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2.
Nat Commun ; 14(1): 4823, 2023 08 10.
Article in English | MEDLINE | ID: mdl-37563129

ABSTRACT

Despite initial responses to hormone treatment, metastatic prostate cancer invariably evolves to a lethal state. To characterize the intra-patient evolutionary relationships of metastases that evade treatment, we perform genome-wide copy number profiling and bespoke approaches targeting the androgen receptor (AR) on 167 metastatic regions from 11 organs harvested post-mortem from 10 men who died from prostate cancer. We identify diverse and patient-unique alterations clustering around the AR in metastases from every patient with evidence of independent acquisition of related genomic changes within an individual and, in some patients, the co-existence of AR-neutral clones. Using the genomic boundaries of pan-autosome copy number changes, we confirm a common clone of origin across metastases and diagnostic biopsies, and identified in individual patients, clusters of metastases occupied by dominant clones with diverged autosomal copy number alterations. These autosome-defined clusters are characterized by cluster-specific AR gene architectures, and in two index cases are topologically more congruent than by chance (p-values 3.07 × 10-8 and 6.4 × 10-4). Integration with anatomical sites suggests patterns of spread and points of genomic divergence. Here, we show that copy number boundaries identify treatment-selected clones with putatively distinct lethal trajectories.


Subject(s)
DNA Copy Number Variations , Prostatic Neoplasms , Male , Humans , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Receptors, Androgen/genetics , Genome , Genomics , Clone Cells/pathology
3.
Res Sq ; 2023 Feb 08.
Article in English | MEDLINE | ID: mdl-36798177

ABSTRACT

Metastatic and high-risk localized prostate cancer respond to hormone therapy but outcomes vary. Following a pre-specified statistical plan, we used Cox models adjusted for clinical variables to test associations with survival of multi-gene expression-based classifiers from 781 patients randomized to androgen deprivation with or without abiraterone in the STAMPEDE trial. Decipher score was strongly prognostic (p<2×10-5) and identified clinically-relevant differences in absolute benefit, especially for localized cancers. In metastatic disease, classifiers of proliferation, PTEN or TP53 loss and treatment-persistent cells were prognostic. In localized disease, androgen receptor activity was protective whilst interferon signaling (that strongly associated with tumor lymphocyte infiltration) was detrimental. Post-Operative Radiation-Therapy Outcomes Score was prognostic in localized but not metastatic disease (interaction p=0.0001) suggesting the impact of tumor biology on clinical outcome is context-dependent on metastatic state. Transcriptome-wide testing has clinical utility for advanced prostate cancer and identified worse outcomes for localized cancers with tumor-promoting inflammation.

4.
Genome Med ; 14(1): 102, 2022 Sep 05.
Article in English | MEDLINE | ID: mdl-36059000

ABSTRACT

BACKGROUND: Genomic copy number alterations commonly occur in prostate cancer and are one measure of genomic instability. The clinical implication of copy number change in advanced prostate cancer, which defines a wide spectrum of disease from high-risk localised to metastatic, is unknown. METHODS: We performed copy number profiling on 688 tumour regions from 300 patients, who presented with advanced prostate cancer prior to the start of long-term androgen deprivation therapy (ADT), in the control arm of the prospective randomised STAMPEDE trial. Patients were categorised into metastatic states as follows; high-risk non-metastatic with or without local lymph node involvement, or metastatic low/high volume. We followed up patients for a median of 7 years. Univariable and multivariable Cox survival models were fitted to estimate the association between the burden of copy number alteration as a continuous variable and the hazard of death or disease progression. RESULTS: The burden of copy number alterations positively associated with radiologically evident distant metastases at diagnosis (P=0.00006) and showed a non-linear relationship with clinical outcome on univariable and multivariable analysis, characterised by a sharp increase in the relative risk of progression (P=0.003) and death (P=0.045) for each unit increase, stabilising into more modest increases with higher copy number burdens. This association between copy number burden and outcome was similar in each metastatic state. Copy number loss occurred significantly more frequently than gain at the lowest copy number burden quartile (q=4.1 × 10-6). Loss of segments in chromosome 5q21-22 and gains at 8q21-24, respectively including CHD1 and cMYC occurred more frequently in cases with higher copy number alteration (for either region: Kolmogorov-Smirnov distance, 0.5; adjusted P<0.0001). Copy number alterations showed variability across tumour regions in the same prostate. This variance associated with increased risk of distant metastases (Kruskal-Wallis test P=0.037). CONCLUSIONS: Copy number alteration in advanced prostate cancer associates with increased risk of metastases at diagnosis. Accumulation of a limited number of copy number alterations associates with most of the increased risk of disease progression and death. The increased likelihood of involvement of specific segments in high copy number alteration burden cancers may suggest an order underlying the accumulation of copy number changes. TRIAL REGISTRATION: ClinicalTrials.gov NCT00268476 , registered on December 22, 2005. EudraCT  2004-000193-31 , registered on October 4, 2004.


Subject(s)
Prostatic Neoplasms , Androgen Antagonists/therapeutic use , DNA Copy Number Variations , Disease Progression , Humans , Male , Prospective Studies , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology
5.
Eur Urol ; 79(3): 325-326, 2021 03.
Article in English | MEDLINE | ID: mdl-33375994

ABSTRACT

Our modification of the traditional Newcastle-Ottawa scale enables urological researchers to effectively appraise and communicate the quality of genetic-based research in urology.


Subject(s)
Urology , Humans
7.
Eur Urol Open Sci ; 20: 37-47, 2020 Jul.
Article in English | MEDLINE | ID: mdl-33000006

ABSTRACT

CONTEXT: Multiparametric magnetic resonance imaging (mpMRI) detects most, but not all, clinically significant prostate cancer. The genetic basis of prostate cancer visibility and invisibility on mpMRI remains uncertain. OBJECTIVE: To systematically review the literature on differential gene expression between mpMRI-visible and mpMRI-invisible prostate cancer, and to use bioinformatic analysis to identify enriched processes or cellular components in genes validated in more than one study. EVIDENCE ACQUISITION: We performed a systematic literature search of the Medline, EMBASE, PubMed, and Cochrane databases up to January 2020 in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement. The primary endpoint was differential genetic features between mpMRI-visible and mpMRI-invisible tumours. Secondary endpoints were explanatory links between gene function and mpMRI conspicuity, and the prognostic value of differential gene enrichment. EVIDENCE SYNTHESIS: We retrieved 445 articles, of which 32 met the criteria for inclusion. Thematic synthesis from the included studies showed that mpMRI-visible cancer tended towards enrichment of molecular features associated with increased disease aggressivity, including phosphatase and tensin homologue (PTEN) loss and higher genomic classifier scores, such as Oncotype and Decipher. Three of the included studies had accompanying publicly available data suitable for further bioinformatic analysis. An over-representation analysis of these datasets revealed increased expression of genes associated with extracellular matrix components in mpMRI-visible tumours. CONCLUSIONS: Prostate cancer that is visible on mpMRI is generally enriched with molecular features of tumour development and aggressivity, including activation of proliferative signalling, DNA damage, and inflammatory processes. Additionally, there appears to be concordant cellular components and biological processes associated with mpMRI conspicuity, as highlighted by bioinformatic analysis of large genetic datasets. PATIENT SUMMARY: Prostate cancer that is detected by magnetic resonance imaging (MRI) tends to have genetic features that are associated with more aggressive disease. This suggests that MRI can be used to assess the likelihood of aggressive prostate cancer, based on tumour visibility.

8.
BMJ Open ; 10(1): e034611, 2020 Jan 27.
Article in English | MEDLINE | ID: mdl-31992607

ABSTRACT

INTRODUCTION: The introduction of multiparametric MRI (mpMRI) has enabled enhanced risk stratification for men at risk of prostate cancer, through accurate prebiopsy identification of clinically significant disease. However, approximately 10%-20% of significant prostate cancer may be missed on mpMRI. It appears that the genomic basis of lesion visibility or invisibility on mpMRI may have key implications for prognosis and treatment. Here, we describe a protocol for the first systematic review and novel bioinformatic analysis of the genomic basis of prostate cancer conspicuity on mpMRI. METHODS AND ANALYSIS: A systematic search of MEDLINE, PubMed, EMBASE and Cochrane databases will be conducted. Screening, data extraction, statistical analysis and reporting will be performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Included papers will be full text articles, written between January 1980 and December 2019, comparing molecular characteristics of mpMRI-visible lesions and mpMRI-invisible lesions at the DNA, DNA-methylation, RNA or protein level. Study bias and quality will be assessed using a modified Newcastle-Ottawa score. Additionally, we will conduct a novel bioinformatic analysis of supplementary material and publicly available data, to combine transcriptomic data and reveal common pathways highlighted across studies. To ensure methodological rigour, this protocol is written in accordance with the PRISMA Protocol 2015 checklist. ETHICS AND DISSEMINATION: Ethical approval will not be required, as this is an academic review of published literature. Findings will be disseminated through publications in peer-reviewed journals, and presentations at national and international conferences. PROSPERO REGISTRATION NUMBER: CRD42019147423.


Subject(s)
Computational Biology/methods , Multiparametric Magnetic Resonance Imaging/methods , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/genetics , Genomics , Humans , Image-Guided Biopsy , Male , Neoplasm Grading , Prostatic Neoplasms/diagnostic imaging , Prostatic Neoplasms/pathology , Research Design , Risk Factors , Tumor Burden , Systematic Reviews as Topic
9.
JCO Precis Oncol ; 4: 882-897, 2020 Nov.
Article in English | MEDLINE | ID: mdl-35050761

ABSTRACT

PURPOSE: The STAMPEDE trial recruits men with newly diagnosed, high-risk, hormone-sensitive prostate cancer. To ascertain the feasibility of targeted next-generation sequencing (tNGS) and the prevalence of baseline genomic aberrations, we sequenced tumor and germline DNA from patients with metastatic prostate cancer (mPCa) starting long-term androgen-deprivation therapy (ADT). METHODS: In a 2-stage approach, archival, formalin-fixed, paraffin-embedded (FFPE) prostate tumor core biopsy samples were retrospectively subjected to 2 tNGS assays. Prospective enrollment enabled validation using tNGS in tumor and germline DNA. RESULTS: In stage 1, tNGS data were obtained from 185 tumors from 287 patients (65%); 98% had de novo mPCa. We observed PI3K pathway aberrations in 43%, due to PTEN copy-number loss (34%) and/or activating mutations in PIK3 genes or AKT (18%) and TP53 mutation or loss in 33%. No androgen receptor (AR) aberrations were detected; RB1 loss was observed in < 1%. In stage 2, 93 (92%) of 101 FFPE tumors (biopsy obtained within 8 months) were successfully sequenced prospectively. The prevalence of DNA damage repair (DDR) deficiency was 14% (somatic) and 5% (germline). BRCA2 mutations and mismatch repair gene mutations were exclusive to high-volume disease. Aberrant DDR (22% v 15%), Wnt pathway (16% v 4%), and chromatin remodeling (16% v 8%) were all more common in high-volume compared with low-volume disease, but the small numbers limited statistical comparisons. CONCLUSION: Prospective genomic characterization is feasible using residual diagnostic tumor samples and reveals that the genomic landscapes of de novo high-volume mPCa and advanced metastatic prostate cancer have notable similarities (PI3K pathway, DDR, Wnt, chromatin remodeling) and differences (AR, RB1). These results will inform the design and conduct of biomarker-directed trials in men with metastatic hormone-sensitive prostate cancer.

10.
Eur Urol Oncol ; 2(1): 1-11, 2019 02.
Article in English | MEDLINE | ID: mdl-30929837

ABSTRACT

BACKGROUND: The prostate cancer (PCa) diagnostic pathway is undergoing a radical change with the introduction of multiparametric magnetic resonance imaging (mpMRI), genomic testing, and different prostate biopsy techniques. It has been proposed that these tests should be used in a sequential manner to optimise risk stratification. OBJECTIVE: To characterise the genomic, epigenomic, and transcriptomic features of mpMRI-visible and -nonvisible PCa in clinically localised disease. DESIGN, SETTING, AND PARTICIPANTS: Multicore analysis of fresh prostate tissue sampled immediately after radical prostatectomy was performed for intermediate- to high-risk PCa. INTERVENTION: Low-pass whole-genome, exome, methylation, and transcriptome profiling of patient tissue cores taken from microscopically benign and cancerous areas in the same prostate. Circulating free and germline DNA was assessed from the blood of five patients. OUTCOME MEASUREMENT AND STATISTICAL ANALYSIS: Correlations between preoperative mpMRI and genomic characteristics of tumour and benign prostate samples were assessed. Gene profiles for individual tumour cores were correlated with existing genomic classifiers currently used for prognostication. RESULTS AND LIMITATIONS: A total of 43 prostate cores (22 tumour and 21 benign) were profiled from six whole prostate glands. Of the 22 tumour cores, 16 were tumours visible and six were tumours nonvisible on mpMRI. Intratumour genomic, epigenomic, and transcriptomic heterogeneity was found within mpMRI-visible lesions. This could potentially lead to misclassification of patients using signatures based on copy number or RNA expression. Moreover, three of the six cores obtained from mpMRI-nonvisible tumours harboured one or more genetic alterations commonly observed in metastatic castration-resistant PCa. No circulating free DNA alterations were found. Limitations include the small cohort size and lack of follow-up. CONCLUSIONS: Our study supports the continued use of systematic prostate sampling in addition to mpMRI, as avoidance of systematic biopsies in patients with negative mpMRI may mean that clinically significant tumours harbouring genetic alterations commonly seen in metastatic PCa are missed. Furthermore, there is inconsistency in individual genomics when genomic classifiers are applied. PATIENT SUMMARY: Our study shows that tumour heterogeneity within prostate tumours visible on multiparametric magnetic resonance imaging (mpMRI) can lead to misclassification of patients if only one core is used for genomic analysis. In addition, some cancers that were missed by mpMRI had genomic aberrations that are commonly seen in advanced metastatic prostate cancer. Avoiding biopsies in mpMRI-negative cases may mean that such potentially lethal cancers are missed.


Subject(s)
Genomics/methods , Multiparametric Magnetic Resonance Imaging/methods , Prostatic Neoplasms/diagnostic imaging , Aged , Humans , Male , Middle Aged , Prostatic Neoplasms/genetics
11.
Mol Oncol ; 11(12): 1687-1697, 2017 12.
Article in English | MEDLINE | ID: mdl-28741788

ABSTRACT

The CellSearch® semiautomated CTC enrichment and staining system has been established as the 'gold standard' for CTC enumeration with CellSearch® CTC counts recognized by the FDA as prognostic for a number of cancers. We and others have gone on to show that molecular analysis of CellSearch® CTCs isolated shortly after CellSearch® enrichment provides another valuable layer of information that has potential clinical utility including predicting response to treatment. Although CellSearch® CTCs can be readily isolated after enrichment, the process of analysing a single CellSearch® patient sample, which may contain many CTCs, is both time-consuming and costly. Here, we describe a simple process that will allow storage of all CellSearch® -enriched cells in glycerol at -20 °C for up to 2 years without any measurable loss in the ability to retrieve single cells or in the genome integrity of the isolated cells. To establish the suitability of long-term glycerol storage for single-cell molecular analysis, we isolated individual CellSearch® -enriched cells by DEPArray™ either shortly after CellSearch® enrichment or following storage of matched enriched cells in glycerol at -20 °C. All isolated cells were subjected to whole-genome amplification (WGA), and the efficacy of single-cell WGA was evaluated by multiplex PCR to generate a Genome Integrity Index (GII). The GII results from 409 single cells obtained from both 'spike-in' controls and clinical samples showed no statistical difference between values obtained pre- and postglycerol storage and that there is no further loss in integrity when DEPArray™-isolated cells are then stored at -80 °C for up to 2 years. In summary, we have established simple yet effective 'stop-off' points along the CTC workflow enabling CTC banking and facilitating selection of suitable samples for intensive analysis once patient outcomes are known.


Subject(s)
Cell Separation/methods , Cryopreservation/methods , Neoplasms/pathology , Neoplastic Cells, Circulating/pathology , Single-Cell Analysis/methods , Cell Count , Colonic Neoplasms/blood , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Female , Genome, Human , Genomics/methods , Humans , Lung Neoplasms/blood , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Neoplasms/blood , Neoplasms/genetics , Neoplastic Cells, Circulating/metabolism , Small Cell Lung Carcinoma/blood , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/pathology
12.
Cancer Res ; 76(7): 1916-25, 2016 04 01.
Article in English | MEDLINE | ID: mdl-26795348

ABSTRACT

The findings from genome-wide association studies hold enormous potential for novel insight into disease mechanisms. A major challenge in the field is to map these low-risk association signals to their underlying functional sequence variants (FSV). Simple sequence study designs are insufficient, as the vast numbers of statistically comparable variants and a limited knowledge of noncoding regulatory elements complicate prioritization. Furthermore, large sample sizes are typically required for adequate power to identify the initial association signals. One important question is whether similar sample sizes need to be sequenced to identify the FSVs. Here, we present a proof-of-principle example of an extreme discordant design to map FSVs within the 2q33 low-risk breast cancer locus. Our approach employed DNA sequencing of a small number of discordant haplotypes to efficiently identify candidate FSVs. Our results were consistent with those from a 2,000-fold larger, traditional imputation-based fine-mapping study. To prioritize further, we used expression-quantitative trait locus analysis of RNA sequencing from breast tissues, gene regulation annotations from the ENCODE consortium, and functional assays for differential enhancer activities. Notably, we implicate three regulatory variants at 2q33 that target CASP8 (rs3769823, rs3769821 in CASP8, and rs10197246 in ALS2CR12) as functionally relevant. We conclude that nested discordant haplotype sequencing is a promising approach to aid mapping of low-risk association loci. The ability to include more efficient sequencing designs into mapping efforts presents an opportunity for the field to capitalize on the potential of association loci and accelerate translation of association signals to their underlying FSVs. Cancer Res; 76(7); 1916-25. ©2016 AACR.


Subject(s)
Breast Neoplasms/genetics , Genetic Variation/genetics , Breast Neoplasms/pathology , Female , Genetic Predisposition to Disease , Haplotypes , Humans , Polymorphism, Single Nucleotide , Risk
13.
Leuk Lymphoma ; 56(2): 301-10, 2015 Feb.
Article in English | MEDLINE | ID: mdl-24798744

ABSTRACT

Splenic marginal-zone lymphoma (SMZL) is a rare tumor that has recently emerged as a prototype for how the interplay between genetics and environment shapes the natural history of lymphomas. Indeed, the recent identification of molecular immunogenetic subgroups within SMZL may prove to be relevant not only for the sub-classification of the disease but also for improved understanding of the underlying biology. In contrast to other B-cell lymphomas, SMZL lacks a characteristic genetic lesion, although the majority of cases harbor genomic aberrations, as recently revealed by high-throughput studies that identified recurrent genetic aberrations, several in pathways related to marginal-zone differentiation and B-cell signaling. Here we provide an overview of recent research into the molecular and cellular biology of SMZL and related disorders, with special emphasis on immunogenetics and genomic aberrations, and discuss the value of molecular and cellular markers for the diagnosis and differential diagnosis of these entities.


Subject(s)
B-Lymphocytes/metabolism , Genetic Predisposition to Disease/genetics , Lymphoma, B-Cell, Marginal Zone/genetics , Splenic Neoplasms/genetics , B-Lymphocytes/pathology , Chromosome Aberrations , DNA Copy Number Variations , Humans , Lymphoma, B-Cell, Marginal Zone/pathology , Mutation , Signal Transduction/genetics , Splenic Neoplasms/pathology
14.
Clin Cancer Res ; 21(18): 4174-4183, 2015 Sep 15.
Article in English | MEDLINE | ID: mdl-25779943

ABSTRACT

PURPOSE: Mounting evidence supports the clinical significance of gene mutations and immunogenetic features in common mature B-cell malignancies. EXPERIMENTAL DESIGN: We undertook a detailed characterization of the genetic background of splenic marginal zone lymphoma (SMZL), using targeted resequencing and explored potential clinical implications in a multinational cohort of 175 patients with SMZL. RESULTS: We identified recurrent mutations in TP53 (16%), KLF2 (12%), NOTCH2 (10%), TNFAIP3 (7%), MLL2 (11%), MYD88 (7%), and ARID1A (6%), all genes known to be targeted by somatic mutation in SMZL. KLF2 mutations were early, clonal events, enriched in patients with del(7q) and IGHV1-2*04 B-cell receptor immunoglobulins, and were associated with a short median time to first treatment (0.12 vs. 1.11 years; P = 0.01). In multivariate analysis, mutations in NOTCH2 [HR, 2.12; 95% confidence interval (CI), 1.02-4.4; P = 0.044] and 100% germline IGHV gene identity (HR, 2.19; 95% CI, 1.05-4.55; P = 0.036) were independent markers of short time to first treatment, whereas TP53 mutations were an independent marker of short overall survival (HR, 2.36; 95 % CI, 1.08-5.2; P = 0.03). CONCLUSIONS: We identify key associations between gene mutations and clinical outcome, demonstrating for the first time that NOTCH2 and TP53 gene mutations are independent markers of reduced treatment-free and overall survival, respectively.


Subject(s)
Lymphoma, B-Cell, Marginal Zone/diagnosis , Lymphoma, B-Cell, Marginal Zone/genetics , Mutation , Splenic Neoplasms/diagnosis , Splenic Neoplasms/genetics , Adult , Aged , Aged, 80 and over , Cohort Studies , DNA Mutational Analysis , DNA-Binding Proteins/genetics , Female , Genetic Markers , High-Throughput Nucleotide Sequencing , Humans , Intracellular Signaling Peptides and Proteins/genetics , Kruppel-Like Transcription Factors/genetics , Male , Middle Aged , Multivariate Analysis , Myeloid Differentiation Factor 88/genetics , Neoplasm Proteins/genetics , Nuclear Proteins/genetics , Prognosis , Receptor, Notch2/genetics , Risk Factors , Transcription Factors/genetics , Treatment Outcome , Tumor Necrosis Factor alpha-Induced Protein 3 , Tumor Suppressor Protein p53/genetics
16.
ESMO Open ; 3(6): e000436, 2018.
Article in English | MEDLINE | ID: mdl-30345083
17.
PLoS One ; 8(12): e83244, 2013.
Article in English | MEDLINE | ID: mdl-24349473

ABSTRACT

The pathogenesis of splenic marginal zone lymphoma (SMZL) remains largely unknown. Recent high-throughput sequencing studies have identified recurrent mutations in key pathways, most notably NOTCH2 mutations in >25% of patients. These studies are based on small, heterogeneous discovery cohorts, and therefore only captured a fraction of the lesions present in the SMZL genome. To identify further novel pathogenic mutations within related biochemical pathways, we applied whole exome sequencing (WES) and copy number (CN) analysis to a biologically and clinically homogeneous cohort of seven SMZL patients with 7q abnormalities and IGHV1-2*04 gene usage. We identified 173 somatic non-silent variants, affecting 160 distinct genes. In additional to providing independent validation of the presence of mutation in several previously reported genes (NOTCH2, TNFAIP3, MAP3K14, MLL2 and SPEN), our study defined eight additional recurrently mutated genes in SMZL; these genes are CREBBP, CBFA2T3, AMOTL1, FAT4, FBXO11, PLA2G4D, TRRAP and USH2A. By integrating our WES and CN data we identified three mutated putative candidate genes targeted by 7q deletions (CUL1, EZH2 and FLNC), with FLNC positioned within the well-characterized 7q minimally deleted region. Taken together, this work expands the reported directory of recurrently mutated cancer genes in this disease, thereby expanding our understanding of SMZL pathogenesis. Ultimately, this work will help to establish a stratified approach to care including the possibility of targeted therapy.


Subject(s)
Chromosomes, Human, Pair 7/genetics , Exome/genetics , Lymphoma, B-Cell, Marginal Zone/genetics , Mutation , Neoplasm Proteins/genetics , Splenic Neoplasms/genetics , Chromosomes, Human, Pair 7/metabolism , DNA Mutational Analysis , Female , Humans , Lymphoma, B-Cell, Marginal Zone/metabolism , Lymphoma, B-Cell, Marginal Zone/pathology , Lymphoma, B-Cell, Marginal Zone/therapy , Male , Neoplasm Proteins/metabolism , Splenic Neoplasms/metabolism , Splenic Neoplasms/pathology , Splenic Neoplasms/therapy
19.
Cancer Epidemiol Biomarkers Prev ; 21(1): 176-81, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22056502

ABSTRACT

BACKGROUND: Multiple genome-wide and candidate gene association studies have been conducted in search of common risk variants for breast cancer. Recent large meta analyses, consolidating evidence from these studies, have been consistent in highlighting the caspase-8 (CASP8) gene as important in this regard. To define a risk haplotype and map the CASP8 gene region with respect to underlying susceptibility variant/s, we screened four genes in the CASP8 region on 2q33-q34 for breast cancer risk. METHODS: Two independent data sets from the United Kingdom and the United States, including 3,888 breast cancer cases and controls, were genotyped for 45 tagging single nucleotide polymorphisms (tSNP) in the expanded CASP8 region. SNP and haplotype association tests were carried out using Monte Carlo-based methods. RESULTS: We identified a three-SNP haplotype across rs3834129, rs6723097, and rs3817578 that was significantly associated with breast cancer (P < 5 × 10(-6)), with a dominant risk ratio and 95% CI of 1.28 (1.21-1.35) and frequency of 0.29 in controls. Evidence for this risk haplotype was extremely consistent across the two study sites and also consistent with previous data. CONCLUSION: This three-SNP risk haplotype represents the best characterization so far of the chromosome upon which the susceptibility variant resides. IMPACT: Characterization of the risk haplotype provides a strong foundation for resequencing efforts to identify the underlying risk variant, which may prove useful for individual-level risk prediction, and provide novel insights into breast carcinogenesis.


Subject(s)
Breast Neoplasms/genetics , Caspase 8/genetics , Adult , Aged , Aged, 80 and over , Breast Neoplasms/enzymology , Case-Control Studies , Female , Genetic Predisposition to Disease , Genome-Wide Association Study , Haplotypes , Humans , Middle Aged , Polymorphism, Single Nucleotide
20.
Curr Protoc Protein Sci ; Chapter 21: Unit 21.11, 2002 May.
Article in English | MEDLINE | ID: mdl-18429230

ABSTRACT

This unit describes the over-expression and purification of active serine proteases and their variants from E. coli inclusion bodies. The strategy includes the folding and purification of a stable zymogen precursor protein, and its later activation with the appropriate convertase to the less stable but active protease. A test to follow the presence of activity in the samples, together with an active-site titration protocol to determine the number of active sites per mole of total protein are provided. It should be emphasized that although most of the protocols described are applied to a specific example, they are fairly representative of the methods and approaches generally used for laboratory-scale preparation of other recombinant serine proteases. The critical steps and how this template protocol can be adapted for the purification of other serine proteases are described.


Subject(s)
Escherichia coli/genetics , Inclusion Bodies/metabolism , Serine Endopeptidases/isolation & purification , Cloning, Molecular/methods , Enzyme Precursors , Methods , Protein Folding , Serine Endopeptidases/genetics
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