA microarray analysis to be at least two-fold under-represented in 85% (28 out of 33) of primary non-small-cell lung carcinomas (NSCLCs). This observation has been validated in an independent series of NSCLCs and adjacent normal tissues by comparative multiplex RT-PCR, and METH-2 mRNA expression was dramatically reduced in all 23 tumour samples analysed. Immunohistochemical analysis of the same sample set demonstrated that METH-2 was strongly expressed in 14 out of 19 normal epithelial sites examined but only one out of 20 NSCLCs. DNA methylation analysis of the proximal promoter region of this gene revealed abnormal hypermethylation in 67% of the adenocarcinomas and 50% of squamous cell carcinomas, indicating that epigenetic mechanisms are involved in silencing this gene in NSCLC. No homozygous deletions of METH-2 were found in lung cancer cell lines. Allelic imbalance in METH-2 was assessed by an intronic single nucleotide polymorphism (SNP) assay and observed in 44% of informative primary samples. In conclusion, the downregulation of METH-2 expression in primary NSCLC, often associated with promoter hypermethylation, is a frequent event, which may be related to the development of the disease.

The alleles of the DNA repair gene O6-alkylguanine-DNA alkyltransferase are expressed at different levels in normal human lung tissue.

O6-Alkylguanine-DNA alkyltransferase (MGMT) confers resistance to many of the mutagenic and toxic effects of certain classes of alkylating agents by repairing the DNA lesions responsible. The levels of expression of this protein are of interest in relation to the prevention and treatment of cancer in man. They vary widely between individuals, and the basis of this variation is not understood. RT-PCR-RFLP analysis of mRNA from normal human lung tissue reveals that the two MGMT alleles are frequently expressed at different levels, indicating that there is a genetic component to inter-individual variation of MGMT levels and that at least some of this variation maps close to or within the MGMT locus.

Cyclin D1 overexpression in bronchial epithelia of patients with lung cancer is associated with smoking and predicts survival.

PURPOSE: Cyclin D1 is overexpressed in almost 60% of resectable non-small-cell lung cancer (NSCLC). In the absence of cyclin D1 gene amplification, overexpression is characterized by allelic imbalanced transcript levels. METHODS: The aims were to study cyclin D1 expression by immunohistochemistry and allelic balance of transcripts in tumor-free bronchial epithelia from patients with resectable NSCLC by using monoclonal antibodies (48 patients and 288 sites), microdissection/reverse transcriptase polymerase chain reaction/restriction fragment length polymorphism analyses (24 patients and 144 sites). Derived data were related to patient characteristics-in particular, smoking habits. RESULTS: In 167 (58%) of 288 sites, cyclin D1 was overexpressed, with cytoplasmic and nuclear sublocalization in 53% and 7% of all sites, respectively. Nuclear overexpression was more frequent in premalignant versus normal or hyperplastic epithelia (55% v 3%; P <.0001). Allele-specific expression imbalances were found in 69 (48%) of 144 sites; in particular, those in which cyclin D1 was overexpressed (P =.004). In 14 (58%) of 24 patients, balanced or imbalanced transcript ratios and degree of expression were consistent at all sites for the same patient, whereas in another 10 patients, transcript balances and cyclin D1 expression patterns varied across the sites. Nuclear cyclin D1 expression in at least one site (14 of 48 patients) was linked to heavy smoking (> 40 pack-years; P =.02) and shorter overall survival (P =.01). CONCLUSION: Allele-specific, probably damage-driven, deregulation of the cyclin D1 gene may precede and perhaps facilitate the spread of preneoplastic clones across the bronchial epithelial surface in a significant number of patients. Cyclin D1 expression at multiple bronchial sites may identify a subgroup of heavy-smoking patients with poor outcome.

Decreased endothelin receptor B expression in large primary uveal melanomas is associated with early clinical metastasis and short survival.

The most devastating aspect of cancer is the metastasis of tumour cells to organs distant from the original tumour site. The major problem facing oncologists treating uveal melanoma, the most common cancer of the eye, is metastatic disease. To lower mortality, it is necessary to increase our understanding of the molecular genetic alterations involved in this process. Using suppression subtractive hybridisation, we have analysed differential gene expression between four primary tumours from patients who have developed clinical metastasis and four primary tumours from patients with no evidence of metastasis to date. We have identified endothelin receptor type B as differentially expressed between these tumours and confirmed this observation using comparative multiplex RT-PCR. In a further 33 tumours, reduced endothelin receptor type B expression correlated with death from metastatic disease. Reduced expression also correlated with other known prognostic indicators, including the presence of epithelioid cells, chromosome 3 allelic imbalance and chromosome 8q allelic imbalance. Endothelin receptor type B expression was also reduced in four out of four primary small cell lung carcinomas compared to normal bronchial epithelium. We also show that the observed down-regulation of endothelin receptor type B in uveal melanoma was not due to gene deletion. Our findings suggest a role for endothelin receptor type B in the metastasis of uveal melanoma and, potentially, in the metastasis of other neural crest tumours.

Alterations of cell cycle regulators are less frequent in advanced non-small cell lung cancer than in resectable tumours.

Prognosis of lung cancer is related to stage of disease at time of diagnosis. In this study we examine alterations of pathways governing the cell cycle, in particular pRb-cyclinD1-p16 alpha and p53-p14ARF, in a series of NSCLC (n=92) at different stages at diagnosis. Using immunohistochemistry, we assessed the expression of the retinoblastoma protein (pRb), cyclin D1, p16 alpha, p53 and p14ARF. Tumours in stage I-IIIA (resectable) were more likely to have alterations in the pRb-cyclinD1-p16 alpha pathway than tumours in advanced stage (IIIB-IV) (90% versus 63%, P=0.002). pRb and p14ARF were more frequently downregulated in resectable tumours (P< or =0.03), and cyclin D1, p16 alpha, and p53 were altered at a similar frequency in resectable and advanced tumours. In 12 patients, metastatic sites (5 lymph node, 3 bone, 2 brain and 2 gastrointestinal metastases) were available for comparison with the primary tumour: 19 altered protein expressions were found to be concordant, six additional alterations (in 4 patients) were found in the metastases only, especially in lymph node metastases (3 patients). Compared with normal protein expression, both pathway alterations were associated with a longer survival (P=0.02). In a multivariate analysis (Cox regression) this difference was not maintained after adjustment for age, stage and tumour differentiation. Cyclin D1 was the sole protein with independent prognostic value in resectable tumours: the relative risk of local relapse was 4.7 in tumours without cyclin D1 overexpression (P=0.02, Cox regression analysis). No protein studied had a predictive significance for response after chemotherapy in non-resectable tumours. These results demonstrate a strong correlation between stage and pathway alterations, cell cycle regulators being less likely altered in advanced NSCLC. Tumours with defects in these control pathways tend therefore to remain localised and to metastasize at a later phase in tumour development. This finding might be an explanation for distinct biological behaviour (e.g. chemotherapy response) of resectable versus advanced disease.

The bmi-1 oncoprotein is differentially expressed in non-small cell lung cancer and correlates with INK4A-ARF locus expression.

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.

Genomic structure and expression profile of LPHH1, a 7TM gene variably expressed in breast cancer cell lines.

Gene identification studies, centred on a region of overlapping loss of heterozygosity in breast tumours within band 1p31.1, lead to the characterisation of LPHH1, a novel human 7TM gene. The coding sequence of LPHH1 extends over a 60 kb region and comprises in excess of 28 exons. Alternative splicing occurs minimally at five positions, four of which are within the coding sequence. The fifth region of alternative splicing occurs at the extreme 5' end of the transcript. A clear tissue specific bias in alternative exon selection is observed to some degree at all five positions, including the extreme 5' region, which raises the possibility of multiple and perhaps tissue specific promoters. One such putative promoter region, which appears to be utilised predominantly in breast cancer cells, has been identified. LPHH1 is highly evolutionarily conserved, with the simplest (19 exon) gene product being 95% identical between human and rat. Comparison of the alternatively spliced exons between three species, where data are available, has so far revealed 100% identity in the encoded peptide sequences, suggesting conservation of a functional aspect of this splicing. Gene expression has been observed in all tissues and cell lines tested, with the exception of lymphoblastoid and multiple myeloma lines, where there appears to be only a very low level of transcription. LPHH1 also appears to be downregulated in human bone marrow. These data are consistent with a role for the gene products in adhesion-mediated signalling. Analysis of a panel of breast tumour cell lines revealed that a number apparently overexpressed the gene whilst others showed very low levels of transcription. In one case, the overexpression correlated with a low level increase in gene copy number in the tumour line. In addition to differences in the overall levels of expression, LPHH1 mRNAs were alternatively spliced to varying degrees with shifts in the major gene product to truncated or altered forms in some lines. No somatic LPHH1 mutations were detected through sequence analysis of four primary breast tumours that showed loss of the adjacent 1p31.1 marker D1S207.

p21 is associated with cyclin D1, p16INK4a and pRb expression in resectable non-small cell lung cancer.

p21 (p21WAF1/CIP1) is involved in cell cycle regulation, as an inhibitor of cyclin dependent kinases (CDK2, CDK4 and CDK6). However, subsequent in vitro studies have suggested that p21 may influence this process by an additional mechanism, in particular through the regulation of cyclin D1 subcellular localisation. This study of primary resectable non-small cell lung cancer (NSCLC) was designed to examine p21 functions in association with the expression of cyclin D1 (including its subcellular localisation), p16INK4a and pRb. p21 expression was examined in 50 NSCLC (stage I-IIIA) and in several normal lung samples all of which had previously been studied for cyclin D1 (DNA, RT-PCR, immunostaining), p16INK4a (DNA, RT-PCR, immunostaining), and pRb (immunostaining). As assessed by immunoblotting and immunostaining, p21 was expressed at low levels in normal lung tissue with immunoreactivity seen in a small number of bronchial epithelial cells only. In NSCLC, p21 expression (> or =10% of positive cells) was observed in 42% (21/50) of cases. High p21 expression was associated with well differentiated tumours (p = 0.01) and cyclin D1 nuclear staining (p = 0.02). Furthermore, we found an inverse correlation with p16INK4a (p = 0.004) and a direct correlation with pRb expression (p = 0.02). Risk of relapse was associated with p16INK4a and p21 status with no relapse in patients with normal p16INK4a and p21. Our results confirm that a large number of NSCLC have a low level of p21 expression. The associations of p21 and nuclear cyclin D1, pRb, p16INK4a support the relevance of pathways linked to lung carcinogenesis that involve p21 but may act in addition to direct CDK inhibition.

Expression of p16INK4a/p16alpha and p19ARF/p16beta is frequently altered in non-small cell lung cancer and correlates with p53 overexpression.

The CDKN2 locus expresses two different mRNA transcripts, designated alpha and beta. The protein product of the alpha transcript is the cell cycle inhibitor and tumour suppressor p16INK4a. The beta 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 significant 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 1alpha 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, fibroblasts, 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 1alpha and these tumours showed normal p19ARF expression. In contrast, no methylation of exon 1alpha was observed in five 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 1alpha were strongly methylated. In tumours, imbalanced multiplex RT-PCR data (p16INK4a

Polymorphism within the cyclin D1 gene is associated with prognosis in patients with squamous cell carcinoma of the head and neck.

We have examined the correlation of a frequent A/G polymorphism within exon 4 of the cyclin D1 gene (CCND1) with genetic susceptibility and clinical outcome in 384 patients with squamous cell carcinoma (SCC) of the head and neck. CCND1 genotype frequencies were similar in the cases and 191 controls. Furthermore, the CCND1 genotype was not associated with susceptibility to SCC of the larynx, pharynx, or oral cavity. The influence of the CCND1 genotype on clinical outcome was also assessed. We found no correlation between genotype and tumor size (T1-T4), the involvement of nodes at presentation, or patient age and gender. However, the distribution of CCND1 genotypes in cases with poorly differentiated tumors was significantly different to that in patients with well-/moderately differentiated tumors (P = 0.016; chi2(2) = 8.71). Homozygosity for CCND1*G (GG genotype) was associated with poorly differentiated tumors (G3). We used Cox's proportional hazards model to investigate the influence of the CCND1 genotype on disease-free interval. CCND1 GG was associated with reduced disease-free interval [P = 0.001; hazard ratio (HR) = 2.95; 95% confidence interval (CI) = 1.54-5.63]. This remained significant after correction for tumor differentiation (P = 0.013; HR = 2.34; 95% CI = 1.2-4.6) and tumor stage (P = 0.005; HR = 2.64; 95% CI = 1.34-5.19). Analysis of the data from patients with tumors at different sites showed that the CCND1 GG genotype was associated with reduced disease-free interval in laryngeal (P = 0.004; HR = 3.63; 95% CI = 1.44-8.83) and pharyngeal (P = 0.006; HR = 3.48; 95% CI = 1.43-8.46) tumors. This is the first report of an association between CCND1 polymorphism and prognosis in SCC of the head and neck. These data show that the CCND1 GG genotype is an independent prognostic indicator of disease-free interval and supports initial observations in non-small cell lung cancer, that polymorphism within CCND1 influences tumor behavior.

Identification of the critical region of 12p over-representation in testicular germ cell tumors of adolescents and adults.

Cytogenetically, testicular germ cell tumors of adolescents and adults (TGCTs) are characterized by gain of 12p-sequences, most often through isochromosome formation (i(12p)). Fluorescence in situ hybridization (FISH) has shown that i(12p))-negative TGCTs also cryptically contain extra 12p-sequences. The consistency of 12p-over-representation in all histological subtypes of TGCTs, including their preinvasive stage, suggests that gain of one or more genes on 12p is crucial in the development of this cancer. So far, studies aimed at the identification of the relevant gene(s) were based on the 'candidate-gene approach'. No convincing evidence in favor of or against a particular gene has been reported. We combined conventional karyotyping, comparative genomic hybridization, and FISH to identify TGCTs with amplifications of restricted regions of 12p. Out of 49 primary TGCTs (23 without i(12p), 13 with and 13 unknown), eight tumors (six without i(12p) and two unknown) showed amplifications corresponding to 12p11.1-p12.1. Using bicolour-FISH, physical mapping, and semi-quantitative polymerase chain reactions, the size of the shortest region of overlap of amplification (SROA) was estimated to be between 1750-3000 kb. In addition, we mapped a number of genes in and around this region. While fourteen known genes could be excluded as candidates based on their location outside this region, we demonstrate that KRAS2, JAW1 and SOX5 genes are localized within the SROA. While KRAS2 and JAW1 map to the proximal border of the SROA, SOX5 maps centrally in the SROA. KRAS2 and JAW1 are expressed in all TGCTs, whereas one 12p amplicon-positive TGCT lacks expression of SOX5. The critical region of 12p over-represented in TGCTs is less than 8% of the total length of the short arm of chromosome 12. It will be helpful in the identification of the gene(s) involved in TGCT-development.

Isolation and characterization of a human homologue of the latrophilin gene from a region of 1p31.1 implicated in breast cancer.

We have identified a region of chromosome 1p31.1 that shows high frequency loss of heterozygosity (LOH) in human breast cancer. This region forms part of a 7 Mb YAC/BAC contig. In order to identify candidate sequences, mutation of which might contribute to the development of disease, we have carried out mapping studies of ESTs localized to 1p31.1. This analysis, coupled with library screening and a modified 5' RACE-PCR strategy, resulted in the identification and characterization of a novel gene (LPHH1) which is located adjacent to the smallest region of overlapping loss (SRO) seen in tumours. The 4209 bp open reading frame of the 7 kb LPHH1 transcript encodes a peptide which shows approximately 65% identity to rat latrophilin, a G-coupled, seven span transmembrane protein, which binds alpha-latrotoxin. In the human sequence, whilst conservation of the transmembrane domain is high, the intra- and extracellular domains show two regions of variable structure, which are presumably generated by alternative splicing. Surprisingly, while expression of the rat gene is tightly restricted to neurological and perhaps some endocrine cells, the human sequence appears to be expressed very widely in all normal tissues tested. Northern and RT-PCR analysis of a panel of tumour cell lines showed that LPHH1 expression was variable, apparently elevated in some lines and absent or markedly reduced in others. Furthermore, characterization of the range of transcripts encoded in a breast tumour cell line, compared to normal breast, suggested that gene product variability was higher in the tumour.

G1 control gene status is frequently altered in resectable non-small cell lung cancer.

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 (CCNDI). 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.

Physical localisation of the breakpoints of a constitutional translocation t(5;6)(q21;q21) in a child with bilateral Wilms' tumour.

A 6 month old boy presented with bilateral Wilms' tumour. Cytogenetic analysis of the lymphocytes from the patient showed a de novo balanced translocation t(5;6)(q21;q21), which was also present in the tumour material as the sole cytogenetic abnormality. To facilitate the identification of the translocation breakpoints, we have established a lymphoblastoid cell line (MA214L) from the patient which maintains the translocation in culture. We have used Genethon microsatellite markers as sequence tagged sites (STSs) to isolate yeast artificial chromosome (YAC) clones to 5q and 6q from human genomic libraries. Using fluorescence in situ hybridisation (FISH) on metaphase preparations of MA214L, we have physically defined the translocation breakpoints between YAC clones on each chromosome arm. The genetic distance separating the flanking YACs on 6q21 is 3 cM, while that on 5q21 is 4 cM. To date this is the first report of these chromosomal regions being implicated in Wilms' tumourigenesis.

Abnormal expression of CCND1 and RB1 in resection margin epithelia of lung cancer patients.

Tumours develop through the accumulation of genetic alterations associated with a progressive increase of the malignant phenotype. In lung cancer, chronic exposure of bronchial epithelium to carcinogens in cigarette smoke may lead to multiple dysplastic and hyperplastic lesions scattered throughout the tracheobronchial tree. Little is known about the genetic alterations in such lesions. This study was carried out to examine cyclin D1 (CCND1) and retinoblastoma (RB1) gene expression in the bronchial epithelium of patients with lung cancer. Lung tumours and their corresponding tumour-free resection margins from 33 patients who underwent resection of non-small-cell lung cancer (NSCLC) were examined by immunostaining with monoclonal antibodies against cyclin D1 (DCS-6; Novocastra) and pRb (NCL Rb-1; Novocastra). Examination of the resection margins revealed four carcinomas in situ, 19 hyperplasias and ten sections showing apparently normal bronchial epithelium. A control group of patients, without lung tumours and who had never smoked, revealed no or weak cyclin D1 and positive pRb staining within bronchial epithelia. Increased cyclin D1 and diminished pRb expression were found in 76% (n = 25) and 27% (n = 9) of the resection margins respectively, and in 12% (n = 4) both cyclin D1 and pRb expression were altered. In the corresponding tumours, 48% (n = 16) were normal, while altered expression was found for cyclin D1 in 33% (n = 11), pRb in 27% (n = 9) and both in 9% (n = 3) of cases. It appears that altered expression of cyclin D1 and pRb is an early event in NSCLC development in almost half of cases analysed. Further investigations are needed to determine the significance of immunostaining of bronchial specimens in individuals at risk of lung cancer, with the possibility that the observations are of importance in the early diagnosis of NSCLC.