1、分子生物学进展肿瘤分子生物学进展肿瘤标志物标志物StatisticsMore than 11 million people are diagnosed with cancer every year.It is estimated that there will be 16 million new cases every year by 2020.From a total of 58 million deaths worldwide in 2005,cancer accounts for 7.6 million(or 13%)of the global mortality.Deaths from
2、 cancer in the world are projected to continue rising,with an estimated 9 million people dying from cancer in 2015 and 11.4 million dying in 2030.In the US in 2006,over 1.4 million new cases of cancer were diagnosed.Over half a million people died from this disease,accounting for approximately 25%of
3、 all deaths in the US each year 分子生物学进展肿瘤标志物2How to Improve the Situation?1.Prevention2.Detection Cancer is a disease of genetic progression that is often associated with specific molecular,genetic and histological changes.The ability to develop biomarkers that can detect the critical components of
4、these hallmarks of cancer together provides a powerful basis for diagnosing,monitoring and predicting outcome and response to treatment.分子生物学进展肿瘤标志物3How to Improve the Situation?The goal of cancer biomarker field is to develop simple non-invasive tests that indicate cancer risk,allow early cancer de
5、tection,classify tumors so that the patient can receive the most appropriate therapy and monitor disease progression,regression and recurrence.3.Treatment分子生物学进展肿瘤标志物4Concept of Cancer Biomarkers1.Definition of biological markers Biological markers(biomarkers)have been defined by Hulka and colleague
6、s(1990)as “cellular,biochemical or molecular alterations that are measurable in biological media such as human tissues,cells,or fluids.”Hulka BS.Overview of biological markers.In:Biological markers in epidemiology(Hulka BS,Griffith JD,Wilcosky TC,eds),pp 315.New York:Oxford University Press,1990.Mor
7、e recently,the definition has been broadened to include“biological characteristics that can be objectively measured and evaluated as an indicator of normal biological processes,pathogenic processes,or pharmacological responses to a therapeutic intervention”Naylor S.Biomarkers:current perspectives an
8、d future prospects.Expert Rev Mol Diagn 3:525529,2003.分子生物学进展肿瘤标志物5Concept of Cancer Biomarkers2.Forms of cancer markersHormones,metabolites，as well as different functional subgroups of proteins such as enzymes,glycoproteins,oncofetal antigens and receptors.Furthermore,other changes in tumors,such a
9、s genetic mutations,amplifications or translocations,and changes in microarray-generated profiles(genetic signatures),are also forms of tumor markers.The markers are produced either by the tumor itself or by other tissues,in response to the presence of cancer or other associated conditions,such as i
10、nflammation.Cancer biomarkers can also be processes such as apoptosis,angiogenesis or proliferation.分子生物学进展肿瘤标志物6Concept of Cancer Biomarkers3.Factors that are ideal for a tumor markerProduced by the tumor cells and enters the circulationPresent at low levels in the serum of healthy individuals and
11、those with benign disease but increases substantially in cancer(preferably in one cancer type only)Easily quantifiable with an inexpensive assayPresent in detectable(or higher than normal)quantities at early or preclinical stagesQuantitative levels of the tumor marker reflect the tumor burdenHigh di
12、agnostic sensitivity(few false negatives)and specificity(few false positives)分子生物学进展肿瘤标志物7Concept of Cancer Biomarkers3.Factors that are ideal for a serological tumor marker分子生物学进展肿瘤标志物8Concept of Cancer Biomarkers4.Types of cancer biomarkers4.1.Diagnostic(screening)biomarker A marker that is used t
13、o detect and identify a given type of cancer in an individual.These markers are expected to have high specificity and sensitivity For example,the presence of BenceJones protein in urine remains one of the strongest diagnostic indicators of multiple myeloma分子生物学进展肿瘤标志物9Concept of Cancer Biomarkers4.2
14、.Prognostic biomarkerThis type of marker is used once the disease status has been established.These biomarkers are expected to predict the probable course of the disease including its recurrence,and they therefore have an important influence on the aggressiveness of therapy.For example,in testicular
15、 teratoma,human chorionic gonadotropin and alfa-fetoprotein levels can discriminate two groups with different survival rates.分子生物学进展肿瘤标志物10Concept of Cancer Biomarkers4.3.Stratification(predictive)biomarkerThis type of marker serves to predict the response to a drug before treatment is started.This
16、marker classifies individuals as likely responders or nonresponders to a particular treatment.These biomarkers mainly arise from array-type experiments that make it possible to predict clinical outcome from the molecular characteristics of a patients tumor.分子生物学进展肿瘤标志物11Current applications of tumor
17、 markers and their limitations分子生物学进展肿瘤标志物12Current applications of tumor markers and their limitations分子生物学进展肿瘤标志物13Cancer biomarkers that are currently in clinical use分子生物学进展肿瘤标志物14Cancer biomarkers that are currently in clinical use分子生物学进展肿瘤标志物15The clinical phases of testing a new cancer drug Ph
18、ase 1 Determinations of toxicity,pharmacokinetics,and optimal dose levels phase 2 Determinations of biologic efficacy Phase 3 Definitive controlled trials of effects on clinical endpoints.For each phase,guidelines exist for subject selection,outcome measures,relevant comparisons for evaluating study
19、 results,and so forth.分子生物学进展肿瘤标志物16Phases of biomarker developmentMargaret SP;et al.University of Washington(2001)1.Preclinical exploratory studies Primary Aims1)To identify leads for potentially useful biomarkers.2)To prioritize identified leads.In this phase,tumor and non-tumor specimens are comp
20、ared.Strategies such as gene expression profiling,mass-spectrometry-based methods and other approaches to biomarker discovery can be usedTo identify genes or clusters of genes(or proteins)that appear to be overexpressed or underexpressed in tumor tissue relative to control tissue.To identify charact
21、eristics unique to tumor tissue that might lead to ideas for clinical tests for detecting cancer.The development of statistical algorithms for selecting promising biomarkers from a large pool of biomarkers is an active area of research.分子生物学进展肿瘤标志物17Phases of biomarker development for early detectio
22、nMargaret SP;et al.University of Washington(2001)1.Preclinical exploratory studies Specimen Selection Tumor tissue from case subjects should be obtained at diagnosis and before treatment because treatment may interfere with the behavior of the biomarker.Noncancer control subjects should be selected
23、so that factors potentially influencing the biomarker,other than the cancer itself,are tightly matched to those of the cancer case subjects.These factors might include age,sex,race,and possibly lifestyle-related characteristics,such as smoking habits.分子生物学进展肿瘤标志物18Phases of biomarker development for
24、 early detectionMargaret SP;et al.University of Washington(2001)1.Preclinical exploratory studies Specimen SelectionFactors should be considered when selecting tumor Specimen1，样品的一致性：取材部位、肿瘤亚型、年龄、性别、种族、生活习惯等。2，样品处理方式的一致性：预处理条件、保存条件（包括时间）、处理条件、操作等。Factors should be considered when selecting nontumor(
25、control)Specimen1，对照样品和肿瘤样品的对等性2，对照样品和肿瘤样品的处理方式的对等性分子生物学进展肿瘤标志物19Phases of biomarker development for early detectionMargaret SP;et al.University of Washington(2001)1.Preclinical exploratory studiesSample Sizes The number depends on the objective of the study and the extent of the variability of the
26、biomarker in the study.The following factors contribute to variability:the number and relative prevalence of the cancer subtypes among the study samplesthe capacities of the biomarkers to discriminate among the different cancer subtypesthe number of biomarkers under studythe number of case and contr
27、ol subjectsand the statistical algorithm used to select promising biomarkers.分子生物学进展肿瘤标志物20Phases of biomarker development for early detectionMargaret SP;et al.University of Washington(2001)2.Assay development and validation Primary Aim1)To estimate the TPR and FPR or ROC curve for the clinical biom
28、arker assay,to assess its ability to distinguish subjects with cancer from subjects without cancer.A clinical assay that uses a specimen of choice(usually something that can be obtained noninvasively)is developed in this phase.The patients assessed in this phase have established disease.The utility
29、of the assay in detecting disease early is not demonstrated in this phase.分子生物学进展肿瘤标志物21Phases of biomarker development for early detectionMargaret SP;et al.University of Washington(2001)3.Retrospective longitudinal clinical repository studiesPrimary Aims1)To evaluate,as a function of time before cl
30、inical diagnosis,the capacity of the biomarker to detect preclinical disease.2)To define criteria for a positive screening test in preparation for phase 4.Repositories of clinical specimens,collected and stored from a cohort of apparently healthy subjects monitored for development of cancer,are used
31、 in phase 3 of the biomarker evaluation.分子生物学进展肿瘤标志物22Phases of biomarker development for early detetionMargaret SP;et al.University of Washington(2001)4.Prospective screening studies Primary Aim To determine the operating characteristics of the biomarker based screening test in a relevant populatio
32、n by determining the detection rate and the false referral rate.In this phase,individuals are screened with the assay and diagnostic procedures are applied to those who screened positive.This can help to establish the tumor stage or the nature of the disease at the time of detection.分子生物学进展肿瘤标志物23Ph
33、ases of biomarker development for early detectionMargaret SP;et al.University of Washington(2001)5.Randomized control trialsPrimary Aim To estimate the reductions in cancer mortality afforded by the screening test.分子生物学进展肿瘤标志物24Strategies and techniques for discovery of cancer biomarkers1.Genomic le
34、vel cancer biomarker discovery1.1.Genomic aberrationSequencing:The Cancer Genome Atlas(TCGA)is applying large-scale genome sequencing technology to identify novel genes involved in cancer pathogenesis.Comparative genomic hybridization(CGH)array-CGH(aCGH)Spectral karyotyping(SKY)1.2.SNPSequencingSNP
35、array1.3.Epigenetic alternations分子生物学进展肿瘤标志物25Strategies and techniques for discovery of cancer biomarkers2.Transcriptional level cancer biomarker discovery2.1.mRNA expression profilecDNA-micro-array,Oligo-micro-array Differential display-PCR(DD-PCR)Serial analysis of gene expression(SAGE),cDNA Libr
36、ary Subtraction,etc.分子生物学进展肿瘤标志物26Strategies and techniques for discovery of cancer biomarkers2.Transcriptional level cancer biomarker discovery2.2.miRNAPotential importance of miRNAs as cancer biomarkers Expression of microRNAs(miRNAs)in various tissues has been associated with a variety of disease
37、s,including cancers.Serum miRNAs contain fingerprints for various diseases.Related techniquesSequencingmiRNA-array分子生物学进展肿瘤标志物27Strategies and techniques for discovery of cancer biomarkers3.Translational level cancer biomarker discovery3.1.Protein(or subtypes:enzymes,antibodies,secreted proteins,etc
38、)2-dimensioal electrophoresis/mass spectrometry(2-DE/MS)Surface-enhanced laser desorption ionization time-of-flight mass spectrometry technology(SELDI-TOP-MS):proteomic pattern diagnosticsMulti-dimensional protein identification technology(MudPIT)/MS分子生物学进展肿瘤标志物28Strategies and techniques for discov
39、ery of cancer biomarkersPrinciple of SELDI-TOF-MS1.One microlitre of raw,unfractionated serum is applied to the surface of a protein-binding chip.2.The chip is rinsed to remove unbound proteins,treated with a MATRIX COMPOUND,washed and dried.3.A laser irradiates and desorbs the adherent proteins.4.T
40、he time-of-flight(TOF)of the ion is detected by an electrode.5.A proteomic signature of the serum is created.分子生物学进展肿瘤标志物29Strategies and techniques for discovery of cancer biomarkers Proteomic pattern diagnostics With this approach,the underlying identity of the individual components of the pattern
41、 is not necessary for its use as a potential diagnostic for disease.分子生物学进展肿瘤标志物30Strategies and techniques for discovery of cancer biomarkers3.Translational level cancer biomarker discovery3.1.Protein(or subtypes:enzymes,antibodies,secreted proteins,etc)2-dimensioal electrophoresis/mass spectrometr
42、y(2-DE/MS)Surface-enhanced laser desorption ionization time-of-flight mass spectrometry technology(SELDI-TOP-MS):proteomic pattern diagnosticsMulti-dimensional protein identification technology(MudPIT)/MS分子生物学进展肿瘤标志物31Strategies and techniques for discovery of cancer biomarkersPrinciple of MudPIT分子生
43、物学进展肿瘤标志物32Current tumor markers under development Contribution of oncoproteomics to cancer biomarker discoveryPublished:2 April 2007Molecular Cancer 2007,6:25分子生物学进展肿瘤标志物34Potential importance of miRNAs as cancer biomarkers1.HistoryDiscovered in Caenorhabditis elegans in 1993 and formally named in
44、2001Have been identified in every plant and animal species examined 2.Features 2.1.General featuresLength:A class of noncoding RNAs,1825 nucleotidesSpeciecs:miRNAs have been identified5 with up to 1,000 predicted.Location:miRNAs are encoded by DNA that may be situated in the exons or introns of gene
45、s or scattered among intergenic DNA分子生物学进展肿瘤标志物35Potential importance of miRNAs as cancer biomarkers2.2.Transcription and maturation(i)nuclear processing into a primary miRNA(pri-miRNA)and then a precursor(pre-miRNA);(ii)export into the cytoplasm;(iii)further processing into mature miRNA;(iv)incorpo
46、ration into an RNA-induced silencing complex(RISC)with an Argonaute protein catalyst 分子生物学进展肿瘤标志物36Potential importance of miRNAs as cancer biomarkers2.3.Function and targets The miRNA-RISC complex hybridizes to nucleotide sequences of varying complementarity in the 3 untranslated region(UTR)of mRNA
47、 and inhibits protein synthesis or degrades the target mRNA Plays key roles in the regulation of fundamental cellular processes分子生物学进展肿瘤标志物37Potential importance of miRNAs as cancer biomarkers 1.Dysregulated expression of microRNAs(miRNAs)in various tissues has been associated with a variety of dise
48、ases,including cancers.2.miRNAs expressed in cancer may act like oncogenes or tumor-suppressor genes by regulating proliferation and/or apoptosis.3.Normal and malignant tissues have specific miRNA signatures and show differential expression across tumor types.4.Overexpression or lack of expression o
49、f specific miRNAs appears to correlate with clinically aggressive or metastatic phenotype.5.miRNA expression has tissue specificity and has been used for tumor classification.Cancer biomarker profiling with microRNAs April 2008,Nature Biotechnology Vol 26/4.分子生物学进展肿瘤标志物38Potential importance of miRN
50、A in cancer1.Here we demonstrate that miRNAs are present in the serum and plasma of humans and other animals such as mice,rats,bovine fetuses,calves,and horses.2.The levels of miRNAs in serum are stable,reproducible,and consistent among individuals of the same species.3.Employing Solexa,we sequenced
