Gene expression profiling in acute leukemias [Elektronische Ressource] : new insights into biology and a global approach to the diagnosis of leukemia using microarray technology / vorgelegt von Alexander Kohlmann

Alexander Kohlmann

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2004
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 14 Mo

Extrait

Gene expression profiling in acute
leukemias: New insights into biology
and a global approach to the
diagnosis of leukemia using
microarray technology








Dissertation
an der Fakultät für Biologie
der Ludwig-Maximilians-Universität
München



vorgelegt von
Alexander Kohlmann
aus Neumarkt i.d. OPf.





München, den 1. Dezember 2004
























Diese Arbeit wurde im Labor für spezielle Leukämie-Diagnostik im
Klinikum der Ludwig-Maximilians-Universität München – Großhadern
unter Anleitung von Herrn Professor Dr. Dr. T. Haferlach durchgeführt.






Erstgutachter: Professor Dr. Thomas Cremer
Zweitgutachter: Professor Dr. Heinrich Leonhardt
Sondervotum: Professor Dr. Dr. Torsten Haferlach

Tag der mündlichen Prüfung: 26. Oktober 2005
IV
Contents

Contents.......................................................................................................IV
List of figures................................................................................................VI
List of tablesVII
Abbreviations .............................................................................................VIII
1. Summary...................................................................................................... 1
2. Introduction................................................................................................. 3
2.1 Microarrays and the era of functional genomics................................... 3
2.2 Leukemia 7
2.3 Gene expression profiling in the field of hematology.......................... 12
2.4 Questions addressed in this work ...................................................... 14
3. Methods and Protocols ............................................................................ 15
3.1 Description of patient samples ........................................................... 15
3.2 Diagnostic procedures ....................................................................... 15
3.3 Microarray target preparation............................................................. 18
3.3.1 Isolation of total RNA .................................................................................... 19
3.3.2 Synthesis of ds cDNA ................................................................................... 21
3.3.3 Cleanup of ds ..................................................................................... 23
3.3.4 Synthesis of biotin-labeled cRNA ................................................................. 25
3.3.5 Cleanup of biotin-labeled cRNA.................................................................... 26
3.3.6 Fragmenting the cRNA ................................................................................. 28
3.3.7 Target hybridization ...................................................................................... 29
3.3.8 Microarrays ................................................................................................... 31
3.3.9 Microarray washing and staining .................................................................. 33
3.3.10 Microarray scanning and image analysis...................................................... 35
3.3.11 Quality assessment....................................................................................... 36
3.3.12 Data sets and scaling procedure 38
3.4 Microarray data analysis .................................................................... 39
3.4.2 Estimation of prediction performance ........................................................... 43
3.4.3 Hierarchical clustering................................................................................... 44
3.4.4 Principal component analysis ....................................................................... 45
3.4.5 Classification of samples based on gene expression patterns..................... 46
3.4.6 Functional gene annotation........................................................................... 48
3.4.7 Network analysis........................................................................................... 49
3.4.8 Software........................................................................................................ 50
4. Results....................................................................................................... 51
4.1 Gene expression profiling in AML ...................................................... 51
4.2 prALL ....................................................... 67
4.3 Gene expression profiling in t(11q23)/MLL leukemias ....................... 73
4.4 Gene expression profiling as potential diagnostic method ................. 82


V
5. Discussion............................................................................................... 107
5.1 Specific patterns in AML with reciprocal rearrangements ................ 107
5.2 Molecular characterization of ALL using microarrays....................... 113
5.3 Novel insights into the biology of t(11q23)/MLL leukemias .............. 116
5.4 Microarray technology as a potential diagnostic platform................. 121
5.5 Concluding remarks ......................................................................... 129
6. References 131
7. Appendix.................................................................................................. 146
7.1 Chemicals, enzymes, and reagents ................................................. 146
7.2 Instrumentation and technical equipment......................................... 148
7.3 Buffers and solutions........................................................................ 149

Publications............................................................................................... 153
Patent applications.................................................................................... 155
Danksagung.............................................................................................. 157
Lebenslauf ................................................................................................ 158

VI
List of figures

Figure 1. Different types of microarray platforms....................................................................................4
Figure 2. Fluorescence image of a microarray (Affymetrix U133A) ........................................................6
Figure 3. Affymetrix chip design .............................................................................................................6
Figure 4. Interlaboratory tests in cytomorphology...11
Figure 5. Gene expression analysis overview ......................................................................................18
Figure 6. Overview about the data analysis workflow...........................................................................39
Figure 7. Hierarchical cluster analysis workflow ...................................................................................44
Figure 8. Principal component analysis workflow .................................................................................45
Figure 9. Multiple-tree model computation ...........................................................................................46
Figure 10. Concept of SVM-based classification ....................................................................................47
Figure 11. Classification task..................................................................................................................47
Figure 12. Network details on node shapes, edge labels, and types......................................................49
Figure 13. Unsupervised hierarchical clustering of AML with t(15;17), t(8;21), and inv(16)....................51
Figure 14. ed PCA of AML with t(15;17), t(8;21), and inv(16)...............................................52
Figure 15. Supervised analysis of AML with t(15;17), t(8;21), and inv(16) .............................................53
Figure 16. PCA of three AML subtypes based on 13 genes...................................................................54
Figure 17. Multiple-tree classifier for AML with t(15;17), t(v(16)..........................................55
Figure 18. Hierarchical clustering using 36 genes from both classifiers .................................................57
Figure 19. Transition from U95Av2 arrays to the U133 array design......................................................58
Figure 20. Morphology of APL samples..................................................................................................59
Figure 21. Class discovery: APL vs. other AML subclasses60
Figure 22. Network visualizing differences when comparing APL to other AML subclasses ..................61
Figure 23. PCA based on genes from the GO category blood coagulation ............................................62
Figure 24. Supervised analysis to discriminate APL subtypes.......................................