Biomedical subjects

V Peter Collins

Publications and source records attributed to V Peter Collins.

At least 19 recordsLinked to original sources

PubMed2006

Detailed assessment of copy number alterations revealing homozygous deletions in 1p and 13q in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is characterized by over-expression of cyclin Dl as a result of the characteristic t(11;14)(q13;q32). However, this translocation alone has proven not to be sufficient for lymphomagenesis, suggesting the involvement of additional alterations. We have characterized 35 cases of MCL by array comparative genomic hybridization with an average resolution of 0.97 Mb distributed over the complete human genome. The most common alterations were losses in 1p13.2-p31.1, 6q16.2-q27, 8p21.3, 9p13.2-p24.3, 9q13-q31.3, 11q14.3-q23.3, 13q14.13-q21.31, 13q33.1-q34, and 22q11.23-q13.33 and gains involving 3q21.2-q29, 7p12.1-p22.3, 8q24.13-q24.23, and 18q21.33-q22.3. Four homozygous deletions were identified in totally three patients; two overlapping at 1p32.3, and two adjacent at 13q32.3. The homozygous deletions at 1p32.3 cover the CDKN2C locus (coding for p18), while the region at 13q32.3 does not encompass any known tumor suppressor genes. A gain in 3q was significantly associated with shorter survival (P=0.047).

Chromosome Deletion↗

PubMed2006

MMASS: an optimized array-based method for assessing CpG island methylation.

We describe an optimized microarray method for identifying genome-wide CpG island methylation called microarray-based methylation assessment of single samples (MMASS) which directly compares methylated to unmethylated sequences within a single sample. To improve previous methods we used bioinformatic analysis to predict an optimized combination of methylation-sensitive enzymes that had the highest utility for CpG-island probes and different methods to produce unmethylated representations of test DNA for more sensitive detection of differential methylation by hybridization. Subtraction or methylation-dependent digestion with McrBC was used with optimized (MMASS-v2) or previously described (MMASS-v1, MMASS-sub) methylation-sensitive enzyme combinations and compared with a published McrBC method. Comparison was performed using DNA from the cell line HCT116. We show that the distribution of methylation microarray data is inherently skewed and requires exogenous spiked controls for normalization and that analysis of digestion of methylated and unmethylated control sequences together with linear fit models of replicate data showed superior statistical power for the MMASS-v2 method. Comparison with previous methylation data for HCT116 and validation of CpG islands from PXMP4, SFRP2, DCC, RARB and TSEN2 confirmed the accuracy of MMASS-v2 results. The MMASS-v2 method offers improved sensitivity and statistical power for high-throughput microarray identification of differential methylation.

Cell Line, Tumor↗

PubMed2006

Application of array CGH on archival formalin-fixed paraffin-embedded tissues including small numbers of microdissected cells.

Array-based comparative genomic hybridisation (aCGH) has diverse applications in cancer gene discovery and translational research. Currently, aCGH is performed primarily using high molecular weight DNA samples and its application to formalin-fixed and paraffin-embedded (FFPE) tissues remains to be established. To explore how aCGH can be reliably applied to archival FFPE tissues and whether it is possible to apply aCGH to small numbers of cells microdissected from FFPE tissue sections, we have systematically performed aCGH on 15 pairs of matched frozen and FFPE astrocytic tumour tissues using a well-established in-house human 1 Mb BAC/PAC genomic array. By spiking tumour DNA with normal DNA, we demonstrated that at least 70% of tumour DNA was required for reliable aCGH analysis. Using aCGH data from frozen tissue as a reference, it was found that only FFPE astrocytic tumour tissues that supported PCR amplification of >300 bp DNA fragment provided high quality, reproducible aCGH data. The presence of necrosis in a tissue specimen had an adverse effect on the quality of aCGH, while fixation in formalin for up to 96 h of fresh tissue did not appear to affect the quality of the result. As little as 10-20 ng DNA from frozen or FFPE tissues could be readily used for aCGH analysis following whole genome amplification (WGA). Furthermore, as few as 2000 microdissected cells from haematoxylin-stained slides of archival FFPE tissues could be successfully used for aCGH investigations when WGA was used. By careful assessment of DNA integrity and review of histology, to exclude necrosis and select specimens with a high proportion of tumour cells, it is feasible to preselect archival FFPE tissues adequate for aCGH analysis. With the help of microdissection and WGA, it is also possible to apply aCGH to histologically defined lesions, such as carcinoma in situ.

Astrocytoma↗

PubMed2006

Differential expression of selected histone modifier genes in human solid cancers.

BACKGROUND: Post-translational modification of histones resulting in chromatin remodelling plays a key role in the regulation of gene expression. Here we report characteristic patterns of expression of 12 members of 3 classes of chromatin modifier genes in 6 different cancer types: histone acetyltransferases (HATs)- EP300, CREBBP, and PCAF; histone deacetylases (HDACs)- HDAC1, HDAC2, HDAC4, HDAC5, HDAC7A, and SIRT1; and histone methyltransferases (HMTs)- SUV39H1and SUV39H2. Expression of each gene in 225 samples (135 primary tumours, 47 cancer cell lines, and 43 normal tissues) was analysedby QRT-PCR, normalized with 8 housekeeping genes, and given as a ratio by comparison with a universal reference RNA. RESULTS: This involved a total of 13,000 PCR assays allowing for rigorous analysis by fitting a linear regression model to the data. Mutation analysis of HDAC1, HDAC2, SUV39H1, and SUV39H2 revealed only two out of 181 cancer samples (both cell lines) with significant coding-sequence alterations. Supervised analysis and Independent Component Analysis showed that expression of many of these genes was able to discriminate tumour samples from their normal counterparts. Clustering based on the normalized expression ratios of the 12 genes also showed that most samples were grouped according to tissue type. Using a linear discriminant classifier and internal cross-validation revealed that with as few as 5 of the 12 genes, SIRT1, CREBBP, HDAC7A, HDAC5 and PCAF, most samples were correctly assigned. CONCLUSION: The expression patterns of HATs, HDACs, and HMTs suggest these genes are important in neoplastic transformation and have characteristic patterns of expression depending on tissue of origin, with implications for potential clinical application.

Breast Neoplasms↗

PubMed2006

Genomic profiling identifies discrete deletions associated with translocations in glioblastoma multiforme.

Glioblastoma multiforme is the most common tumor arising in the central nervous system. Patients with these tumors have limited treatment options and their disease is invariably fatal. Molecularly targeted agents offer the potential to improve patient treatment, however the use of these will require a fuller understanding of the genetic changes in these complex tumors. In this study, we identify copy number changes in a series of glioblastoma multiforme tumors and cell lines by applying high-resolution microarray comparative genomic hybridization. Molecular cytogenetic characterization of the cell lines revealed that copy number changes define translocation breakpoints. We focused on chromosome 6 and further characterized three regions of copy number change associated with translocations including a discrete deletion involving IGF2R, PARK2, PACRG and QKI and an unbalanced translocation involving POLH, GTPBP2 and PTPRZ1.

Adult↗

PubMed2006

Small regions of overlapping deletions on 6q26 in human astrocytic tumours identified using chromosome 6 tile path array-CGH.

Deletions of chromosome 6 are a common abnormality in diverse human malignancies including astrocytic tumours, suggesting the presence of tumour suppressor genes (TSG). In order to help identify candidate TSGs, we have constructed a chromosome 6 tile path microarray. The array contains 1,780 clones (778 P1-derived artificial chromosome and 1,002 bacterial artificial chromosome) that cover 98.3% of the published chromosome 6 sequences. A total of 104 adult astrocytic tumours (10 diffuse astrocytomas, 30 anaplastic astrocytomas (AA), 64 glioblastomas (GB)) were analysed using this array. Single copy number change was successfully detected and the result was in general concordant with a microsatellite analysis. The pattern of copy number change was complex with multiple interstitial deletions/gains. However, a predominance of telomeric 6q deletions was seen. Two small common and overlapping regions of deletion at 6q26 were identified. One was 1,002 kb in size and contained PACRG and QKI, while the second was 199 kb and harbours a single gene, ARID1B. The data show that the chromosome 6 tile path array is useful in mapping copy number changes with high resolution and accuracy. We confirmed the high frequency of chromosome 6 deletions in AA and GB, and identified two novel commonly deleted regions that may harbour TSGs.

Astrocytoma↗

PubMed2006

Genomic analysis of pilocytic astrocytomas at 0.97 Mb resolution shows an increasing tendency toward chromosomal copy number change with age.

Brain tumors are the most common solid tumors of childhood, accounting for over 20% of cancers in children under 15 years of age. Pilocytic astrocytomas (PAs), World Health Organization grade I, are one of the most frequently occurring childhood brain tumors, yet little is known about genetic changes characterizing this entity. We have used microarray comparative genomic hybridization at 0.97 Mb resolution to study a series of PAs (n = 44). No copy number abnormality was seen in 64% of cases at this resolution. However, whole chromosomal gain (median 5 chromosomes affected) occurred in 32% of tumors. The most frequently affected chromosomes were 5 and 7 (11 of 44 cases each) followed by 6, 11, 15, and 20 (greater than 10% of cases each). Findings were confirmed by fluorescence in situ hybridization and microsatellite analysis in a subset of tumors. Chromosomal gain was significantly more frequent in PAs from patients over 15 years old (p = 0.03, Fisher exact test). The number of chromosomes involved was also significantly greater in the older group (p = 0.02, Mann-Whitney U test). One case (2%) showed a region of gain on chromosome 3 and one (2%) a deletion on 6q as their sole abnormalities. This is the first genomewide study to show this nonrandom pattern of genetic alteration in pilocytic astrocytomas.

Adolescent↗

PubMed2005

High-resolution array-CGH profiling of germline and tumor-specific copy number alterations on chromosome 22 in patients affected with schwannomas.

Schwannomas may develop sporadically or in association with NF2 and schwannomatosis. The fundamental aberration in schwannomas is the bi-allelic inactivation of the NF2 gene. However, clinical and molecular data suggest that these tumors share a common pathogenetic mechanism related to as yet undefined 22q-loci. Linkage studies in schwannomatosis, a condition related to NF2, have defined a candidate 22q-locus and excluded the NF2 gene as the causative germline mutation. Thus, analysis of aberrations in schwannomas may lead to the identification of putative gene(s) involved in the development of schwannoma/schwannomatosis. We profiled a series of 88 schwannomas and constitutional DNA using a tiling path chromosome 22 array. Array-CGH is a suitable method for high-resolution discrimination between germline and tumor-specific aberrations. Previously reported frequencies of 22q-associated deletions in schwannomas display large discrepancies, ranging from 30% to 80%. We detected heterozygous deletions in 53% of schwannomas and the predominant pattern was monosomy 22. In addition, three tumors displayed terminal deletions and four harbored overlapping interstitial deletions of various sizes encompassing the NF2 gene. When profiling constitutional DNA, we identified eight loci that were affected by copy number variation (CNV). Some of the identified CNVs may not be phenotypically neutral and the possible role of these CNVs in the pathogenesis of schwannomas should be studied further. We observed a correlation between the breakpoint position, present in tumor and/or constitutional DNA and the location of segmental duplications. This association implicates these unstable regions in rearrangements occurring both in meiosis and mitosis.

Chromosomes, Human, Pair 22↗

PubMed2005

Clinical significance of EGFR amplification and the aberrant EGFRvIII transcript in conventionally treated astrocytic gliomas.

The aim of this study was to evaluate the clinical value of assessing epidermal growth factor receptor (EGFR) amplification and the common 5' rearrangement of EGFR resulting in the EGFRvIII transcript in astrocytic gliomas. Data from 221 tumours were correlated with patient survival. The majority of previous studies evaluated amplification alone and provided contradictory results. Amplification was analysed by a densitometry of Southern blot analysis or quantitative polymerase chain reaction (PCR). EGFR transcripts were examined by reverse transcription PCR and subsequent sequencing. A ribonuclease (RNase) protection assay was carried out on a subgroup to confirm PCR results. Amplification of EGFR was found in 41% (65/160) of glioblastomas (GBs) and 10% (4/41) of anaplastic astrocytomas (AAs). The EGFRvIII rearrangement was identified in 54% (35/65) of GBs and 75% (3/4) of AAs with amplification, as well as in 8% (8/95) of GBs and 5% (2/37) of AAs without amplification (confirmed by RNase protection assay). There were no abnormalities of the EFGR or its transcript in grade II astrocytoma (AII). We found no significant association between EGFR amplification or rearrangement, and age or survival in the 160 GB patients. We noted a tendency towards decreased survival with any EGFR abnormality in the 41 patients with AAs. This was most marked in the five cases with the EGFRvIII transcript (p=0.069), but these were significantly older than those without (p=0.023). No abnormalities of EGFR were identified in AII patients. We conclude that neither EGFR amplification nor the presence of the EGFRvIII transcript predicts patient outcome in conventionally treated GBs. However, in AAs, although uncommon, EGFR aberrations appear to be associated with shorter survival.

Adult↗

PubMed2005

Frequent promoter hypermethylation and low expression of the MGMT gene in oligodendroglial tumors.

Allelic losses on the chromosome arms 1p and 19q have been associated with favorable response to chemotherapy and good prognosis in anaplastic oligodendroglioma patients, but the molecular mechanisms responsible for this relationship are as yet unknown. The DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT) may cause resistance to DNA-alkylating drugs commonly used in the treatment of anaplastic oligodendrogliomas and other malignant gliomas. We report on the analysis of 52 oligodendroglial tumors for MGMT promoter methylation, as well as mRNA and protein expression. Using sequencing of sodium bisulfite-modified DNA, we determined the methylation status of 25 CpG sites within the MGMT promoter. In 46 of 52 tumors (88%), we detected MGMT promoter hypermethylation as defined by methylation of more than 50% of the sequenced CpG sites. Real-time reverse transcription-PCR showed reduced MGMT mRNA levels relative to non-neoplastic brain tissue in the majority of tumors with hypermethylation. Similarly, immunohistochemical analysis showed either no or only small fractions of MGMT positive tumor cells. MGMT promoter hypermethylation was significantly more frequent and the percentage of methylated CpG sites in the investigated MGMT promoter fragment was significantly higher in tumors with loss of heterozygosity on chromosome arms 1p and 19q as compared to tumors without allelic losses on these chromosomes arms. Taken together, our data suggest that MGMT hypermethylation and low or absent expression are frequent in oligodendroglial tumors and likely contribute to the chemosensitivity of these tumors.

Adolescent↗

PubMed2005

Replication timing of human chromosome 6.

Genomic microarrays have been used to assess DNA replication timing in a variety of eukaryotic organisms. A replication timing map of the human genome has already been published at a 1Mb resolution. Here we describe how the same method can be used to assess the replication timing of chromosome 6 with a greater resolution using an array of overlapping tile path clones. We report the replication timing map of the whole of chromosome 6 in general, and the MHC region in particular. Positive correlations are observed between replication timing and a number of genomic features including GC content, repeat content and transcriptional activity.

Cell Line↗

PubMed2005

Complex chromosome 22 rearrangements in astrocytic tumors identified using microsatellite and chromosome 22 tile path array analysis.

Many studies have reported chromosome 22 as being abnormal in astrocytic tumors. In an attempt to map precisely the abnormal region or regions that potentially harbor tumor-suppressor genes or oncogenes, we constructed a chromosome 22 tile path array covering 82% of 22q with the use of 441 chromosome 22 clones. A 10-Mb whole-genome array consisting of 270 clones from all autosomes was included in the array. A total of 126 astrocytic tumors-5 diffuse astrocytomas (A), 29 anaplastic astrocytomas (AA), and 92 glioblastomas (GB)-were examined for chromosome 22 alterations both by microsatellite analysis (using 28 markers to identify allelic imbalance) and with the tile path array. The results showed that chromosome 22 alterations in astrocytic tumors could be complex. A number of tumors had a combination of deletions with and without reduplication of the retained chromosome, as well as copy number gains and amplifications. In two glioblastomas, overlapping homozygous deletions were identified that involved three genes (DEPDC5/KIAA0645, YWHAH, C22ORF24/HSN44A4A). The terminal region telomeric to the clone RP3-398C22 appeared to be the most frequently deleted region. The estimated incidence of any chromosome 22 alteration was 5% in A, 33% in AA, and 38% in GB. This study demonstrated the advantages of combining array comparative genomic hybridization and microsatellite analysis in elucidating complex genomic rearrangements in primary human tumor tissue. Supplementary material for this article can be found on the Genes, Chromosomes and Cancer website at http://www.interscience.wiley.com/jpages/1045-2257/suppmat/index.html.

Astrocytoma↗

PubMed2004

Molecular pathogenesis of astrocytic tumours.

Our current knowledge of the molecular pathogenesis of the diffuse adult astrocytic tumours is vast if compared to 20 years ago, yet we are far from understanding the details of this process at the molecular level and using such an understanding to logically and specifically treat patients' tumours. In other astrocytic tumours we have little or no knowledge of the molecular processes. This article will attempt to summarise the histological classification criteria and genetic data for all the astrocytic tumours. The current World Health Organisation classification lists six entities, some with subgroups. Common problems associated with the diagnosis of these tumours are outlined. While the molecular findings are not as yet used clinically, we are approaching a time when the histological investigation will have to be supplemented with molecular data to ensure the best choice of treatment for the patient and as an accurate indicator of prognosis.

Adult↗

PubMed2003

Short postoperative survival for glioblastoma patients with a dysfunctional Rb1 pathway in combination with no wild-type PTEN.

PURPOSE: Glioblastoma (GB, WHO grade IV) is the most common primary brain tumor in adults. Survival is typically <1 year but varies between a few months and a couple of years. The aim of the study was to find novel genetic prognostic factors in a well-defined GB series. EXPERIMENTAL DESIGN: The survival data on 129 GBs were correlated with the results of a detailed analysis of 9 genes. These included 3 genes coding for proteins in the p53 pathway (i.e., TP53, p14(ARF), and MDM2), 4 in the Rb1 pathway (i.e., CDKN2A, CDKN2B, RB1, and CDK4), as well as PTEN and epidermal growth factor receptor. RESULTS: We found that abnormalities in any of the four genes (CDKN2A, CDKN2B, RB1, and CDK4) coding for components of the Rb1 pathway were associated with shorter survival (P = 0.002). In combination with loss of wild-type PTEN, the association was even stronger (P < 0.001), the median survival being 166 days as compared with the group without these abnormalities where the median postoperative survival was 437 days. The survival difference remained statistically significant in Cox' regression analysis adjusting for age (P = 0.012). CONCLUSIONS: The findings indicate that knowledge of the molecular genetic abnormalities in GBs provides important data in assessing individual patients. As additional advances in our understanding of the molecular genetics and cell biology of gliomas are made, in addition to providing prognostic information, such data may also provide targets for innovative therapy in the individual case.

Adolescent↗

PubMed2003

Expression of the coxsackie and adenovirus receptor in human astrocytic tumors and xenografts.

The sensitivity of human tissues and tumors to infection with type C adenoviruses correlates with the expression of the human coxsackie B- and adenovirus receptor, hCAR. HCAR is heterogeneously expressed in various tissues and types of human cancer cells, which has implications for the use of adenoviruses as vectors in cancer gene therapy. Using immunoblotting, real-time PCR, FACS-analysis and sensitivity to infection with adenovirus-lacZ, we analyzed the expression level of hCAR in glioma Grade IV cell lines. With real-time PCR, we also analyzed hCAR expression in primary human astrocytomas of different malignancy grades, as well as in their xenograft derivatives. Analysis of a set of 10 cell lines showed great variation in hCAR expression. Susceptibility to Ad5lacZ correlated well with hCAR expression, whereas no correlation was observed with the expression of alphavbeta3/alphavbeta5 integrins, proposed to function as co-receptors for adenoviruses. A great variation of CAR expression was also observed in primary astrocytomas of different malignancy grades. The mean value of CAR expression was significantly lower in 22 Grade IV tumors as compared to the values for 6 Grade II (p = 0.01) and 6 Grade III (p = 0.01) tumors. When the hCAR expression in 11 xenografts derived from Grade IV gliomas were compared to the levels detected in the original parental tumors, a mean 12-fold higher expression was seen in the xenografts (P = 0.01). Two xenografts with low hCAR expression grew considerably faster than the hCAR-expressing cells. Our results have relevance for the use of adenoviruses in gene therapy against astrocytomas.

Adenoviridae↗

PubMed2003

High-resolution profiling of an 11 Mb segment of human chromosome 22 in sporadic schwannoma using array-CGH.

Previous low-resolution schwannoma studies have reported diverse frequencies (30-80%) of 22q deletions, involving the neurofibromatosis-2 tumor suppressor (NF2) gene. We constructed an array spanning 11 million base pairs of 22q encompassing the NF2 gene, with 100% coverage and an average resolution of 58 kb. Moreover, the 220 kb genomic sequence encompassing the NF2 gene was covered by 13 cosmids to further enhance the resolution of analysis. The rationale of this array-CGH study was to map and size 22q deletions around the NF2 gene in sporadic schwannoma using a reliable method with maximal resolution. We studied tumor and constitutional DNA from 47 patients and detected heterozygous deletions in 21 (45%) tumors, which could be classified into three profiles. The predominant profile (12/21) was a continuous deletion of the 11 Mb segment, consistent with monosomy 22. The second profile, comprising five schwannomas, was also in agreement with a continuous 11 Mb heterozygous deletion. However, these displayed a distinctly different level of deletion when compared to the first profile, suggesting a considerable amount of normal tissue in the tumor samples. This is the first report demonstrating the sensitivity of array-CGH to discriminate such samples. The third profile was composed of four cases displaying interstitial deletions of various sizes. Two of these did not encompass the NF2 locus, which further emphasize the importance of other loci in schwannoma development. This is the first high-resolution study performed on a large series of tumors, using an array continuously covering 1/3 of a human chromosome. Our findings warrant further studies of an extended tumor series on a full 22q genomic array, to better define additional, putative 22q-located loci important for schwannoma development. Our array also provides a new diagnostic tool for analysis of NF2 gene deletions in patients affected with neurofibromatosis-2.

Adult↗

PubMed2002

Cellular mechanisms targeted during astrocytoma progression.

Astrocytomas are the commonest type of primary brain tumour. Four malignancy grades are recognized with very different prognosis. The most malignant and commonest form in adults is called glioblastoma and has a median survival with modern treatment of less than one year. Over the last 20 years, molecular genetic and cell biological data have helped identify some of the genes affected during oncogenesis and progression. This will be briefly reviewed. Many significant observations have been made but we are still far from understanding the neoplastic astrocyte at the molecular level. This is reflected in inconclusive attempts to date to use current molecular knowledge in providing additional prognostic information and in the design of molecular based treatments. However, it seems reasonable to assume that further understanding of oncogenesis and progression at the molecular level will provide a basis for improved clinical assessment and individually tailored treatment.

Astrocytoma↗

PubMed2002

A full-coverage, high-resolution human chromosome 22 genomic microarray for clinical and research applications.

We have constructed the first comprehensive microarray representing a human chromosome for analysis of DNA copy number variation. This chromosome 22 array covers 34.7 Mb, representing 1.1% of the genome, with an average resolution of 75 kb. To demonstrate the utility of the array, we have applied it to profile acral melanoma, dermatofibrosarcoma, DiGeorge syndrome and neurofibromatosis 2. We accurately diagnosed homozygous/heterozygous deletions, amplifications/gains, IGLV/IGLC locus instability, and breakpoints of an imbalanced translocation. We further identified the 14-3-3 eta isoform as a candidate tumor suppressor in glioblastoma. Two significant methodological advances in array construction were also developed and validated. These include a strictly sequence defined, repeat-free, and non-redundant strategy for array preparation. This approach allows an increase in array resolution and analysis of any locus; disregarding common repeats, genomic clone availability and sequence redundancy. In addition, we report that the application of phi29 DNA polymerase is advantageous in microarray preparation. A broad spectrum of issues in medical research and diagnostics can be approached using the array. This well annotated and gene-rich autosome contains numerous uncharacterized disease genes. It is therefore crucial to associate these genes to specific 22q-related conditions and this array will be instrumental towards this goal. Furthermore, comprehensive epigenetic profiling of 22q-located genes and high-resolution analysis of replication timing across the entire chromosome can be studied using our array.

Chromosome Mapping↗