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Atypical lipomatous tumor (ALT) is an intermediate malignant mesenchymal tumor that is characterized by supernumerary ring chromosomes and/or giant rod-shaped marker chromosomes (RGMC). Fluorescence in situ hybridization (FISH) and molecular genetic analyses have disclosed that the RGMCs always contain amplified sequences from the long arm of chromosome 12. Typically, RGMCs are the sole clonal changes and so far no deletions or other morphologic aberrations of the two normal-appearing chromosomes 12 that invariably are present have been detected. The mechanisms behind the formation of the RGMCs are unknown, but it could be hypothesized that RGMC formation is preceded by trisomy 12 or, alternatively, that ring formation of one chromosome 12 is followed by duplication of the remaining homolog. The latter scenario would always result in isodisomy for the two normal-appearing chromosomes 12, whereas the former would yield isodisomy in one-third of the cases. In order to investigate these possible mechanisms behind ring formation, we studied polymorphic loci on chromosome 12 in 14 cases of ALT showing one or more supernumerary ring chromosomes and few or no other clonal aberrations at cytogenetic analysis. The molecular genetic analyses showed that the tumor cells always retained both parental copies of chromosome 12, thus refuting the trisomy 12 and duplication hypotheses. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Clinical genetic laboratories often require a comprehensive analysis of chromosomal rearrangements/structural variants (SVs), from large events like translocations and inversions to supernumerary ring/marker chromosomes and small deletions or duplications. Understanding the complexity of these events and their clinical consequences requires pinpointing breakpoint junctions and resolving the derivative chromosome structure. This task often surpasses the capabilities of short-read sequencing technologies. In contrast, long-read sequencing techniques present a compelling alternative for clinical diagnostics. Here, Genomic Medicine Sweden-Rare Diseases has explored the utility of HiFi Revio long-read genome sequencing (lrGS) for digital karyotyping of SVs nationwide. The 16 samples from 13 families were collected from all Swedish healthcare regions. Prior investigations had identified 16 SVs, ranging from simple to complex rearrangements, including inversions, translocations, and copy number variants. We have established a national pipeline and a shared variant database for variant calling and filtering. Using lrGS, 14 of the 16 known SVs are detected. Of these, 13 are mapped at nucleotide resolution, and one complex rearrangement is only visible by read depth. Two Chromosome 21 rearrangements, one mosaic, remain undetected. Average read lengths are 8.3-18.8 kb with coverage exceeding 20× for all samples. De novo assembly results in a limited number of phased contigs per individual (N50 6-86 Mb), enabling direct characterization of the chromosomal rearrangements. In a national pilot study, we demonstrate the utility of HiFi Revio lrGS for analyzing chromosomal rearrangements. Based on our results, we propose a 5-year plan to expand lrGS use for rare disease diagnostics in Sweden., (© 2024 Eisfeldt et al.; Published by Cold Spring Harbor Laboratory Press.)

The physiological response of a cell to stimulation depends on its proteome configuration. Therefore, the abundance variation of regulatory proteins across unstimulated single cells can be associatively linked with their response to stimulation. Here we developed an approach that leverages this association across individual cells and nuclei to systematically identify potential regulators of biological processes, followed by targeted validation. Specifically, we applied this approach to identify regulators of nucleocytoplasmic protein transport in macrophages stimulated with lipopolysaccharide (LPS). To this end, we quantified the proteomes of 3,412 individual nuclei, sampling the dynamic response to LPS treatment, and linking functional variability to proteomic variability. Minutes after the stimulation, the protein transport in individual nuclei correlated strongly with the abundance of known protein transport regulators, thus revealing the impact of natural protein variability on functional cellular response. We found that simple biophysical constraints, such as the quantity of nuclear pores, partially explain the variability in LPS-induced nucleocytoplasmic transport. Among the many proteins newly identified to be associated with the response, we selected 16 for targeted validation by knockdown. The knockdown phenotypes confirmed the inferences derived from natural protein and functional variation of single nuclei, thus demonstrating the potential of (sub-)single-cell proteomics to infer functional regulation. We expect this approach to generalize to broad applications and enhance the functional interpretability of single-cell omics data., Competing Interests: Competing interests: N.S. is a founding director and CEO of Parallel Squared Technology Institute, which is a nonprofit research institute.

TP53 is the most frequently mutated gene in human cancer. This gene shows not only loss-of-function mutations but also recurrent missense mutations with gain-of-function activity. We have studied the primary bone malignancy osteosarcoma, which harbours one of the most rearranged genomes of all cancers. This is odd since it primarily affects children and adolescents who have not lived the long life thought necessary to accumulate massive numbers of mutations. In osteosarcoma, TP53 is often disrupted by structural variants. Here, we show through combined whole-genome and transcriptome analyses of 148 osteosarcomas that TP53 structural variants commonly result in loss of coding parts of the gene while simultaneously preserving and relocating the promoter region. The transferred TP53 promoter region is fused to genes previously implicated in cancer development. Paradoxically, these erroneously upregulated genes are significantly associated with the TP53 signalling pathway itself. This suggests that while the classical tumour suppressor activities of TP53 are lost, certain parts of the TP53 signalling pathway that are necessary for cancer cell survival and proliferation are retained. In line with this, our data suggest that transposition of the TP53 promoter is an early event that allows for a new normal state of genome-wide rearrangements in osteosarcoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Objectives: The aim of this study was to investigate genetic outcomes, analyze the family experience, and describe the process of implementing genetic sequencing for children with profound sensorineural hearing loss (SNHL) at a tertial audiological center in southern Sweden., Design: This is a prospective pilot study including eleven children with profound bilateral SNHL who underwent cochlear implant surgery. Genetic diagnostic investigation was performed with whole exome sequencing (WES) complemented with XON-array to identify copy number variants, using a manually curated gene panel incorporating 179 genes associated with non-syndromic and syndromic SNHL. Mitochondrial DNA (mtDNA) from blood was examined separately. A patient reported experience measures (PREM) questionnaire was used to evaluate parental experience. We also describe here the process of implementing WES in an audiology department., Results: Six female and five male children (mean 3.4 years, SD 3.5 years), with profound bilateral SNHL were included. Genetic variants of interest were found in six subjects (55%), where three (27%) could be classified as pathogenic or likely pathogenic. Among the six cases, one child was found to have a homozygous pathogenic variant in MYO7A and two children had homozygous likely pathogenic variants in SLC26A4 and PCDH15, respectively. One was carrying a compound heterozygote frameshift variant of uncertain significance (VUS) on one allele and in trans, a likely pathogenic deletion on the other allele in PCDH15. Two subjects had homozygous VUS in PCDH15 and ADGRV1, respectively. In five of the cases the variants were in genes associated with Usher syndrome. For one of the likely pathogenic variants, the finding was related to Pendred syndrome. No mtDNA variants related to SNHL were found. The PREM questionnaire revealed that the families had difficulty in fully understanding the results of the genetic analysis. However, the parents of all eleven (100%) subjects still recommended that other families with children with SNHL should undergo genetic testing. Specifically addressed referrals for prompt complementary clinical examination and more individualized care were possible, based on the genetic results. Close clinical collaboration between different specialists, including physicians of audiology, audiologists, clinical geneticists, ophthalmologists, pediatricians, otoneurologists, physiotherapists and hearing habilitation teams was initiated during the implementation of the new regime. For all professionals involved, a better knowledge of the diversity of the genetic background of hearing loss was achieved., Conclusions: Whole exome sequencing and XON-array using a panel of genes associated with SNHL had a high diagnostic yield, added value to the families, and provided guidance for further examinations and habilitation for the child. Great care should be taken to thoroughly inform parents about the genetic test result. Collaborations between departments were intensified and knowledge of hearing genomics was increased among the staff., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Mosaic genome-wide paternal uniparental disomy (GW-pUPD) is a rarely recognised disorder. The phenotypic manifestations of multilocus imprinting defects (MLIDs) remain unclear. We report of an apparently non-syndromic infant with severe congenital hyperinsulinism (CHI) and diffuse pancreatic labelling by 18F*-DOPA-PET/CT leading to near-total pancreatectomy. The histology was atypical with pronounced proliferation of endocrine cells comprising >70% of the pancreatic tissue and a small pancreatoblastoma. Routine genetic analysis for CHI was normal in the blood and resected pancreatic tissue. At two years' age, Beckwith-Wiedemann Syndrome (BWS) stigmata emerged, and at five years a liver tumour with focal nodular hyperplasia and an adrenal tumour were resected. pUPD was detected in 11p15 and next in the entire chromosome 11 with microsatellite markers. Quantitative fluorescent PCR with amplification of chromosome-specific DNA sequences for chromosomes 13, 18, 21 and X indicated GW-pUPD. A next generation sequencing panel with 303 SNPs on 21 chromosomes showed pUPD in both blood and pancreatic tissue. The mosaic distribution of GW-pUPD ranged from 31 to 35% in blood and buccal swap to 74% in the resected pancreas, 80% in a non-tumour liver biopsy, and 100% in the liver focal nodular hyperplasia and adrenal tumour. MLID features included transient conjugated hyperbilirubinaemia and lack of macrosomia from BWS (pUPD6); and behavioural and psychomotor manifestations of Angelman Syndrome (pUPD15) on follow-up. In conclusion, atypical pancreatic histology in apparently non-syndromic severe CHI patients may be the first clue to BWS and multi-syndromal CHI from GW-pUPD. Variations in the degree of mosaicism between tissues explained the phenotype., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Wilms tumors in patients with constitutional WT1 mutations are examples of Knudson's tumor suppressor paradigm, with somatic inactivation of the second allele occurring through 11p loss of heterozygosity. The time point of this second hit has remained unknown. We analyzed seven Wilms tumors from two patients with constitutional WT1 mutations by whole exome sequencing and genomic array. All tumors exhibited wild type WT1 loss through uniparental isodisomy. Each tumor had a unique genomic breakpoint in 11p, typically accompanied by a private activating mutation of CTNNB1. Hence, convergent evolution rather than field carcinogenesis underlies multifocal tumors in WT1 mutation carriers., (© 2018 Wiley Periodicals, Inc.)

A major challenge to personalized oncology is that driver mutations vary among cancer cells inhabiting the same tumor. Whether this reflects principally disparate patterns of Darwinian evolution in different tumor regions has remained unexplored 1-5 . We mapped the prevalence of genetically distinct clones over 250 regions in 54 childhood cancers. This showed that primary tumors can simultaneously follow up to four evolutionary trajectories over different anatomic areas. The most common pattern consists of subclones with very few mutations confined to a single tumor region. The second most common is a stable coexistence, over vast areas, of clones characterized by changes in chromosome numbers. This is contrasted by a third, less frequent, pattern where a clone with driver mutations or structural chromosome rearrangements emerges through a clonal sweep to dominate an anatomical region. The fourth and rarest pattern is the local emergence of a myriad of clones with TP53 inactivation. Death from disease was limited to tumors exhibiting the two last, most dynamic patterns.

PurposePREPL deficiency causes neonatal hypotonia, ptosis, neonatal feeding difficulties, childhood obesity, xerostomia, and growth hormone deficiency. Different recessive contiguous gene deletion syndromes involving PREPL and a variable combination of SLC3A1 (hypotonia-cystinuria syndrome), CAMKMT (atypical hypotonia-cystinuria syndrome), and PPM1B (2p21 deletion syndrome) have been described. In isolated PREPL deficiency, previously described only once, the absence of cystinuria complicates the diagnosis. Therefore, we developed a PREPL blood assay and further delineated the phenotype.MethodsClinical features of new subjects with PREPL deficiency were recorded. The presence of PREPL in lymphocytes and its reactivity with an activity-based probe were evaluated by western blot.ResultsFive subjects with isolated PREPL deficiency, three with hypotonia-cystinuria syndrome, and two with atypical hypotonia-cystinuria syndrome had nine novel alleles. Their IQs ranged from 64 to 112. Adult neuromuscular signs included ptosis, nasal dysarthria, facial weakness, and variable proximal and neck flexor weakness. Autonomic features are prevalent. PREPL protein and reactivity were absent in lymphocytes from subjects with PREPL deficiency, but normal in the clinically similar Prader-Willi syndrome.ConclusionPREPL deficiency causes neuromuscular, autonomic, cognitive, endocrine, and dysmorphic clinical features. PREPL is not deficient in Prader-Willi syndrome. The novel blood test should facilitate the confirmation of PREPL deficiency.

Subtelomeric 1q43q44 microdeletions cause a syndrome associating intellectual disability, microcephaly, seizures and anomalies of the corpus callosum. Despite several previous studies assessing genotype-phenotype correlations, the contribution of genes located in this region to the specific features of this syndrome remains uncertain. Among those, three genes, AKT3, HNRNPU and ZBTB18 are highly expressed in the brain and point mutations in these genes have been recently identified in children with neurodevelopmental phenotypes. In this study, we report the clinical and molecular data from 17 patients with 1q43q44 microdeletions, four with ZBTB18 mutations and seven with HNRNPU mutations, and review additional data from 37 previously published patients with 1q43q44 microdeletions. We compare clinical data of patients with 1q43q44 microdeletions with those of patients with point mutations in HNRNPU and ZBTB18 to assess the contribution of each gene as well as the possibility of epistasis between genes. Our study demonstrates that AKT3 haploinsufficiency is the main driver for microcephaly, whereas HNRNPU alteration mostly drives epilepsy and determines the degree of intellectual disability. ZBTB18 deletions or mutations are associated with variable corpus callosum anomalies with an incomplete penetrance. ZBTB18 may also contribute to microcephaly and HNRNPU to thin corpus callosum, but with a lower penetrance. Co-deletion of contiguous genes has additive effects. Our results confirm and refine the complex genotype-phenotype correlations existing in the 1qter microdeletion syndrome and define more precisely the neurodevelopmental phenotypes associated with genetic alterations of AKT3, ZBTB18 and HNRNPU in humans.

Myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN-U) are rare genetically heterogeneous hematologic diseases associated with older age and a poor prognosis. If the disease progresses into acute myeloid leukemia (AML), it is often refractory to treatment. To gain insight into genetic alterations associated with disease progression, whole exome sequencing and single nucleotide polymorphism arrays were used to characterize the bone marrow and blood samples from a 39-year-old woman at MDS/MPN-U diagnosis and at AML progression, in which routine genetic diagnostics had not identified any genetic alterations. The data revealed the presence of a partial tandem duplication of the MLL gene as the only detectable copy number change and 11 non-silent somatic mutations, including DNMT3A R882H and NRAS G13D. All somatic lesions were present both at initial MDS/MPN-U diagnosis and at AML presentation at similar mutant allele frequencies. The patient has since had two extramedullary relapses and is at high risk of a future bone marrow relapse. A directed ex vivo drug sensitivity analysis showed that the patient's AML cells are sensitive to, for example, the MEK inhibitor trametinib and the proteasome inhibitor bortezomib, indicating that she may benefit from treatment with these drugs. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents. Alveolar (ARMS) and embryonal (ERMS) histologies predominate, but rare cases are classified as spindle cell/sclerosing (SRMS). For treatment stratification, RMS is further subclassified as fusion-positive (FP-RMS) or fusion-negative (FN-RMS), depending on whether a gene fusion involving PAX3 or PAX7 is present or not. We investigated 19 cases of pediatric RMS using high resolution single-nucleotide polymorphism (SNP) array. FP-ARMS displayed, on average, more structural rearrangements than ERMS; the single FN-ARMS had a genomic profile similar to ERMS. Apart from previously known amplification (e.g., MYCN, CDK4, and MIR17HG) and deletion (e.g., NF1, CDKN2A, and CDKN2B) targets, amplification of ERBB2 and homozygous loss of ASCC3 or ODZ3 were seen. Combining SNP array with cytogenetic data revealed that most cases were polyploid, with at least one case having started as a near-haploid tumor. Further bioinformatic analysis of the SNP array data disclosed genetic heterogeneity, in the form of subclonal chromosomal imbalances, in five tumors. The outcome was worse for patients with FP-ARMS than ERMS or FN-ARMS (6/8 vs. 1/9 dead of disease), and the only children with ERMS showing intratumor diversity or with MYOD1 mutation-positive SRMS also died of disease. High resolution SNP array can be useful in evaluating genomic imbalances in pediatric RMS., (© 2015 Wiley Periodicals, Inc.)

Neuroblastoma is a childhood tumour with heterogeneous characteristics and children with metastatic disease often have a poor outcome. Here we describe the establishment of neuroblastoma patient-derived xenografts (PDXs) by orthotopic implantation of viably cryopreserved or fresh tumour explants of patients with high risk neuroblastoma into immunodeficient mice. In vivo tumour growth was monitored by magnetic resonance imaging and fluorodeoxyglucose-positron emission tomography. Neuroblastoma PDXs retained the undifferentiated histology and proliferative capacity of their corresponding patient tumours. The PDXs expressed neuroblastoma markers neural cell adhesion molecule, chromogranin A, synaptophysin and tyrosine hydroxylase. Whole genome genotyping array analyses demonstrated that PDXs retained patient-specific chromosomal aberrations such as MYCN amplification, deletion of 1p and gain of chromosome 17q. Thus, neuroblastoma PDXs recapitulate the hallmarks of high-risk neuroblastoma in patients. PDX-derived cells were cultured in serum-free medium where they formed free-floating neurospheres, expressed neuroblastoma gene markers MYCN, CHGA, TH, SYP and NPY, and retained tumour-initiating and metastatic capacity in vivo. PDXs showed much higher degree of infiltrative growth and distant metastasis as compared to neuroblastoma SK-N-BE(2)c cell line-derived orthotopic tumours. Importantly, the PDXs presented with bone marrow involvement, a clinical feature of aggressive neuroblastoma. Thus, neuroblastoma PDXs serve as clinically relevant models for studying and targeting high-risk metastatic neuroblastoma., (© 2014 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.)

Genetic differences among neoplastic cells within the same tumour have been proposed to drive cancer progression and treatment failure. Whether data on intratumoral diversity can be used to predict clinical outcome remains unclear. We here address this issue by quantifying genetic intratumoral diversity in a set of chemotherapy-treated childhood tumours. By analysis of multiple tumour samples from seven patients we demonstrate intratumoral diversity in all patients analysed after chemotherapy, typically presenting as multiple clones within a single millimetre-sized tumour sample (microdiversity). We show that microdiversity often acts as the foundation for further genome evolution in metastases. In addition, we find that microdiversity predicts poor cancer-specific survival (60%; P=0.009), independent of other risk factors, in a cohort of 44 patients with chemotherapy-treated childhood kidney cancer. Survival was 100% for patients lacking microdiversity. Thus, intratumoral genetic diversity is common in childhood cancers after chemotherapy and may be an important factor behind treatment failure.

Background: Urothelial carcinoma (UC) is characterized by nonrandom chromosomal aberrations, varying from one or a few changes in early-stage and low-grade tumors, to highly rearranged karyotypes in muscle-invasive lesions. Recent array-CGH analyses have shed further light on the genomic changes underlying the neoplastic development of UC, and have facilitated the molecular delineation amplified and deleted regions to the level of specific candidate genes. In the present investigation we combine detailed genomic information with expression information to identify putative target genes for genomic amplifications., Methods: We analyzed 38 urothelial carcinomas by whole-genome tiling resolution array-CGH and high density expression profiling to identify putative target genes in common genomic amplifications. When necessary expression profiling was complemented with Q-PCR of individual genes., Results: Three genomic segments were frequently and exclusively amplified in high grade tumors; 1q23, 6p22 and 8q22, respectively. Detailed mapping of the 1q23 segment showed a heterogeneous amplification pattern and no obvious commonly amplified region. The 6p22 amplicon was defined by a 1.8 Mb core region present in all amplifications, flanked both distally and proximally by segments amplified to a lesser extent. By combining genomic profiles with expression profiles we could show that amplification of E2F3, CDKAL1, SOX4, and MBOAT1 as well as NUP153, AOF1, FAM8A1 and DEK in 6p22 was associated with increased gene expression. Amplification of the 8q22 segment was primarily associated with YWHAZ (14-3-3-zeta) and POLR2K over expression. The possible importance of the YWHA genes in the development of urothelial carcinomas was supported by another recurrent amplicon paralogous to 8q22, in 2p25, where increased copy numbers lead to enhanced expression of YWHAQ (14-3-3-theta). Homozygous deletions were identified at 10 different genomic locations, most frequently affecting CDKN2A/CDKN2B in 9p21 (32%). Notably, the latter occurred mutually exclusive with 6p22 amplifications., Conclusion: The presented data indicates 6p22 as a composite amplicon with more than one possible target gene. The data also suggests that amplification of 6p22 and homozygous deletions of 9p21 may have complementary roles. Furthermore, the analysis of paralogous regions that showed genomic amplification indicated altered expression of YWHA (14-3-3) genes as important events in the development of UC.

The t(1;19)(q23;p13), one of the most common translocations in childhood and adult acute lymphoblastic leukaemias (ALLs), usually results in fusion of exons 1-16 of TCF3 (previously E2A) and exons 3-9 of PBX1. However, some t(1;19)-positive ALLs are negative for this chimaera. We here report an alternative TCF3/PBX1 transcript, fusing exon 17 of TCF3 with exon 5 of PBX1, in a paediatric t(1;19)-positive ALL. The different breakpoints made this hybrid undetectable by reverse transcription polymerase chain reaction using standard TCF3 and PBX1 primers. Hence, ALLs with t(1;19) that test negative for TCF3/PBX1 should be analysed further before excluding this alternative fusion. Furthermore, we have characterised the genomic translocation breakpoints in eight TCF3/PBX1-positive ALLs; four cases with a balanced t(1;19) and four with an unbalanced der(19)t(1;19). It has previously been suggested that the breakpoints are clustered, particularly in TCF3, and that N-nucleotides are frequently present in the fusion junctions. Three of seven investigated TCF3 intron 16 breakpoints were within the previously described 14 base pair-cluster, and all but two junctions harboured N-nucleotides. The PBX1 breakpoints were more dispersed, although still clustered in two regions. This confirms that most t(1;19) rearrangements may arise by a combination of illegitimate V(D)J recombination and non-homologous end joining.

Purpose: Chromosomal instability (CIN) is believed to have an important role in the pathogenesis of urothelial cancer (UC). The aim of this study was to evaluate whether disturbances of mitotic segregation contribute to CIN in UC, if these processes have any effect on the course of disease, and how deregulation of these mechanisms affects tumor cell growth., Experimental Design: We developed molecular cytogenetic methods to classify mitotic segregation abnormalities in a panel of UC cell lines. Mitotic instabilities were then scored in biopsies from 52 UC patients and compared with the outcome of tumor disease. Finally, UC cells were exposed in vitro to a telomerase inhibitor to assess how this affects mitotic stability and cell proliferation., Results: Three distinct chromosome segregation abnormalities were identified: (a) telomere dysfunction, which triggers structural rearrangements and loss of chromosomes through anaphase bridging; (b) sister chromatid nondisjunction, which generates discrete chromosomal copy number variations; and (c) supernumerary centrosomes, which cause dramatic shifts in chromosome copy number through multipolar cell division. Chromosome segregation errors were already present in preinvasive tumors and a high rate mitotic instability was an independent predictor of poor survival. However, induction of even higher levels of the same segregation abnormalities in UC cells by telomerase inhibition in vitro led to reduced tumor cell proliferation and clonogenic survival., Conclusion: Several distinct chromosome segregation errors contribute to CIN in UC, and the rate of such mitotic errors has a significant effect on the clinical course. Efficient tumor cell proliferation may depend on the tight endogenous control of these processes.

Background: Cohort studies have reported increased risks of cardiopulmonary mortality from long-term air pollution exposure, but the evidence is limited and inconclusive. We studied the association between long-term exposure to source-specific air pollution and myocardial infarction (MI) in a case-control study of first-time MI cases and population controls age 45 to 70 years in Stockholm county in 1992 to 1994., Methods: Home addresses during several decades were combined with historical emission databases and dispersion models to obtain annual mean levels of pollutants from traffic and heating during 30 years for 1397 cases and 1870 controls. Nitrogen dioxide (NO2), carbon monoxide (CO), and particulate matter with an aerodynamic diameter less than 10 microm (PM10) were used as indicators of traffic emissions and sulfur dioxide (SO2) as an indicator of emissions from residential heating., Results: There was no association between long-term average air pollution exposure and overall MI, but an increased risk of fatal MI was suggested, especially for out-of-hospital death. After adjustment for cardiovascular risk factors, the odds ratio for fatal MI associated with a 5th to 95th percentile difference in 30-year average exposure was 1.51 (95% confidence interval = 0.96-2.16) for NO2, 1.22 (0.98-1.52) for CO, 1.39 (0.94-2.07) for PM10, and 1.24 (0.77-2.02) for SO2. For out-of-hospital death, the odds ratio related to NO2 exposure was 2.17 (1.05-4.51)., Conclusions: This study provides some support for an association between long-term air pollution exposure and fatal cardiovascular disease.

Telomere dysfunction has been associated with chromosomal instability in colorectal carcinoma, but the consequences of telomere-dependent instability for chromosome integrity and clonal evolution have been little explored. We show here that abnormally short telomeres lead to a wide spectrum of mitotic disturbances in colorectal cancer cell lines, including anaphase bridging, whole-chromosome lagging, and mitotic multipolarity. These abnormalities were found in both the presence and absence of microsatellite instability. The mean telomere length varied extensively between cells from the same tumor, allowing the establishment of tumor cell subpopulations with highly different frequencies of mitotic disturbances. Anaphase bridging typically resulted in either inter-centromeric chromatin fragmentation or centromere detachment, leading to pericentromeric chromosome rearrangements and loss of whole chromosomes, respectively. There was a strong correlation between anaphase bridges and multipolar mitoses, and the induction of dicentric chromosomes by gamma irradiation and telomerase inhibition led to an elevated frequency of multipolar mitotic spindles, suggesting that multipolarity could result from polyploidization triggered by anaphase bridging. Chromatid segregation in multipolar mitoses was close to random, resulting in frequent nullisomies and nonviable daughter cells. In contrast, there was a high clonogenic survival among cells having gone through anaphase bridging in bipolar mitoses. Bridging of telomere-deficient chromosomes could thus be a major mutational mechanism in colorectal cancer, whereas mitotic multipolarity appears to be a secondary phenomenon that rarely, if ever, contributes to clonal evolution.

DNA copy number alterations are believed to play a major role in the development and progression of human neoplasms. Although most of these genomic imbalances have been associated with dysregulation of individual genes, their large-scale transcriptional consequences remain unclear. Pancreatic carcinomas frequently display gene copy number variation of entire chromosomes as well as of chromosomal subregions. These changes range from homozygous deletions to high-level amplifications and are believed to constitute key genetic alterations in the cellular transformation of this tumor type. To investigate the transcriptional consequences of the most drastic genomic changes, that is, genomic amplifications, and to analyse the genome-wide transcriptional effects of DNA copy number changes, we performed expression profiling of 29 pancreatic carcinoma cell lines and compared the results with matching genomic profiling data. We show that a strong association between DNA copy numbers and mRNA expression levels is present in pancreatic cancer, and demonstrate that as much as 60% of the genes within highly amplified genomic regions display associated overexpression. Consequently, we identified 67 recurrently overexpressed genes located in seven precisely mapped commonly amplified regions. The presented findings indicate that more than one putative target gene may be of importance in most pancreatic cancer amplicons.

Background: It is difficult to assess the orientation of the acetabular component on routine radiographs. We present a method for determining the spatial orientation of the acetabular component after total hip arthroplasty (THA) using computed tomography., Patients and Methods: Two CT-scans, 10 min apart, were obtained from each of 10 patients after THA. Using locally developed software, two independent examiners measured the orientation of the acetabular component in relation to the pelvis. The measurements were repeated after one week. To be independent of the patient position during scanning, the method involved two steps. Firstly, a 3D volumetric image of the pelvis was brought into a standard pelvic orientation, then the orientation of the acetabular component was measured. The orientation of the acetabular component was expressed as operative anteversion and inclination relative to an internal pelvic reference coordinate system. To evaluate precision, we compared measurements across pairs of CT volumes between observers and trials., Results: Mean absolute interobserver angle error was 2.3 degrees for anteversion (range 0-6.6 degrees), and 1.1 degrees for inclination (range 0-4.6 degrees). For interobserver measurements, the precision, defined as one standard deviation, was 2.9 degrees for anteversion, and 1.5 degrees for inclination. A Student's t-test showed that the overall differences between the examiners, trials, and cases were not significant. Data were normally distributed and were not dependent on examiner or trial., Interpretation: We conclude that the implant angles of the acetabular component in relation to the pelvis could be detected repeatedly using CT, independently of patient positioning.

Pancreatic carcinomas display highly complex chromosomal abnormalities, including many structural and numerical aberrations. There is ample evidence indicating that some of these abnormalities, such as recurrent amplifications and homozygous deletions, contribute to tumorigenesis by altering expression levels of critical oncogenes and tumor suppressor genes. To increase the understanding of gene copy number changes in pancreatic carcinomas and to identify key amplification/deletion targets, we applied genome-wide array-based comparative genomic hybridization to 31 pancreatic carcinoma cell lines. Two different microarrays were used, one containing 3,565 fluorescence in situ hybridization-verified bacterial artificial chromosome clones and one containing 25,468 cDNA clones representing 17,494 UniGene clusters. Overall, the analyses revealed a high genomic complexity, with several copy number changes detected in each case. Specifically, 60 amplicons at 32 different locations were identified, most frequently located within 8q (8 cases), 12p (7 cases), 7q (5 cases), 18q (5 cases), 19q (5 cases), 6p (4 cases), and 8p (4 cases). Amplifications of 8q and 12p were mainly clustered at 8q23-24 and 12p11-12, respectively, whereas amplifications on other chromosome arms were more dispersed. Furthermore, our analyses identified several novel homozygously deleted segments located to 9p24, 9p21, 9q32, 10p12, 10q22, 12q24, and 18q23. The individual complexity and aberration patterns varied substantially among cases, i.e., some cell lines were characterized mainly by high-level amplifications, whereas others showed primarily whole-arm imbalances and homozygous deletions. The described amplification and deletion targets are likely to contain genes important in pancreatic tumorigenesis.

Transforming growth factor beta-1 (TGFB1)-induced gene expression was studied in five pancreatic carcinoma cell lines and one known TGFB1-sensitive cell line (HaCaT) by use of high-density filter-based cDNA microarrays representing over 4,000 human genes. The results indicate a complex cellular response to TGFB1 with 10% of the investigated genes showing altered expression after 3 or 48 hr of TGFB1 exposure. The tumor cell lines displayed a gradually inversed gene expression pattern, which correlated with reduced sensitivity to TGFB1, as compared to the HaCaT cell line. In the HaCaT cells, several proapoptotic genes showed increased expression in response to TGFB1, whereas the expression of antiapoptotic genes was decreased. In contrast, two pancreatic carcinoma cell lines, previously found to be growth stimulated by TGFB1, displayed an expression pattern opposite to that of these genes. Similarly, the expression of other functional groups of genes, such as cell cycle and transcription factor related genes, was almost completely reversed in these two tumor cell lines. Importantly, three of the five investigated pancreatic carcinoma cell lines responded to TGFB1, although they had SMAD4 inactivations, suggesting that the observed gene expression changes in these cell lines must be accomplished by SMAD-independent pathways., (Copyright 2003 Wiley-Liss, Inc.)

Previous cytogenetic and comparative genomic hybridization (CGH) analyses have shown that the gain of chromosome arm 12p is frequent in pancreatic carcinomas. We investigated 15 pancreatic carcinoma cell lines using CGH, fluorescence in situ hybridization (FISH), and semiquantitative polymerase chain reaction (PCR) to characterize 12p amplifications in detail. The CGH analysis revealed gains of 12p in four of the cell lines and local amplification within 12p11-12 in six cell lines. By FISH analysis, using precisely mapped YAC clones, the commonly amplified region was found to be approximately 5 Mb. The amplified segment extended from YAC 753f12, covering the KRAS2 locus, to YAC 891f1, close to the centromere. A semiquantitative PCR methodology was used to estimate genomic copy numbers of 14 precisely mapped expressed sequence tags (ESTs) and sequence-tagged sites, located within this interval. The level of amplification ranged from two- to 12-fold. The produced gene copy profiles revealed a 3.5-Mb segment with various local amplifications. This region includes KRAS2 and ranges from D12S1617 to sts-N38796. Two of the cell lines (primary and metastatic tumor from the same patient) showed amplification peaks within the distal region of this segment, two had peaks within the proximal region, one showed subpeaks in both regions, and one displayed amplification of the entire region. Chromosome segment-specific cDNA array analysis of 29 expressed sequences within the whole interval between D12S1617 and sts-N38796 indicated overexpression of four ESTs, two corresponding to DEC2 and PPFIBP1, and two to ESTs with unknown function. Expression analysis of these and of KRAS2 showed specific overexpression in the six cell lines with local 12p amplifications. These findings indicate two target regions within the 3.5-Mb segment in 12p11-12, one proximal including PPFIBP1, and one distal including KRAS2., (Copyright 2002 Wiley-Liss, Inc.)

Although mechanisms for chromosomal instability in tumors have been described in animal and in vitro models, little is known about these processes in man. To explore cytogenetic evolution in human tumors, chromosomal breakpoint profiles were constructed for 102 pancreatic carcinomas and 140 osteosarcomas, two tumor types characterized by extensive genomic instability. Cases with few chromosomal alterations showed a preferential clustering of breakpoints to the terminal bands, whereas tumors with many changes showed primarily interstitial and centromeric breakpoints. The terminal breakpoint frequency was negatively correlated to telomeric TTAGGG repeat length, and fluorescence in situ hybridization with telomeric TTAGGG probes consistently indicated shortened telomeres and >10% of chromosome ends lacking telomeric signals. Because telomeric dysfunction may lead to formation of unstable ring and dicentric chromosomes, mitotic figures were also evaluated. Anaphase bridges were found in all cases, and fluorescence in situ hybridization demonstrated extensive structural rearrangements of chromosomes, with terminal transferase detection showing fragmented DNA in 5-20% of interphase cells. Less than 2% of cells showed evidence of necrosis or apoptosis, and telomerase was expressed in the majority of cases. Telomeric dysfunction may thus trigger chromosomal fragmentation through persistent bridge-breakage events in pancreatic carcinomas and osteosarcomas, leading to a continuous reorganization of the tumor genome. Telomerase expression is not sufficient for completely stabilizing the chromosome complement but may be crucial for preventing complete genomic deterioration and maintaining cellular survival.

Alteration of the transforming growth factor beta (TGFB) signalling pathway is important in pancreatic carcinogenesis, as shown by the frequent inactivation of the downstream target SMAD4. We recently analysed a series of pancreatic carcinoma cell lines with respect to alterations of five SMAD genes involved in TGFB signalling, and showed that SMAD4 was structurally rearranged in 42% of these. This pathway may, however, also be affected by alterations of genes whose products regulate the activation of TGFB as well as of TGFB receptor genes. We therefore studied the expression of UPA, UPAR, IGF2R, ALK5 (TGFBR1), TGFBR2, TGFBR3, ENG, ALK1, TGFB1, TGFB2, and TGFB3 in a series of 14 pancreatic carcinoma cell lines. We also analysed ALK5 and TGFBR2 for mutations, cell surface localisation of TGFBR2 and ENG, and TGFB1 response. No mutations of ALK5 or TGFBR2 were found. However, 4 cell lines were methylated within the ALK5 promoter region. ALK5 expression was strongly reduced in 9 cases, whereas TGFBR2 expression was increased in 12 of the cell lines. The TGFB signalling associated receptors ENG and ALK1 were co-expressed in 4 of the cell lines. There was no evidence for disruption of the UPAR-IGF2R TGFB activating pathway. The response to TGFB1 was analysed in 12 cell lines, and 6 of these (50%) showed increased proliferation. The cell lines stimulated by TGFB showed frequent mutations of SMAD4, KRAS2, and TP53, as well as frequent absence of CDKN2B expression. These results suggest that the ALK5-SMAD4 part of the TGFB signalling pathway is a major target for inactivation in pancreatic carcinomas, that the expression of TGFBR2, TGFBR3, and receptors involved in TGFB activation are maintained, and that alterations of components of the TGFB signalling pathway may be accompanied by a positive effect of TGFB on cell growth.