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1、分子生物学进展肿瘤标志物,分子生物学进展肿瘤标志物,Statistics,More 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. De
2、aths from 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 approx
3、imately 25% of all deaths in the US each year,分子生物学进展肿瘤标志物,2,StatisticsMore than 11 million,How to Improve the Situation?,PreventionDetection Cancer is a disease of genetic progression that is often associated with specific molecular, genetic and histological changes.The ability to develop biomarker
4、s that can detect the critical components of 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,3,How to Improve the Situation?P,How to Improve the Situation?,The goal of cancer biomarker field is to
5、develop simple non-invasive tests that indicate cancer risk, allow early cancer detection, 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,4,How to Improve the Situation?T,Concept of Ca
6、ncer Biomarkers,Definition of biological markers Biological markers (biomarkers) have been defined by Hulka and colleagues (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 mark
7、ers. In: Biological markers in epidemiology (Hulka BS, Griffith JD, Wilcosky TC, eds), pp 315. New York: Oxford University Press, 1990.More recently, the definition has been broadened to include “biological characteristics that can be objectively measured and evaluated as an indicator of normal biol
8、ogical processes, pathogenic processes, or pharmacological responses to a therapeutic intervention ” Naylor S. Biomarkers: current perspectives and future prospects. Expert Rev Mol Diagn 3:525529, 2003.,分子生物学进展肿瘤标志物,5,Concept of Cancer BiomarkersDe,Concept of Cancer Biomarkers,2. Forms of cancer mar
9、kersHormones, metabolites，as well as different functional subgroups of proteins such as enzymes, glycoproteins, oncofetal antigens and receptors. Furthermore, other changes in tumors, such as genetic mutations, amplifications or translocations, and changes in microarray-generated profiles (genetic s
10、ignatures), 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 inflammation. Cancer biomarkers can also be processes such as apoptosis, angiogenesis or proliferation.,
11、分子生物学进展肿瘤标志物,6,Concept of Cancer Biomarkers2.,Concept of Cancer Biomarkers,3. 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 those with benign disease but increases substantially in cancer (
12、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 diagnostic sensitivity (few false negatives) and specificity (f
13、ew false positives),分子生物学进展肿瘤标志物,7,Concept of Cancer Biomarkers3.,Concept of Cancer Biomarkers,3. Factors that are ideal for a serological tumor marker,分子生物学进展肿瘤标志物,8,Concept of Cancer Biomarkers3.,Concept of Cancer Biomarkers,4. Types of cancer biomarkers4.1. Diagnostic (screening) biomarkerA marke
14、r that is used to detect and identify a given type of cancer in an individual. These markers are expected to have high specificity and sensitivityFor 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,9,Concept of
15、Cancer Biomarkers4.,Concept of Cancer Biomarkers,4.2. 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
16、the aggressiveness of therapy. For example, in testicular 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,10,Concept of Cancer Biomarkers4.,Concept of Cancer Biomarkers,4.3. Stratification (predictive) biomark
17、erThis type of marker serves to predict the response to a drug before treatment is started. This 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
18、from the molecular characteristics of a patients tumor.,分子生物学进展肿瘤标志物,11,Concept of Cancer Biomarkers4.,Current applications of tumor markers and their limitations,分子生物学进展肿瘤标志物,12,Current applications of tumor,Current applications of tumor markers and their limitations,分子生物学进展肿瘤标志物,13,Current applica
19、tions of tumor,Cancer biomarkers that are currently in clinical use,分子生物学进展肿瘤标志物,14,Cancer biomarkers that are cur,Cancer biomarkers that are currently in clinical use,分子生物学进展肿瘤标志物,15,Cancer biomarkers that are cur,The clinical phases of testing a new cancer drug,Phase 1Determinations of toxicity, p
20、harmacokinetics, and optimal dose levelsphase 2Determinations of biologic efficacyPhase 3Definitive controlled trials of effects on clinical endpoints.For each phase, guidelines exist for subject selection, outcome measures, relevant comparisons for evaluating study results, and so forth.,分子生物学进展肿瘤标
21、志物,16,The clinical phases of testing,Phases 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
22、 are compared. 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 id
23、entify characteristics 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,17,Phases of biomarker developmen,Ph
24、ases of biomarker development for early detectionMargaret SP; et al. University of Washington(2001),1. Preclinical exploratory studiesSpecimen SelectionTumor tissue from case subjects should be obtained at diagnosis and before treatment because treatment may interfere with the behavior of the biomar
25、ker.Noncancer control subjects should be selected 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
26、 habits.,分子生物学进展肿瘤标志物,18,Phases of biomarker developmen,Phases of biomarker development for early detectionMargaret SP; et al. University of Washington(2001),1. Preclinical exploratory studiesSpecimen 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
27、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 (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,19,Phases of biomarker developmen,Phases of biomarker development for early detectionMargaret SP; et al. University of Washington(2001),Pre
28、clinical exploratory studiesSample Sizes The number depends on the objective of the study and the extent of the variability of the biomarker in the study. The following factors contribute to variability: the number and relative prevalence of the cancer subtypes among the study samplesthe capacities
29、of the biomarkers to discriminate among the different cancer subtypesthe number of biomarkers under studythe number of case and control subjectsand the statistical algorithm used to select promising biomarkers.,分子生物学进展肿瘤标志物,20,Phases of biomarker developmen,Phases of biomarker development for early
30、detectionMargaret SP; et al. University of Washington(2001),2. Assay development and validation Primary AimTo estimate the TPR and FPR or ROC curve for the clinical biomarker assay, to assess its ability to distinguish subjects with cancer from subjects without cancer.A clinical assay that uses a sp
31、ecimen 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 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,21,Phases of biomarker developmen,Phas
32、es 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 clinical diagnosis, the capacity of the biomarker to detect preclinical disease.2) To de
33、fine 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 in phase 3 of the biomarker evaluation.,分子生物学进展肿瘤标志物,22,Phases of biomarker devel
34、opmen,Phases of biomarker development for early detetionMargaret SP; et al. University of Washington(2001),4. Prospective screening studies Primary AimTo determine the operating characteristics of the biomarker based screening test in a relevant population by determining the detection rate and the f
35、alse 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,23,Phases of biomarker developmen,Phases of b
36、iomarker development for early detectionMargaret SP; et al. University of Washington(2001),5. Randomized control trialsPrimary AimTo estimate the reductions in cancer mortality afforded by the screening test.,分子生物学进展肿瘤标志物,24,Phases of biomarker developmen,Strategies and techniques for discovery of c
37、ancer biomarkers,Genomic level 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 kar
38、yotyping (SKY)1.2. SNPSequencingSNP array1.3. Epigenetic alternations,分子生物学进展肿瘤标志物,25,Strategies and techniques for,Strategies and techniques for discovery of cancer biomarkers,2. Transcriptional level cancer biomarker discovery2.1. mRNA expression profilecDNA-micro-array, Oligo-micro-array Differen
39、tial display-PCR (DD-PCR)Serial analysis of gene expression (SAGE),cDNA Library Subtraction, etc.,分子生物学进展肿瘤标志物,26,Strategies and techniques for,Strategies and techniques for discovery of cancer biomarkers,2. Transcriptional level cancer biomarker discovery2.2. miRNAPotential importance of miRNAs as
40、cancer biomarkers Expression of microRNAs (miRNAs) in various tissues has been associated with a variety of diseases,including cancers. Serum miRNAs contain fingerprints for various diseases. Related techniquesSequencingmiRNA-array,分子生物学进展肿瘤标志物,27,Strategies and techniques for,Strategies and techniq
41、ues for discovery of cancer biomarkers,3. Translational level cancer biomarker discovery3.1. Protein (or subtypes: enzymes, antibodies, secreted proteins, etc)2-dimensioal electrophoresis/mass spectrometry(2-DE/MS)Surface-enhanced laser desorption ionization time-of-flight mass spectrometry technolo
42、gy (SELDI-TOP-MS): proteomic pattern diagnosticsMulti-dimensional protein identification technology (MudPIT)/MS,分子生物学进展肿瘤标志物,28,Strategies and techniques for,Strategies and techniques for discovery of cancer biomarkers,Principle of SELDI-TOF-MS,One microlitre of raw, unfractionated serum is applied
43、to the surface of a protein-binding chip.The chip is rinsed to remove unbound proteins, treated with a MATRIX COMPOUND, washed and dried.A laser irradiates and desorbs the adherent proteins.The time-of-flight (TOF) of the ion is detected by an electrode. A proteomic signature of the serum is created
44、.,分子生物学进展肿瘤标志物,29,Strategies and techniques for,Strategies and techniques for discovery of cancer biomarkers,Proteomic pattern diagnosticsWith this approach, the underlying identity of the individual components of the pattern is not necessary for its use as a potential diagnostic for disease.,分子生物学进
45、展肿瘤标志物,30,Strategies and techniques for,Strategies and techniques for discovery of cancer biomarkers,3. Translational level cancer biomarker discovery3.1. Protein (or subtypes: enzymes, antibodies, secreted proteins, etc)2-dimensioal electrophoresis/mass spectrometry(2-DE/MS)Surface-enhanced laser d
46、esorption 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,31,Strategies and techniques for,Strategies and techniques for discovery of cancer biomarkers,Principle of MudPIT
47、,分子生物学进展肿瘤标志物,32,Strategies and techniques for,分子生物学进展肿瘤标志物培训课件,Current 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,34,Current tumor markers under de,Potential importance of miRNAs as canc
48、er biomarkers,1. HistoryDiscovered in Caenorhabditis elegans in 1993 and formally named in 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 p
49、redicted.Location: miRNAs are encoded by DNA that may be situated in the exons or introns of genes or scattered among intergenic DNA,分子生物学进展肿瘤标志物,35,Potential importance of miRNAs,Potential importance of miRNAs as cancer biomarkers,2.2. Transcription and maturation(i) nuclear processing into a prima
50、ry miRNA (pri-miRNA) and then a precursor (pre-miRNA);(ii) export into the cytoplasm;(iii) further processing into mature miRNA;(iv) incorporation into an RNA-induced silencing complex (RISC) with an Argonaute protein catalyst,分子生物学进展肿瘤标志物,36,Potential importance of miRNAs,Potential importance of mi
