张敏跃分子生物学进展肿瘤标志物.ppt

1、,Discovery of Cancer Biomarkers 张敏跃 南京大学生命科学院,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

2、global mortality. Deaths 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,

3、accounting for approximately 25% of all deaths in the US each year,How to Improve the Situation?,Prevention 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 c

4、ritical components of these hallmarks of cancer together provides a powerful basis for diagnosing, monitoring and predicting outcome and response to treatment.,How to Improve the Situation?,The goal of cancer biomarker field is to develop simple non-invasive tests that indicate cancer risk, allow ea

5、rly cancer detection, classify tumors so that the patient can receive the most appropriate therapy and monitor disease progression, regression and recurrence. 3. Treatment,Concept of Cancer Biomarkers,Definition of biological markers Biological markers (biomarkers) have been defined by Hulka and col

6、leagues (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 Uni

7、versity 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, pathogenic processes, or pharmacological responses to a therapeutic intervention ” Naylor S. Bio

8、markers: current perspectives and future prospects. Expert Rev Mol Diagn 3:525529, 2003.,Concept of Cancer Biomarkers,2. Forms of cancer markers Hormones, metabolites，as well as different functional subgroups of proteins such as enzymes, glycoproteins, oncofetal antigens and receptors. Furthermore,

9、other changes in tumors, such as 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

10、other associated conditions, such as inflammation. Cancer biomarkers can also be processes such as apoptosis, angiogenesis or proliferation.,Concept of Cancer Biomarkers,3. Factors that are ideal for a tumor marker Produced by the tumor cells and enters the circulation Present at low levels in the s

11、erum of healthy individuals and those with benign disease but increases substantially in cancer (preferably in one cancer type only) Easily quantifiable with an inexpensive assay Present in detectable (or higher than normal) quantities at early or preclinical stages Quantitative levels of the tumor

12、marker reflect the tumor burden High diagnostic sensitivity (few false negatives) and specificity (few false positives),Concept of Cancer Biomarkers,3. Factors that are ideal for a serological tumor marker,Concept of Cancer Biomarkers,4. Types of cancer biomarkers 4.1. Diagnostic (screening) biomark

13、er A marker that is used to 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,Concept of Canc

14、er Biomarkers,4.2. Prognostic biomarker This 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

15、 example, in testicular teratoma, human chorionic gonadotropin and alfa-fetoprotein levels can discriminate two groups with different survival rates.,Concept of Cancer Biomarkers,4.3. Stratification (predictive) biomarker This type of marker serves to predict the response to a drug before treatment

16、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 patients tumor.,Current applications of

17、tumor markers and their limitations,Current applications of tumor markers and their limitations,Cancer biomarkers that are currently in clinical use,Cancer biomarkers that are currently in clinical use,The clinical phases of testing a new cancer drug,Phase 1 Determinations of toxicity, pharmacokinet

18、ics, 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 results, and so forth.,Phases of biomark

19、er developmentMargaret SP; et al. University of Washington (2001),1. Preclinical exploratory studies Primary Aims 1) 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 p

20、rofiling, mass-spectrometry-based methods and other approaches to biomarker discovery can be used To 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

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

22、01),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 so that factors potentially influencing the

23、 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.,Phases of biomarker development for early detectionMargaret SP; et al. University of

24、 Washington(2001),1. Preclinical exploratory studies Specimen Selection Factors should be considered when selecting tumor Specimen 1，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 (control) Specimen 1，对照样品和肿瘤样品的对等性 2，对照样品

25、和肿瘤样品的处理方式的对等性,Phases of biomarker development for early detectionMargaret SP; et al. University of Washington(2001),Preclinical exploratory studies Sample 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 co

26、ntribute to variability: the number and relative prevalence of the cancer subtypes among the study samples the capacities of the biomarkers to discriminate among the different cancer subtypes the number of biomarkers under study the number of case and control subjects and the statistical algorithm u

27、sed to select promising biomarkers.,Phases of biomarker development for early detectionMargaret SP; et al. University of Washington(2001),2. Assay development and validation Primary Aim To estimate the TPR and FPR or ROC curve for the clinical biomarker assay, to assess its ability to distinguish su

28、bjects 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 of the assay in detecting disease early is not

29、 demonstrated in this phase.,Phases of biomarker development for early detectionMargaret SP; et al. University of Washington(2001),3. Retrospective longitudinal clinical repository studies Primary Aims 1) To evaluate, as a function of time before clinical diagnosis, the capacity of the biomarker to

30、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 in phase 3 of the biomarker evaluation.,Phas

31、es 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 population by determining the detection rate and the false refe

32、rral 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.,Phases of biomarker development for early detectionMargaret SP; et

33、 al. University of Washington(2001),5. Randomized control trials Primary Aim To estimate the reductions in cancer mortality afforded by the screening test.,Strategies and techniques for discovery of cancer biomarkers,Genomic level cancer biomarker discovery 1.1. Genomic aberration Sequencing: The Ca

34、ncer 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. SNP Sequencing SNP array 1.3. Epigenetic alternations,Strategies and techniq

35、ues for discovery of cancer biomarkers,2. Transcriptional level cancer biomarker discovery 2.1. mRNA expression profile cDNA-micro-array, Oligo-micro-array Differential display-PCR (DD-PCR) Serial analysis of gene expression (SAGE), cDNA Library Subtraction, etc.,Strategies and techniques for discov

36、ery of cancer biomarkers,2. Transcriptional level cancer biomarker discovery 2.2. miRNA Potential importance of miRNAs as cancer biomarkers Expression of microRNAs (miRNAs) in various tissues has been associated with a variety of diseases,including cancers. Serum miRNAs contain fingerprints for vari

37、ous diseases. Related techniques Sequencing miRNA-array,Strategies and techniques for discovery of cancer biomarkers,3. Translational level cancer biomarker discovery 3.1. Protein (or subtypes: enzymes, antibodies, secreted proteins, etc) 2-dimensioal electrophoresis/mass spectrometry(2-DE/MS) Surfa

38、ce-enhanced laser desorption ionization time-of-flight mass spectrometry technology (SELDI-TOP-MS): proteomic pattern diagnostics Multi-dimensional protein identification technology (MudPIT)/MS,Strategies and techniques for discovery of cancer biomarkers,Principle of SELDI-TOF-MS,One microlitre of r

39、aw, 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 and dried. A laser irradiates and desorbs the adherent proteins. The time-of-flight (TOF) of the ion is detected by an electrode. A prot

40、eomic signature of the serum is created.,Strategies and techniques for discovery of cancer biomarkers,Proteomic pattern diagnostics With this approach, the underlying identity of the individual components of the pattern is not necessary for its use as a potential diagnostic for disease.,Strategies a

41、nd techniques for discovery of cancer biomarkers,3. Translational level cancer biomarker discovery 3.1. 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 spectrom

42、etry technology (SELDI-TOP-MS): proteomic pattern diagnostics Multi-dimensional protein identification technology (MudPIT)/MS,Strategies and techniques for discovery of cancer biomarkers,Principle of MudPIT,Strategies and techniques for discovery of cancer biomarkers,4. Post-translational modificati

43、ons of proteins (cleavage products, altered glycosylation, etc) 4.1. Cleavage products of tumour-derived proteins have been proposed as potential cancer biomarkers. 4.2. Altered protein glycosylation in cancer is another source of potential cancer biomarkers. Identification of glycosylated proteins

44、relies on various glyco-capture strategies, a means of glycosylated-protein sub-selection by affinity chromatography. Electron-transfer dissociation (电子转移解离)allows labile modifications to remain intact while obtaining peptide sequence information, enabling the study of modifications such as glycosyl

45、ation and phosphorylation.,Current tumor markers under development,Contribution of oncoproteomics to cancer biomarker discoveryPublished: 2 April 2007 Molecular Cancer 2007, 6:25,Potential importance of miRNAs as cancer biomarkers,1. History Discovered in Caenorhabditis elegans in 1993 and formally

46、named in 2001 Have been identified in every plant and animal species examined 2. Features 2.1. General features Length: A class of noncoding RNAs, 1825 nucleotides Speciecs: miRNAs have been identified5 with up to 1,000 predicted. Location: miRNAs are encoded by DNA that may be situated in the exons

47、 or introns of genes or scattered among intergenic DNA,Potential importance of miRNAs as cancer biomarkers,2.2. 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

48、miRNA; (iv) incorporation into an RNA-induced silencing complex (RISC) with an Argonaute protein catalyst,Potential importance of miRNAs as cancer biomarkers,2.3. Function and targets The miRNA-RISC complex hybridizes to nucleotide sequences of varying complementarity in the 3 untranslated region (U

49、TR) of mRNA and inhibits protein synthesis or degrades the target mRNA Plays key roles in the regulation of fundamental cellular processes,Potential importance of miRNAs as cancer biomarkers,Dysregulated expression of microRNAs (miRNAs) in various tissues has been associated with a variety of diseas

50、es, 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 types. Overexpression or lack of expression of sp

51、ecific 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 26/4.,Potential importance of miRNA in cancer,Here

52、 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 consistent among individuals of the same species. Employing Solexa, we sequenced all serum m

53、iRNAs 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 cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints for various

54、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 biomarkers for diagnosis of cancer and other diseases. Cell Research, 2008, 18:997-1006.

55、,Organizations,1. The International Cancer Biomarkers Consortium (ICBC) (/science/international_biomarker) Under the leadership of Dr. Lee Hartwell, President and Director of Fred Hutchinson Cancer Research Center the ICBC is pioneering a new model for biomarker discovery and developing

56、 technologies and methodologies to make it possible.,Organizations,1. The International Cancer Biomarkers Consortium (ICBC) (/science/international_biomarker) The goal of the International Cancer Biomarker Consortium (ICBC) is to advance medical research and improve patient outcomes by

57、discovering biomarkers (indicators) for multiple types of cancer. Through a large-scale effort similar to the Human Genome Project, the consortium aims to facilitate highly coordinated research and by leveraging resources and expertise from around the world to overcome the current obstacles in bioma

58、rker research.,Organizations,1. The International Cancer Biomarkers Consortium (ICBC) (/science/international_biomarker) At the same time, the ICBC will provide a structure for international teams to work together on global issues such as adoption of data standards and the sharing data

59、as well as on scientific details such as the logistics of tissue sample sharing and investigation of mouse models of cancer.,Organizations,2. National Institute of Healths (NIH) National Cancer Institute (NCI) () Cancer Biomarkers Research Group This group promotes and supports research to identify, develop, and validate biological markers for earlier cancer detection and risk assessment. The group integrates basic and clinical science studies along with computational, statistical and epidemiologic approaches for a comprehensive understanding of biomarkers. It coordinate