分子生物学进展肿瘤标志物培训课件

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. Death

2、s 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 approxima

3、tely 25% of all deaths in the US each year 分子生物学进展肿瘤标志物2StatisticsMore than 11 millionHow 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 biomarkers that

4、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3How to Improve the Situation?PHow to Improve the Situation?The goal of cancer biomarker field is to develop simp

5、le 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4How to Improve the Situation?TConcept of Cancer BiomarkersD

6、efinition 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 markers. In: Biologic

7、al 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 biological processes,

8、 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5Concept of Cancer BiomarkersDeConcept of Cancer Biomarkers2. Forms of cancer markersHormones, metaboli

9、tes，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 signatures), are also f

10、orms 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.分子生物学进展肿瘤标志物6Concept of

11、 Cancer Biomarkers2.Concept 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 those with benign disease but increases substantially in cancer (preferably in one cancer ty

12、pe 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 (few false positives)分子生物学进展肿

13、瘤标志物7Concept of Cancer Biomarkers3.Concept 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 Biomarkers3.Concept of Cancer Biomarkers4. Types of cancer biomarkers4.1. Diagnostic (screening) biomarkerA marker that is used to detect and identify

14、 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9Concept of Cancer Biomarkers4.Concept of Cancer Bio

15、markers4.2. 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 the aggressiveness of therapy. For example

16、, 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10Concept of Cancer Biomarkers4.Concept of Cancer Biomarkers4.3. Stratification (predictive) biomarkerThis type of marker serves to predict the res

17、ponse 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 from the molecular characteristics of a patient

18、s tumor.分子生物学进展肿瘤标志物11Concept of Cancer Biomarkers4.Current applications of tumor markers and their limitations分子生物学进展肿瘤标志物12Current applications of tumor Current applications of tumor markers and their limitations分子生物学进展肿瘤标志物13Current applications of tumor Cancer biomarkers that are currently in cl

19、inical use分子生物学进展肿瘤标志物14Cancer biomarkers that are curCancer biomarkers that are currently in clinical use分子生物学进展肿瘤标志物15Cancer biomarkers that are curThe clinical phases of testing a new cancer drugPhase 1Determinations of toxicity, pharmacokinetics, and optimal dose levelsphase 2Determinations of b

20、iologic 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志物16The clinical phases of testingPhases of biomarker developmentMa

21、rgaret 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 compared. Strategies such as gene expression profiling, mass-spect

22、rometry-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 characteristics unique to tumor tissue that might lead to ideas

23、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 developmenPhases of biomarker development for early detectionMargaret SP; et al. Univer

24、sity 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 biomarker.Noncancer control subjects should be selected so that factors potentiall

25、y 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 developmenPhases of biomarker deve

26、lopment 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 select

27、ing 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19Phases of biomarker developmenPhases of biomarker development for early detectionMargaret SP; et al. University of Washington(2001)Preclinical exploratory studiesSample Sizes The number depends on the objective of the st

28、udy 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 of the biomarkers to discriminate among the different cancer subtypesthe number of bio

29、markers 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20Phases of biomarker developmenPhases of biomarker development for early detectionMargaret SP; et al. University of Washington(2001)2. Assay development and valida

30、tion 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 specimen of choice (usually something that can be obtained noninvasively) is developed in thi

31、s 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21Phases of biomarker developmenPhases of biomarker development for early detectionMargaret SP; et al. University of Washington(20

32、01)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 define criteria for a positive screening test in preparation for phase 4.Repositories of clinical

33、 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22Phases of biomarker developmenPhases of biomarker development for early detetionMargaret SP; et al. University of Washingto

34、n(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 false referral rate.In this phase, individuals are screened with the assay and diagnostic procedures

35、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23Phases of biomarker developmenPhases of biomarker development for early detectionMargaret SP; et al. University of Washington(2001)5. Randomized

36、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24Phases of biomarker developmenStrategies and techniques for discovery of cancer biomarkersGenomic level cancer biomarker discovery1.1. Genomic aberrationSequencing: The Cancer Genome

37、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 array1.3. Epigenetic alternations分子生物学进展肿瘤标志物25Strategies and techniques f

38、or Strategies 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 Library Subtraction, etc.分子生物学进展肿瘤标志物26Str

39、ategies and techniques for Strategies 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 diseases,i

40、ncluding 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 Strategies and techniques for discovery of cancer biomarkers3. Translational level cancer biomarker discovery3.1. Protein (or subtypes: enzymes

41、, antibodies, secreted proteins, etc)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

42、物28Strategies and techniques for Strategies and techniques for discovery of cancer biomarkersPrinciple of SELDI-TOF-MSOne microlitre of raw, unfractionated serum is applied to the surface of a protein-binding chip.The chip is rinsed to remove unbound proteins, treated with a MATRIX COMPOUND, washed

43、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.分子生物学进展肿瘤标志物29Strategies and techniques for Strategies and techniques for discovery of cancer biomarkersProteomic pattern diag

44、nosticsWith this approach, the underlying identity of the individual components of the pattern is not necessary for its use as a potential diagnostic for disease.分子生物学进展肿瘤标志物30Strategies and techniques for Strategies and techniques for discovery of cancer biomarkers3. Translational level cancer biom

45、arker 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 technology (SELDI-TOP-MS): proteomic pattern diagnosticsMulti-dimensional protein

46、 identification technology (MudPIT)/MS分子生物学进展肿瘤标志物31Strategies and techniques for Strategies and techniques for discovery of cancer biomarkersPrinciple of MudPIT分子生物学进展肿瘤标志物32Strategies and techniques for 分子生物学进展肿瘤标志物培训课件Current tumor markers under developmentContribution of oncoproteomics to cancer

47、 biomarker discoveryPublished: 2 April 2007Molecular Cancer 2007, 6:25分子生物学进展肿瘤标志物34Current tumor markers under dePotential importance of miRNAs as cancer biomarkers1. HistoryDiscovered in Caenorhabditis elegans in 1993 and formally named in 2001Have been identified in every plant and animal species

48、 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 genes or scattered among intergenic DNA分子生物学进展肿瘤标志物35Pot

49、ential importance of miRNAsPotential 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) incorporation into an

50、 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 miRNAsPotential importance of miRNAs as cancer biomarkers2.3. Function and targetsThe miRNA-RISC complex hybridizes to nucleotide sequences of varying complementarity in the 3 untranslated

51、region (UTR) of mRNA and inhibits protein synthesis or degrades the target mRNAPlays key roles in the regulation of fundamental cellular processes分子生物学进展肿瘤标志物37Potential importance of miRNAsPotential importance of miRNAs as cancer biomarkers Dysregulated expression of microRNAs (miRNAs) in various t

52、issues has been associated with a variety of diseases, including cancers.miRNAs expressed in cancer may act like oncogenes or tumor-suppressor genes by regulating proliferation and/or apoptosis. Normal and malignant tissues have specific miRNA signatures and show differential expression across tumor

53、 types.Overexpression or lack of expression of specific miRNAs appears to correlate with clinically aggressive or metastatic phenotype.miRNA expression has tissue specificity and has been used for tumor classification.Cancer biomarker profiling with microRNAs April 2008, Nature Biotechnology Vol 26/

54、4.分子生物学进展肿瘤标志物38Potential importance of miRNAsPotential importance of miRNA in cancerHere 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. The levels of miRNAs in serum are stable, reproducible, and con

55、sistent among individuals of the same species. Employing Solexa, we sequenced all serum miRNAs of healthy Chinese subjects and found over 100 and 91 serum miRNAs in male and female subjects, respectively. We also identified specific expression patterns of serum miRNAs for lung cancer, colorectal can

56、cer, and diabetes, providing evidence that serum miRNAs contain fingerprints for various diseases. Through these analyses, we conclude that serum miRNAs can serve as potential biomarkers for the detection of various cancers and other diseases. Characterization of microRNAs in serum: a novel class of

57、 biomarkers for diagnosis of cancer and other diseases. Cell Research, 2008, 18:997-1006. 分子生物学进展肿瘤标志物39Potential importance of miRNA Organizations1. The International Cancer Biomarkers Consortium (ICBC) (/science/international_biomarker) Under the leadership of Dr. Lee Hartwell, President and Direc

58、tor of Fred Hutchinson Cancer Research Center the ICBC is pioneering a new model for biomarker discovery and developing technologies and methodologies to make it possible. 分子生物学进展肿瘤标志物40Organizations分子生物学进展肿瘤标志物40Organizations1. The International Cancer Biomarkers Consortium (ICBC) (/science/interna

59、tional_biomarker)The goal of the International Cancer Biomarker Consortium (ICBC) is to advance medical research and improve patient outcomes by discovering biomarkers (indicators) for multiple types of cancer. Through a large-scale effort similar to the Human Genome Project, the consortium aims to

60、facilitate highly coordinated research and by leveraging resources and expertise from around the world to overcome the current obstacles in biomarker research.分子生物学进展肿瘤标志物41Organizations分子生物学进展肿瘤标志物41Organizations1. The International Cancer Biomarkers Consortium (ICBC) (/science/international_biomar