KML ur ett kliniskt perspektiv Standardiserad mätning av BCR-ABL1

KML ur ett kliniskt perspektiv Standardiserad mätning av BCR-ABL1 Hans Ehrencrona Genetiska kliniken Labmedicin Skåne Lund

Leukemi-incidens Leukemityp Nya fall i Sverige 2008* Totalt KML KLL AML ALL 869 81 436 275 77 *Cancerförekomst i Sverige 2008. Socialstyrelsen.

KML - Epidemiologi c:a 90 nyinsjuknade med KML per år i Sverige Medianålder vid diagnos: 60-65 år Män något överrepresenterade Man:kvinna ratio = 1.3:1 85% diagnostiseras under kronisk fas

KML - Naturalförlopp Kronisk fas Accelererad fas Progression Blastkris Mediantid 5 6 år Stabil sjukdom Mediantid 6 9 månader Medianöverlevnad 3 6 månader Trötthet; Förhöjt antal vita blodkroppar; mjältförstoring; viktförlust Symptom Komplett svikt av immunsystemet; blödningar

Hur ställs diagnosen KML? Ur Svensk Förening för Hematologis nationella riktlinjer: KML misstänks utifrån avvikande blodvärden (leukocytos, trombocytos), förekomst av/symtom från förstorad mjälte eller allmänsymtom såsom trötthet, avmagring och svettningar. Cirka 1/3 av patienterna är asymtomatiska vid diagnos. Diagnosen KML ställs genom morfologisk bedömning av blodoch benmärgsutstryk, samt påvisande av BCR-ABL1-fusionen i celler från benmärg eller blod. http://www.sfhem.se/kml/

KML: Blodutstryk Normalt blod KML i kronisk fas

KML-behandling fram till 2001 Busulfan/hydroxyurea på 80-talet Från 90-talet interferon till de som tålde det Benmärgstransplantation om möjligt Annars palliation Överlevnaden förbättrades. En hel del patienter botades.

OVERALL SURVIVAL FROM DIAGNOSIS 1,00 ALL PATIENTS, CML 84 AND CML 89 0,75 0,50 0,25 CML 89 (n=251) CML 84 (n=179) 0,00 0 p=0,0001 1 2 3 4 5 6 7 8 9 10 11 12 13 14 YEARS

Nowell and Hungerford, Science, 1960, 132: 1497

Philadelphiatranslokationen t(9;22) vid KML Rowley, J.D. Nature 243, 290-293 (1973)

Genfusionen BCR-ABL1 vid t(9;22)

BCR-ABL1 Signal Transduction Pathways Cytoskeletal proteins BCR-ABL1 PI3K Stat5 Shc DOK Ras Raf AKT Decreased adhesion; Enhanced mobility BAD BAD BCLX L BCLX L 14-3-3 14-3-3 Mitochondria MAP kinase cascade Survival; Proliferation; Differentiation;

BCR-ABL1 och imatinib (Glivec)

Cellular selectivity of Glivec (imatinib) Kinase v-abl1 p210 bcr-abl1 p190 bcr-abl1 TEL-Abl1 PDGF receptor TEL-PDGF receptor c-kit Flt-3 c-fms and v-fms EGF receptor c-erbb2 Insulin receptor IGF-I receptor v-src JAK-2 IC 50 [ 0.1-0.3 0.25 0.25 0.35 0.1 0.15 0.1 >10 >10 >100 >100 >100 >100 >10 >100

IRIS-studien Komplett cytogenetisk respons (CCyR)

Patienter i Glivec-armen (IRIS-studien). N=553 1,00 OVERALL SURVIVAL FROM DIAGNOSIS ALL PATIENTS, CML 84 AND CML 89 0,75 0,50 0,25 0,00 0 p=0,0001 1 CML 89 (n=251) CML 84 (n=179) 2 3 4 5 6 7 8 9 10 11 12 13 14 YEARS

Baccarani et al., Blood. 2006;108:1809-1820 Baccarani et al., JCO, 2009 vol. 27 (35) pp. 6041-6051

2009 European LeukemiaNet Recommendations Initial Treatment of Ph+ CML in CP Imatinib (Glivec) 400 mg is the recommended initial treatment Treatment goal: Major Molecular Remission (MMR)

2009 European LeukemiaNet Recommendations Ph+ CML Treatment Response Definitions Haematologic Response (HR) Cytogenetic Response (CgR) Molecular Response (MolR) [BCR-ABL1 to control gene ratio according to International Scale (IS)] Complete (CHR) Platelets: <450 x 10 9 /L WBCC*: <10 x 10 9 /L Differential without immature granulocytes and <5% basophils Nonpalpable spleen Complete (CCgR) Partial (PCgR) Minor Ph+ 0% Ph+ 1-35% Ph+ 36-65% "Complete" Major (MMolR) Transcripts nonquantifiable and nondetectable (sensitivity <0.01%) 0.1% Minimal Ph+ 66-95% None Ph+ >95% Major = Partial + Complete *WBCC = White blood cell count. Standardised baseline represents 100% on IS; 0.1% 3-log reduction from standard baseline.

BCR-ABL1 Diagnostics: Choosing the Technique to Match the Objective Test Target Tissue Sensitivity (%)* Use Cytogenetics Ph chromosome BM 1 10 Confirm diagnosis of CML Evaluate karyotypic abnormalities other than Ph chromosome - clonal evolution (ACA) and clonal changes in Ph negative cells (OCA)) Evaluate Cytogenetic Response (CR) FISH Juxtaposition of BCR and ABL1 PB/BM 0.5 5 Confirm diagnosis of CML Detection of atypical (cytogenetically negative or complex) BCR-ABL1 rearrangements RT-PCR BCR-ABL1 mrna PB/BM 0.0001 0.001 Routine measurement of minimal residual disease (MRD) Determine the breakpoints of the fusion genes

Total number of leukemic cells Relationship between number of leukemic cells, BCR-ABL1 transcripts and response criteria in CML 10 12 Diagnosis, pretreatment or hematologic relapse 100 10 11 Complete hematologic response (CHR) 10 10 10 10 9 10 8 Complete cytogenetic response (CCyR) Major molecular response (MMR) 1 0.1 0.01 (according to International Scale) BCR-ABL1/ABL1 % 10 7 0.001 10 6 Undetectable transcript (>5-6 log reduction) 0.0001

2009 European LeukemiaNet Recommendations ELN Definitions of Response on First-Line Imatinib Optimal Response Suboptimal Response Failure Warnings Baseline NA NA NA High Risk CCA/Ph+ * 3 Months CHR and at least minor CyR (Ph+ 65%) 6 Months At least PCyR (Ph+ 35%) No CyR (Ph+ > 95%) Less than CHR NA Less than PCyR (Ph+ > 35%) 12 Months CCyR PCyR (Ph+ 1-35%) No CyR (Ph+ > 95%) Less than PCyR (Ph+ > 35%) 18 Months MMR Less than MMR Less than CCyR NA NA Less than MMR Any time Stable or improving MMR Loss of MMR Mutations Loss of CHR Loss of CCyR Mutations CCA/Ph+ * Any rise in transcript levels CCA/Ph- * CCA/Ph+ = Clonal chromosome abnormalities in Ph+ cells; CCA/Ph+ is a warning factor at diagnosis although its occurrence during treatment (ie, clonal progression) is a marker of treatment failure. Two consecutive cytogenetic tests are required and must show the same CCA in at least two Ph cells. MMR indicates a ratio of BCR-ABL1 to ABL1 or other housekeeping genes, 0.1% on the international scale. BCR-ABL1 kinase domain mutations still sensitive to imatinib. BCR-ABL1 kinase domain mutations poorly sensitive to imatinib. Baccarani et al., JCO, 2009 vol. 27 (35) pp. 6041-6051

% alive Time to CCyR Does Not Affect Long-Term Outcomes for Pts on IM Therapy: Overall Survival by Time to CCyR 100 90 80 70 60 50 40 30 20 10 0 Time to CCyR <=6 months >6 <=12 months >12 <=18 months >18 months No CCyR *P = 0.55 for OS rates among pts who achieved a CCyR 0 12 24 36 48 60 72 Months since start of treatment Guilhot F. et al, Blood. 2007; 110, 11. Abstract 27. ASH 2007 Oral Presentation

% CCyR >3 months Probability of CCyR by Cytogenetic Response at 3 months 100 90 80 70 60 50 40 30 20 10 0 (n=159) (n=35) (n=25) (n=33) 0 3 6 9 12 15 18 21 24 27 30 33 Months since randomization CyR at 3 months (Ph+) 1-35% 36-65% 66-95% >95%

% without progression Progression-free Survival on First-line Imatinib by Molecular Response (MR) at 12 months 100 90 80 70 60 p<0.001 50 40 30 20 10 No CCyR <3 log reduction >=3 log reduction Estimated rate (95% CI) at 42 months: n=138 75% (67-84) n= 94 90% (84-97) n=136 98% (96-100) 0 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 Months since randomization 27

2009 European LeukemiaNet Recommendations ELN Definitions of Response on First-Line Imatinib Optimal Response Suboptimal Response Failure Warnings Baseline NA NA NA High Risk CCA/Ph+ * 3 Months CHR and at least minor CyR (Ph+ 65%) 6 Months At least PCyR (Ph+ 35%) No CyR (Ph+ > 95%) Less than CHR NA Less than PCyR (Ph+ > 35%) 12 Months CCyR PCyR (Ph+ 1-35%) No CyR (Ph+ > 95%) Less than PCyR (Ph+ > 35%) 18 Months MMR Less than MMR Less than CCyR NA NA Less than MMR Any time Stable or improving MMR Loss of MMR Mutations Loss of CHR Loss of CCyR Mutations CCA/Ph+ * Any rise in transcript levels CCA/Ph- * CCA/Ph+ = Clonal chromosome abnormalities in Ph+ cells; CCA/Ph+ is a warning factor at diagnosis although its occurrence during treatment (ie, clonal progression) is a marker of treatment failure. Two consecutive cytogenetic tests are required and must show the same CCA in at least two Ph cells. MMR indicates a ratio of BCR-ABL1 to ABL1 or other housekeeping genes, 0.1% on the international scale. BCR-ABL1 kinase domain mutations still sensitive to imatinib. BCR-ABL1 kinase domain mutations poorly sensitive to imatinib. Baccarani et al., JCO, 2009 vol. 27 (35) pp. 6041-6051

Recommendations for Monitoring Molecular Response and Rising BCR-ABL1 Levels Molecular Monitoring every 3 months 1 Achieve MMR: screen every 6 months 1 No MMR? Sequence for TKI mutations Persistent MMR Highly promising 2 Rising RQ-PCR Repeat at 1-3 month intervals 2 > 10x rise is concerning 2 mutational testing 1. Baccarani et al., JCO, 2009 vol. 27 (35) pp. 6041-6051. 2. Radich JP, et al. Blood. 2009;114:3376-3381.

2009 European LeukemiaNet Recommendations Monitoring Treatment Response Baccarani et al., JCO, 2009 vol. 27 (35) p. 6045

Standardization - what for? Standardized BCR-ABL1 monitoring has shown to predict for response (IRIS study, common baseline) Hughes et al., NEJM 2003; Druker et al., NEJM 2006 Huge variety of (semiquantitative) molecular methods leads to considerable variation of results Noncomparability of results Confusion of doctors and patients Suboptimal treatment decisions

Standardization - how? Unification of all preanalytic and analytic steps? (RNA extraction, cdna synthesis, PCR protocol, housekeeping gene, evaluation method) give up established methods Use of a uniform plasmid as external control? (housekeeping gene) Adaptation to the `International Scale by sample exchange with standardized lab? (minimal change of established methods necessary) degree of freedom

Müller et al., Leukemia, 2009 vol. 23 (11) pp. 1957-1963

How to transmit the `International Scale? Lab A (central lab, approved CF) Lab B (participating lab) Two major ways to align results between two labs: 1) Send `reference material from lab A to lab B which has been adequately characterized in lab A align results 2) Send aliquots of patient samples from lab B to lab A align results

Current standardization approach to transmit the International Scale to 61 European labs Sample preparation using 5 buffy coats from healthy volunteers (~5*10 9 WBC) and PB of one CML patient at diagnosis (b3a2 pos.) 12 samples per lab, 4 dilutions (10%, 1%, 0.1%, 0.01%) + 2 test samples 10 (20) million WBC per sample, 1ml Trizol harmonized: RNA extraction via Trizol not harmonized: cdna synthesis, RQ-PCR, plasmids, evaluation method

Variety of Techniques RNA extraction: Trizol n=31, Qiagen (variants) n=16, Other n=9 Platforms: LightCycler n=16, TaqMan n=33, Rotorgene n=4, Stratagene n=4 Control genes: total ABL1 n=46, GUSB n=4, B2M n=2, G6PD n=4, PBGD n=1 Reference material: Ipsogen plasmids n=28 pme-2 plasmids n=7 local in-house plasmids n=11 RNA calibrator n=4

Lab X Dilution samples: Linearity of results compared to reference results linear results n=52 non linear results n=6 Lab X Calculation of CF

BCR-ABL1 BCR-ABL1 levels in 58 labs pre and post conversion 1000 100 10 CV 1.32 1.30 1.10 1.02 0.20 0.24 0.31 0.41 1 0.1 MMR 0.01 0.001 0.0001 Level 1 Level 2 Level 3 Level 4 Level 1 Level 2 Level 3 Level 4 reference results BCR-ABL1 L pre conversion BCR-ABL1 IS post conversion

Validation n=48 of the participating labs sent aliquots of 25-30 patient samples (median n=28; WBC in Trizol) to the central lab. BCR-ABL1 expression should cover a wide range (0.001%-10% BCR-ABL1 IS ). Comparison of results (Lab A vs. Lab B): i) using the CFs from the dilution sample round ii) using the CFs from the patient samples evaluation (Bland-Altman difference plots)

Validation samples: Improvement of concordance After conversion using the CFs (derived from dilution samples) the median proportion of samples per laboratory were within an x-fold range: 2-fold range (0.5-2.0): 52% 3-fold range (0.33-3.0): 76% 5-fold range (0.2-5.0): 94% Recalculation of the CFs by incorporating validation samples (Bland-Altman) led to significantly higher concordance rates: 2-fold range (0.5-2.0): 72% p=0.0002 3-fold range (0.33-3.0): 90% p=0.0002 5-fold range (0.2-5.0): 96% p=0.0011

Standardization of BCR-ABL1 quantification in Europe - 2002 Iceland Sweden Finland Norway Estonia Portugal Ireland Spain Denmark Netherlands United Kingdom France Belgium Luxembourg Switzerland Germany Czech Rep Poland Slovakia Austria Hungary Slovenia Croatia Italy Latvia Lithuania Bosnia Serbia and Herzegovina Belarus Romania Bulgaria Ukraine Moldova Macedonia Russia Greece Montenegro Turkey Albania Countries with a ref lab with validated CF Israel

Standardization of BCR-ABL1 quantification in Europe - 2006 Iceland Sweden Finland Norway Estonia Portugal Ireland Spain Denmark Netherlands United Kingdom France Belgium Luxembourg Switzerland Germany Czech Rep Poland Slovakia Austria Hungary Slovenia Croatia Italy Latvia Lithuania Bosnia Serbia and Herzegovina Belarus Romania Bulgaria Ukraine Moldova Macedonia Russia Greece Montenegro Turkey Albania Countries with a ref lab with validated CF Israel

Standardization of BCR-ABL1 quantification in Europe - 2010 Iceland Sweden Finland Norway Estonia Portugal Ireland Spain Denmark Netherlands United Kingdom France Belgium Luxembourg Switzerland Germany Czech Rep Poland Slovakia Austria Hungary Slovenia Croatia Italy Latvia Lithuania Bosnia Serbia and Herzegovina. Albania Belarus Romania - Greece Bulgaria Ukraine Moldova Macedonia Montenegro Russia Turkey Countries with a ref lab with validated CF Countries using a ref lab in a different country Countries with a ref lab validation pending Israel

Preliminära resultat från det svenska standardiseringsarbetet Efter tillämpning av preliminära konversionsfaktorer från spädningsserierna låg medianen av prover för svenska laboratorier (Umeå, Solna, Huddinge, Lund) inom en x-faldig avvikelse jämfört med referenslaboratoriet i Uppsala: Tvåfaldig (0.5-2.0): 56% (Europa 52%) Trefaldig (0.33-3.0): 76% (Europa 76%) Femfaldig (0.2-5.0): 96% (Europa 94%) Etablering av nya konversionsfaktorer genom att inkorporera patientproverna (enl Bland-Altman) medförde påtagligt förbättrade resultat: Tvåfaldig (0.5-2.0): 85% (Europa 72%) Trefaldig (0.33-3.0): 96% (Europa 90%) Femfaldig (0.2-5.0): 100% (Europa 96%)

Rapportering av BCR-ABL1 enligt den internationella skalan i Sverige Laboratorium Konversionsfaktor (kontrollgen) Datum för införande i klinisk rutin Uppsala (referenslab) 0,6003 (GUS) Januari 2009 Umeå 0,3649 (ABL1) September 2010 Stockholm Solna: 0,4682 (GUS) Mars 2010 Stockholm Huddinge 0,4538 (ABL1) Maj 2010 Linköping Pågående Ej infört Göteborg Pågående Ej infört Lund 0,3765 (GUS) Oktober 2010 (prel)

BCR-ABL1 standardisering nästa steg? C:a 3500 ampuller frystorkade celler har producerats från respektive fyra spädningar över intervallet 0,01-10% BCR-ABL1. Dessa har erhållit definierade värden enligt den internationella skalan efter tester i tio europeiska laboratorier. Huvudansvariga: National Genetics Reference Laboratory och National Institute for Biological Standards and Control, UK.

KML ur ett kliniskt perspektiv Standardiserad mätning av BCR-ABL1 Hans Ehrencrona Genetiska kliniken Labmedicin Skåne Lund