Tetraspanins CD9 and CD151, epidermal growth factor receptor and cyclooxygenase-2 expression predict malignant progression in oral epithelial dysplasia.

BACKGROUND: Prognostic biomarkers aim to improve on the current inadequate method of histological assessment to identify patients with oral epithelial dysplasia at greatest risk of malignant transformation. We aimed to assess the prognostic ability of six protein biomarkers linked to the epidermal growth factor receptor (EGFR) pathway, including three tetraspanins, in a large multicentre oral dysplasia cohort. METHODS: One hundred and forty-eight cases with varying degrees of epithelial dysplasia underwent immunohistochemical assessment for CD9, CD151, CD82, EGFR, Her-2, and COX-2. Scoring was performed independently by two observers. Univariate analyses using both logistic and Cox regression models and a multivariate regression were performed. RESULTS: Malignant progression was significantly greater in those cases with decreased expression of CD9 (P=0.02), and increased expression of CD151 (P=0.02), EGFR (P=0.04), and COX-2 (P=0.003). Histological grade (P=0.0002) and morphology (P=0.03) were also prognostic, whereas smoking and alcohol were not. The optimal combination by backward-variable selection was of histological grade (hazard ratio (HR) 1.64; 95% CI 1.12, 2.40), COX-2 overexpression (HR 1.12; 1.02, 1.24) and CD9 underexpression (HR 0.88; 0.80, 0.97). CD82 and Her-2 demonstrated no prognostic ability. CONCLUSION: This is the first study of the expression and prognostic potential of the tetraspanins in oral dysplasia. A combination of certain biomarkers with clinical factors appeared to improve the accuracy of determining the risk of malignancy in individuals with oral dysplasia. These findings may also offer potential new therapeutic approaches for this condition.

Diagnosis of copy number variation by Illumina next generation sequencing is comparable in performance to oligonucleotide array comparative genomic hybridisation.

Array comparative genomic hybridisation (aCGH) profiling is currently the gold standard for genetic diagnosis of copy number. Next generation sequencing technologies provide an alternative and adaptable method of detecting copy number by comparing the number of sequence reads in non-overlapping windows between patient and control samples. Detection of copy number using the BlueGnome 8×60k oligonucleotide aCGH platform was compared with low resolution next generation sequencing using the Illumina GAIIx on 39 patients with developmental delay and/or learning difficulties who were referred to the Leeds Clinical Cytogenetics Laboratory. Sensitivity and workflow of the two platforms were compared. Customised copy number algorithms assessed sequence counts and detected changes in copy number. Imbalances detected on both platforms were compared. Of the thirty-nine patients analysed, all eleven imbalances detected by array CGH and confirmed by FISH or Q-PCR were also detected by CNV-seq. In addition, CNV-seq reported one purported pathogenic copy number variant that was not detected by array CGH. Non-pathogenic, unconfirmed copy number calls were detected by both platforms; however few were concordant between the two. CNV-seq offers an alternative to array CGH for copy number analysis with resolution and future costs comparable to conventional array CGH platforms and with less stringent sample requirements.

Microdissection molecular copy-number counting (microMCC)--unlocking cancer archives with digital PCR.

Most cancer genomes are characterized by the gain or loss of copies of some sequences through deletion, amplification or unbalanced translocations. Delineating and quantifying these changes is important in understanding the initiation and progression of cancer, in identifying novel therapeutic targets, and in the diagnosis and prognosis of individual patients. Conventional methods for measuring copy-number are limited in their ability to analyse large numbers of loci, in their dynamic range and accuracy, or in their ability to analyse small or degraded samples. This latter limitation makes it difficult to access the wealth of fixed, archived material present in clinical collections, and also impairs our ability to analyse small numbers of selected cells from biopsies. Molecular copy-number counting (MCC), a digital PCR technique, has been used to delineate a non-reciprocal translocation using good quality DNA from a renal carcinoma cell line. We now demonstrate microMCC, an adaptation of MCC which allows the precise assessment of copy number variation over a significant dynamic range, in template DNA extracted from formalin-fixed paraffin-embedded clinical biopsies. Further, microMCC can accurately measure copy number variation at multiple loci, even when applied to picogram quantities of grossly degraded DNA extracted after laser capture microdissection of fixed specimens. Finally, we demonstrate the power of microMCC to precisely interrogate cancer genomes, in a way not currently feasible with other methodologies, by defining the position of a junction between an amplified and non-amplified genomic segment in a bronchial carcinoma. This has tremendous potential for the exploitation of archived resources for high-resolution targeted cancer genomics and in the future for interrogating multiple loci in cancer diagnostics or prognostics.

Clonal divergence in lung cancer development is associated with allelic loss on chromosome 4.

Patients who receive curative treatment for lung cancer can develop additional lung tumors that may or may not be related to the original tumor and thus require different clinical management. If a subsequent tumor has a pattern of allele loss, revealed by allelotype analysis, overlapping that of the first tumor, it is believed to be a local recurrence or metastasis. In this case history, we present loss of heterozygosity analyses of the original primary tumor, and two second primary tumors occurring in the ipsilateral and the contra-lateral lungs. The allelotyping suggests that these tumors are all clonally related but concordance is not complete. Our interpretation is that the original primary tumor and the two new primary tumors have developed to full malignancy independently, but are clonally related, possibly via a clone of motile progenitor cells. Deletion mapping of DNA from biopsies of this patient delineated a region in 4p16 that we had previously shown to be lost in the transition from carcinoma in situ to invasive tumor. We identified a minimally deleted region encompassing six genes including two candidate tumor suppressor genes, CRMP1 a lung cancer metastasis-suppressing gene and PPP2R2C a gene for a regulatory subunit of the PP2 complex known to suppress tumorigenesis, particularly viral induced transformation.

Lung cancer . 3: Fluorescence bronchoscopy: clinical dilemmas and research opportunities.

Recent developments in the detection of pre-invasive lesions of the large airways by fluorescence bronchoscopy are reviewed and the clinical and biological implications discussed.

Inadequate lung development and bronchial hyperplasia in mice with a targeted deletion in the Dutt1/Robo1 gene.

Chromosome 3 allele loss in preinvasive bronchial abnormalities and carcinogen-exposed, histologically normal bronchial epithelium indicates that it is an early, possibly the first, somatic genetic change in lung tumor development. Candidate tumor suppressor genes have been isolated from within distinct 3p regions implicated by heterozygous and homozygous allele loss. We have proposed that DUTT1, nested within homozygously deleted regions at 3p12-13, is the tumor suppressor gene that deletion-mapping and tumor suppression assays indicate is located in proximal 3p. The same gene, ROBO1 (accession number ), was independently isolated as the human homologue of the Drosophila gene, Roundabout. The gene, coding for a receptor with a domain structure of the neural-cell adhesion molecule family, is widely expressed and has been implicated in the guidance and migration of axons, myoblasts, and leukocytes in vertebrates. A deleted form of the gene, which mimics a naturally occurring, tumor-associated human homozygous deletion of exon 2 of DUTT1/ROBO1, was introduced into the mouse germ line. Mice homozygous for this targeted mutation, which eliminates the first Ig domain of Dutt1/Robo1, frequently die at birth of respiratory failure because of delayed lung maturation. Lungs from these mice have reduced air spaces and increased mesenchyme, features that are present some days before birth. Survivors acquire extensive bronchial epithelial abnormalities including hyperplasia, providing evidence of a functional relationship between a 3p gene and the development of bronchial abnormalities associated with early lung cancer.

Molecular evidence for derivation of metastatic cells from minor subclones of primary clear renal cell carcinomas.

The production of metastases depends on changes in a large number of genes. It is also connected with the interaction of tumor cells with the environment. It has been reported that primary tumor clone domination is also an important factor in metastasizing, and in many neoplasms dominating clones are the metastatic forerunners. Up to now it is unknown whether domination of a given clone in a primary renal cell carcinoma is a crucial factor in forming metastases or rather presence or absence of specific genes imposes the major advantage in the metastatic process. To study the presence or absence of the duplication and mitotic nondisjunction event as one of the phenomenon in the creation of metastases, as well as possible derivation of metastatic cells from the minor subclone of primary tumor, we examined three metastatic renal clear-cell carcinomas in which by comparative genomic hybridization we detected the loss of 3p in the primary tumor and two copies of 3p in the corresponding metastasis. Loss of heterozygosity analyses using markers for 3p25 (D3S1038), 3p21.1 (D3S1295), and 3p14.2 (D3S1481) proved heterozygosity of at least two 3p loci in all metastatic tumors, which indicates the absence of mitotic nondisjunction event as a cause of occurrence of two copies of 3p in metastases. Our results suggest that in some of the clear-cell renal carcinomas metastatic cells may derive from minor subclones of primary tumors.

Cell cycle checkpoint evasion and protracted cell cycle arrest in X-irradiated small-cell lung carcinoma cells.

PURPOSE: To determine the longevity and dose-dependence of acute X-irradiation-induced cell cycle perturbations in a panel of seven small-cell lung carcinoma (SCLC) cell lines (COR-L32B, COR-L51B, COR-L88B, COR-L96C, COR-L103, COR-L266B, COR-L279), assessed for TP53 tumour suppressor gene status and showing characteristically long population doubling periods. MATERIALS AND METHODS: Cell lines were screened for abnormalities in TP53. Cell cycle arrest and nuclear fragmentation were determined by flow cytometry under culture conditions that minimized the propensity of SCLC cells to form multicellular aggregates. A faster growing SCLC cell line (NCI-H69) and two breast tumour cell lines were used as controls. RESULTS: NCI-H69 and five of the COR-SCLC cell lines showed clear evidence of TP53 abnormalities and the cycle arrest responses of the breast tumour cell lines established the effects of TP53 mutation on G1/S checkpoint loss. All SCLC lines, at 24 h after low dose irradiation, showed abrogation of the G1/S checkpoint together with a range of expression of a protracted G2/M delay. G2/M delay progressed in all panel cell lines up to 48 h post-irradiation while NCI-H69 showed significant recovery for the dose range 75-600cGy. Only NCI-H69 and one panel line showed dose-dependent progression to complete nuclear DNA fragmentation. CONCLUSIONS: The culture method permits the measurement of cell cycle effects that reflect the TP53 status of SCLC cells. G1/S checkpoint failure, long-term radiation-induced G2 arrest, highly muted apoptotic responses and delayed recovery appear to be typical responses of the recently derived COR-SCLC lines. The results imply that low levels of unrepaired DNA damage, induced at clinically relevant doses, can persist for days in SCLC cells with long cell cycle traverse times, and can remain capable of checkpoint activation with implications for S phase-targeted therapies.

Multi-step evolution of lung cancer.

In an attempt to improve the poor survival rates for lung cancer, therapeutic strategies require a deeper understanding of the biological events contributing to the formation and progression of the disease. In particular, the importance of studying the different stages of lung cancer including early pre-neoplasia is being recognised and studies examining genetic changes in pre-invasive and invasive lesions are being used to identify key events in tumorigenesis.

Elucidation of the mechanism of homozygous deletion of 3p12-13 in the U2020 cell line reveals the unexpected involvement of other chromosomes.

Homozygous deletions in tumor cells have been useful in the localization and validation of tumor suppressor genes. We have described a homozygous deletion in a lung cancer cell line (U2020) which is located within the most proximal of the three regions on the short arm of chromosome 3 believed to be lost in lung cancer development. Construction of a YAC contig map indicates that the deletion spans around 8 Mb, but no large deletion was apparent on conventional cytogenetic analysis of the cell line. To investigate this paradox, whole chromosome, arm-specific, and regional paints have been used. This analysis has revealed that genetic loss has occurred by complex rearrangements of chromosomes 3, rather than simple interstitial deletion. These studies emphasize the power of molecular cytogenetics to disclose unsuspected tumor-specific translocations within the extremely complex karyotypes characteristic of solid tumors.

A combined approach of conventional and molecular cytogenetics for detailed karyotypic analysis of the small cell lung carcinoma cell line U2020.

Until recently the ability to analyze complex karyotypic rearrangements was totally dependent upon light microscopy of G-banded chromosomes. Developments in the area of molecular cytogenetics have revolutionized such analysis, making it possible to determine the nature of complex rearrangements. An extensive analysis has been made of the small cell lung carcinoma (SCLC) cell line U2020, using a combined approach of conventional and molecular cytogenetics, enabling a highly detailed karyotype to be constructed revealing rearrangements previously undetected by G-banding alone. This approach offers the opportunity to reassess other tumor karyotypes, particularly those of high complexity found in solid tumors, for tumor-specific consistent rearrangements indecipherable by conventional karyotyping.

Homozygous deletions at 3p12 in breast and lung cancer.

We have constructed a physical map of the region homozygously deleted in the U2020 cell line at 3p12, including the location of putative CpG islands. Adjacent to one of these islands, we have identified and cloned a new gene (DUTT1) and used probes from this gene to detect two other homozygous deletions occurring in lung and breast carcinomas: the smallest deletion is within the gene itself and would result in a truncated protein. The DUTT1 gene is a member of the neural cell adhesion molecule family, although its widespread expression suggests it plays a less specialized role compared to other members of the family.

The DUTT1 gene, a novel NCAM family member is expressed in developing murine neural tissues and has an unusually broad pattern of expression.

A new member of the NCAM family mapping to 3p12 has been isolated and predicted to be arranged in five immunoglobulin-like domains and three fibronectin-like domains which are particularly homologous to L1. There is a transmembrane domain and a long cytoplasmic region with no detectable homology to other sequences. Although less closely related to DCC, another family member, both share a loop of positively charged amino acids within the first immunoglobulin domain, unique to these two members of this very large gene family. Preliminary studies of expression in mouse embryos support an inferred role in neural development, but the observation of widespread gene expression in adult human tissues indicates that this protein has additional functions to those performed in neural cells.

Investigation of the genes for RET and its ligand complex, GDNF/GFR alpha-I, in small cell lung carcinoma.

RET is a receptor tyrosine kinase expressed in neuroendocrine cells and in tumors of these cell types. RET activation may be mediated by a ligand complex comprising glial cell line-derived neurotrophic factor (GDNF) and GDNF family receptor alpha-1 (GFR alpha-1). Activating RET mutations are found in the inherited cancer syndrome multiple endocrine neoplasia type 2 and in a subset of the related sporadic tumors, medullary thyroid carcinoma and pheochromocytoma, both being derived from neuroendocrine tissues. In one small study, mutations were identified in another tumor with neuroendocrine features, small cell lung carcinoma (SCLC). To determine whether RET mutations contribute to the pathogenesis of SCLC, we examined a panel of 54 SCLC cell lines. No mutations were identified in RET exons 10, 11, and 13-16, regions previously implicated in SCLC or other neuroendocrine tumors. We further examined the expression pattern of RET and the genes encoding the components of its ligand complex GDNF and GFR alpha-1, in 21 SCLC lines by using RT-PCR. Although we found no consistent pattern of expression for these three genes, RET was expressed in 57% of SCLC lines. Thus, although RET mutations appear unlikely to be an important step in the tumorigenesis of SCLC, the frequent expression of this gene suggests that RET may have a mitogenic role in a subset of SCLC cell lines.

Genome wide search for genetic damage in p53 transgenic mouse lung tumours reveals consistent loss of chromosome 4.

The tumour which develops most frequently in mice carrying a p53 Val135 transgene is adenocarcinoma of the lung. We established 10 cell lines from these tumours and investigated their karyotypes by detailed cytogenetic analysis using a complete set of mouse chromosome-specific paints. Consistent loss of chromosome 4 material was noted in 9 out of 10 cell lines; this loss was detected in tetraploid but not diploid cells of the same cell line, suggesting that mouse chromosome 4 plays a critical role in the progression of lung adenocarcinomas. Other frequently observed chromosome aberrations involved chromosomes 7, 5 and 8. Atypical bronchial epithelium was observed together with lung tumours and in tumour-free, apparently normal lungs indicating that mouse lung tumours induced due to the presence of a mutant p53 transgene may develop via pre-invasive lesions and thus may be effective models for the study of lung tumour progression.

Losses at 3p common deletion sites in subtypes of kidney tumours: histopathological correlations.

Deletions of the short arm of chromosome 3 (3p) have been recognized as characteristic features of clear cell renal cell carcinomas (clear cell RCC). We analysed 55 clear-cell RCCs and 30 non-clear-cell kidney tumours (10 papillary and 7 chromophobic RCCs, 11 oncocytomas and 2 collecting duct carcinomas) in loss of heterozygosity (LOH) studies using microsatellite markers for previously observed regions of common deletions on 3p in kidney tumours (3p25, 3p21.3, 3p14.2 and 3p12-13). Alterations were found in all 55 cases of clear-cell RCCs at two to four of the 3p regions. Extensive losses were not found in non-clear-cell tumours except for collecting duct carcinomas; 1 of 10 papillary RCCs showed interstitial deletion limited to a single 3p21.3 locus. LOH analyses using microsatellite markers for regions of common deletions at 3p may be of value in differential diagnosis of kidney tumours.

Clonal evolution of lung tumors.

Lung tumors, particularly squamous cell carcinomas, are believed to develop through a series of morphological abnormalities, driven by underlying somatic genetic changes. One way of studying this process is to analyze candidate somatic genetic changes in samples of squamous metaplasia and bronchial dysplasia of varying degrees of severity as well as tumor from the same patient. This assumes a clonal relationship between these lesions. In this article, we provide evidence that adjacent, physically distinct bronchial abnormalities are clonally related. This has been achieved using a plaque assay technique to detect the same p53 mutation, present throughout a tumor specimen, in a small proportion of cells in an adjacent squamous metaplasia. In addition, we have obtained two dysplasia samples from a tumor-free patient over a 9-month interval. The earlier sample had one p53 mutation, whereas the later sample has to p53 mutations on different alleles. Thus, the pattern of clonal evolution detected in the parallel samples mimics the pattern seen in longitudinal samples and supports the analysis of synchronously collected samples for the study of tumor progression.

Deletion mapping of the short arm of chromosome 3 in Merkel cell carcinoma.

Little is known about the biology of Merkel cell carcinoma (MCC), also called small cell carcinoma of the skin. MCC has similarities with small cell lung cancer (SCLC): both are neuroendocrine malignancies with early metastasis to distant sites and a poor prognosis. Small cell lung cancer biopsies are known to have frequent losses on chromosome 3 in the region 3p21, yet MCCs have not been reported to have 3p deletions by karyotypic analysis. Considering the similarities between SCLC and MCC, we investigated 26 MCC tumours for loss of heterozygosity (LOH) on 3p. First, RFLP analysis was performed using PCR with nine primer sets from six loci. Second, 25 tumours were examined by microsatellite analysis for 3p markers D3S1289 and D3S1285 and SST on 3q. All 26 tumours were informative at one or more loci; of these, 18 (69%) demonstrated LOH for at least one marker on the short arm. For all informative loci the frequency of LOH was greater than 30% (range 33-75%). In a cell line derived from one tumour, it was possible to demonstrate rearrangement of chromosome 3 by in situ hybridisation. No LOH was seen in 15 informative cases for the 3q locus SST. A region 3p13-p21.1, centered on the marker D3S2, was deleted in all tumours demonstrating LOH, with a secondary deletion involving D3S30 detected in some tumours at 3p13. Our results indicate that LOH on 3p is a common occurrence in MCC; however, three tumours for which DNA was also available from a corresponding cell line suggest there may be a subset of MCC whose genesis is independent of deletions of 3p.

Sequential molecular genetic changes in lung cancer development.

Epithelial tumours develop through a sequence of pre-invasive lesions of increasing disarray driven by underlying somatic genetic changes. We have studied the occurrence of the two most common somatic genetic changes associated with lung cancer in a series of premalignant bronchial lesions representing different stages in lung tumorigenesis. We present evidence that allele loss on chromosome 3 precedes damage to the p53 gene. Damage to chromosome 3 itself appears to be sequential in that the pattern of allele loss seen in dysplasia is often much more discrete than in invasive tumours. This implies that preneoplastic lesions may be a useful source of material for deletion mapping studies aimed at localising the position of tumour suppressor genes. We illustrate this by the comparison of an interstitial deletion described in this study with a homozygous deletion we have described previously, which has resulted in a better definition of the localisation of a tumour suppressor gene believed to be involved in lung cancer development.

Chromosome specific paints from a high resolution flow karyotype of the mouse.

Chromosomes from antigen stimulated B-cells from spleens of inbred mice have been separated using flow cytometry into 18 distinguishable peaks. Using locus-specific oligonucleotides and fluorescence in situ hybridization to banded metaphase spreads, 15 individual chromosomes were identified: 1, 2, 3, 6, 7, 8, 9, 11, 12, 16, 17, 18, 19, X and Y. The remaining six chromosomes, occurring as pairs in three peaks, 4 with 5, 10 with 13, and 14 with 15, were resolved by flow sorting chromosomes from mice carrying an appropriate homozygous translocation and 4, 5 and 14 have been isolated in this way. This is the first demonstration of how a complete set of mouse chromosome paints can be produced.