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How is monitoring being made

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Presentation on theme: "How is monitoring being made"— Presentation transcript:

1 How is monitoring being made
Susanne Schnittger How is monitoring being made

2 Methods Cytogenetics FISH PCR Quantitative PCR Sequencing

3 Philadelphia-Translocation BCR-ABL-Rearrangement
1 2 3

4 BCR-ABL-Fusion Philadelphia Chromosome BCR-ABL fusion gene
BCR-ABL fusion transcript BCR-ABL fusion protein Philadelphia Chromosome

5 Methods Cytogenetics FISH PCR Quantitative PCR Sequencing

6 Cell Culture

7

8 Metaphase wieviele

9 Sorting of Chromosomes (Step 1)

10 Sorting of Chromosomes (Step 2)

11 Normal Karyogram

12 Karyotype: 46,XY,t(9;22)(q34;q11)

13 Time Table Cytogenetics
Metaphasefining Salt incubation Colcemid Arrival Staining Report day 1 day 2 day 3 day 4 day 5 Incubation at 37°C Hypotonic treatment Centifugations Fixation Preparation of slides Evaluation

14 Advantages/Disadvantages Chromosome Analysis
Advantage - Overview comprizing all Chomosome aberrations - no previous knowledge required Disadvantage - resolution in very limited (Mbp) - vital cells required - time consuming

15 Methods Cytogenetics FISH (Fluorescence in situ hybridization) PCR Quantitative PCR Sequencing

16 FISH technique Fluorescence labled probe Single stranded DNA
Chromosome Single stranded DNA Fluorescence labled probe

17 FISH with Gene-specific Probes
Chromosom 9 Chromosom 22 9q+ 22q- Translokation ABL BCR Metaphase Interphase

18 Advantages/Disadvantages FISH
Advantage - cultivation not obligatory - no vital cells - fast (within 4 h) - more sensitive than cytogenetics - detection of Ph-, BCR-ABL+ cases Disadvantage - only BCR-ABL detectable - no detection of additional aberrations

19 Methods Cytogenetics FISH PCR (Polymerase Chain Reaction) Quantitative PCR Sequencing

20 BCR-ABL Detection 9 22 9q+ 22q- (Ph) Fusionsprotein BCR BCR-ABL ABL
ABL-BCR mRNA „Fusionstranscript“ BCR ABL ABL Ib Ia XI a2 m-bcr bcr a3 a4 a5 a6 a7 a8 a9 a10 BCR e1 e13 e15 e16 e23 e2 e6 e7 e12 e17 e18 e20 e19 b2 b3 b4 b5 b1 c1 c2 c4 c3 e3 e4 e5 M-BCR

21 Polymerase Chain Reaction (PCR)
Master copy

22 Polymerase Chain Reaction (PCR)
Template 1. Denaturation after 20 Cykles: > 1 Million Molecules 2. Annealing after 35 Cyklen: > 30 Billion Molecules 3. Elongation Agarosegele Stabilisation of p53 in the nucleus when DNA is damaged. Direct interaction with the tumorsuppresso ARF to regulate the cell cycle by sequestering the protein in the cytoplasm. Other functions: nucleic acid binding, ATP binding, stimuation of DANN polymerase a binding. Muation are in Exon 12 in the very 3´region, just in front of the untranslated region Hellgrau untranslatierte region

23 Detection of Fusionsgenen

24 BCR-ABL Multiplex PCR a2 a3 ABL BCR m-bcr M-bcr µ-bcr p190 p210 p230
1% 98% b2a2 b3a2 b2a3 b3a3 -C C C P P2 P3 P4 P5 P P P8 P9 MW H2O p210 p190 BCR e1a2 b3a2 b2a2

25 Time Table PCR Arrival Multiplex PCR Report stabilisation
5 Mio cells/vial day 1 am day 1 pm day 2 am day 2 pm Sample Processing RNA isolation Evaluation

26 Methods Cytogenetics FISH PCR Quantitative PCR (RQ-PCR) Sequencing

27 Minimal Residual Disease (MRD)
10 12 8 6 4 2 Time from therapy start Number of leukemic cells Relapse Cure CR MRD

28 BCR-ABL nested PCR 1.Reaction Agarosegel 2. Reaction Agarosegel

29 Quantitative PCR (RQ-PCR)

30 Report (MMR) Quantitative Real time PCR: Date LabID. Sample Assay
%BCR-ABL/ABL ABL-Copies(+) PB BCR-ABL_P210 36,021 11410 KM 5,292 52760 0,302 87604 0,038 110882 0,044 66053 0,041 38169

31 Nested PCR (CMR)

32 Suboptimal Response Quantitative Real time PCR: Date LabID. Sample
Assay %BCR-ABL/ABL ABL-Copies(+) PB BCR-ABL_P210 92,639 56650 36,607 5600 4,904 26100 2,717 7058 3,075 3350 0,470 3282 1,628 34310

33 Resistance Primary Resistance Secondary Resistance

34 Methods Cytogenetics FISH PCR Quantitative PCR Sequencing

35 BCR-ABL Mutations BCR ABL BCR ABL ABL-R

36 Method Sanger Sequencing
896g>a ggg>gag G250E/Gly250Glu

37 Mutation Analysis MLL (2005-2011)
E459K E255V + T315I 2 E292A S417Y E255V + F359V A269A A397P E255K + Q252H 3 H396R L387M E255K + M351T E255K + T315I L387F E255G + F359V Y253H + T315I F359C + H396R G254G G250E + H396R F359I + F350V Y253H + F359V L248V M351T + L359V Y253H + F311I L248_K274del M351I Y253H + E279K 4 E279K M351H Y253F + E255K F359C G321E Y253H + F317L 5 D276G F317L + L364I G250E + Y253F G250E + T315I F317L + M351T G250E + T315I + F359I M244V + T315I F317I G250E + Q252H 6 T315I + F359V G250E + M388L Q252H T315I + F359C G250E + F359V 8 E255V T315I + E355Q G250E + E255K 13 M351T F315L Y253H V299L + F359V K247fs 15 F359V V299L + L273M K245E + L284S 16 V299L K294S_insFPQ M244V + E355A 20 G250E I293V + H396R 21 M244V L284V K247R 34 E255K E281Q F359I F317L L273M F311I 94 T315I n Mutation Mutations: n=438 Patients: n=378 2 Mutations: n=60 (16%)

38 Report Mutations Report of mutation at DNA Level g.A58758G, g.T58795C, g.A58796T (U ) Report of mutation at amino acid level M244V (Met244Val), Y253F (Tyr253Phe), Y253H (Tyr253His) Mutation burden amount of mutated/unmutated BCR-ABL transcripts Hughes et al., Blood, 2006

39 Mutation Analysis Material: Peripheral Blood Turn around: 2-3 days
Sensitivity: 10%

40 Summary Method Sensitivity Detection Application Cytogenetics 1/25
Ph+ cells diagnosis FISH 1:200 BCR-ABL+ cells Multiplex PCR 1: fusion transcript BCR-ABL type RQ-PCR 1:10.000 quantification Nested PCR 1: most sensitive detection Sequencing 1:10 resistance

41 Invitation to MLL Lab-Tour
Tomorrow Sunday 4-6 p.m.

42 Immunphänotypisierung
Munich Leukemia Laboratory (MLL) Zytomorphologie Zytogenetik/FISH Molekulargenetik NGS/Microarrays Torsten Haferlach Richard Schabath Eva Ammerlahn Sandra Eckstein Katharina Hermus Carina Holdenried Stefanie Jansa Katja Macijewski Yvonne Schneider Laura Urmetzer Jana Wagenbreth A.-Ch. Weichselbaumer Claudia Haferlach Sandra Böker Sabine Denzel Silvia Finkböck Claudia Hepperger Jinda Holzwarth Doris Kestner Verena Patterer Marietta Truger Sarah Volkert Juliane Brendel Anna-Maria Brucks Sabina Bursch Jennifer Eland Sylvia Erdle Annika Eßer Eyleen Haubner Annika Heger Annette Königsbauer Stephanie Maier Susann Marschner Alida März Alexandra Mohr Nicole Mudrack Katja Niklaus Maximiliane Oppl Isabel Penzler Sandra Pöstgen Cornelia Pröger Isabelle Rast Franziska Schäfer Nadine Schedler Martin Schröter Ulrike Schulz Karin Seige Kathrin Stadler Marita Staller Melanie Zenger Kathleen Zieschang Susanne Schnittger Frank Dicker Christiane Eder Annette Fasan Sabine Jeromin Manja Meggendorfer Isabel Schiemann Simone Weber Katharina Bayer Jessica Brust Meike Büchler Heidi Eitner Ina Hochrein Rabea Konietschke Angelika Koles Julia Niederreiter Louisa Noël Carina Schrauder Elisabeth Sirch Antje Stiel Madlen Ulke Nicole Wendland Alexander Kohlmann Claudine Bergmann Vera Großmann Andreas Kowarsch Niroshan Nadarajah Ulrike Schöck Sandra Weißmann Lucia Böck Stefanie Burkhart Katrin Butschalowski Stefan Harbich Jennifer Kienast Franziska Pötzinger Sonja Schindela Kimberly Steuer Elisa Stopp Feyza Ucaroglu Immunphänotypisierung Organisation Wolfgang Kern Frauke Bellos Sabrina Bauer Sabrina Baumann Angelika Eckelt Nicole Fietzeck Anja Glück Elena Goubermann Franziska Krakau Rita Lapping Maja Lumpe Evelyn Seif Christine Stangl Antonia Wagner Carina Zanggl Tamara Alpermann Peter Baumann Romina Biasi Dirk Bursch Elke Erhard Claudia Eßbauer Julia Hägel Katrin Iltzhöfer Sandra Nawroth Franziska Prantl Tina Riedel Ulrike Riese Sandra Schulze Sabrina Weiß Wenke Worseg Qualitätsmanagement Janine Elosge Monika Trnobransky

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