STATUS OF DETECTION OF MINIMAL RESIDUAL DISEASE (MRD) IN ACUTE LYMPHOBLASTIC LEUKEMIAS

1. STATUS OF DETECTION OF MINIMAL RESIDUAL DISEASE (MRD) IN ACUTE LYMPHOBLASTIC LEUKEMIAS DEPT OF MOL ONCOLOGY CANCER INSTITUTE (WIA) ADYAR, CHENNAI - 600 020

2. INTRODUCTION • ALL MOST COMMON PEDIATRIC MALIGNANCY REGISTERED AT CANCER INSTITUTE (WIA) • 30-40% T-ALL- WESTERN STUDIES – 85% B-ALL AND 15% T-ALL • MCP 841 –80-90 % ACHIEVE CR BUT 30-40% RELAPSE • PERSISTENCE OF LOW NUMBERS OF RESIDUAL LEUKEMIC CELLS – NOT DETECTABLE BY CONVENTIONAL CYTOMORPHOLOGICAL METHODS (1-5%) • NEED FOR A SPECIFIC AND SENSITIVE METHOD TO DETECT/DEFINE MOLECULAR REMISSION/ RELAPSE (MINIMAL RESIDUAL DISEASE)

3. DETECTION OF MINIMAL RESIDUAL DISEASE IN T-ALL • WHY DETECT MRD? • HELP ANTEDATE RELAPSE. • THERAPY STRATIFICATION • RISK STRATIFY PATIENTS • ASSESS RESPONSE TO TREATMENT • INTRODUCTION OF NEWER FORMS OF • BIOLOGICAL THERAPY WHEN TUMOUR • LOAD IS LOW • EVALUATION AS A PROGNOSTIC MARKER

4. SENSITIVITY OF THE TECHNIQUES IN DETECTION OF MRD

5. MARKERS USED FOR MRDIN ALL • PCR ANALYSIS OF CLONE SPECIFIC • JUNCTIONAL REGIONS OF TCR  AND  GENE • REARRANGEMENTS • PCR ANALYSIS OF BREAKPOINT FUSION • TRANSCRIPTS OF LEUKEMIA SPECIFIC • CHROMOSOMAL ABERRATIONS (BCR-ABL, • TEL-AML,E2A-PBX, MLL-AF4. TAL-1 DELETION) • MULTI PARAMETER FLOW CYTOMETRY • QUALITATIVE AND QUANTITATIVE

6. TCRgANDd GENE REARRANGEMNTS DIVERSITY OF TCR BY T CELL DIFFERENTIATION-CORTICAL THYMOCYTES--V-D-J RECOMBINATION Germline J1 V  V  D  J 1 V 1-J1 V1-J 1 Rearranged JUNCTIONAL REGION JUNCTIONAL REGION T-ALL ARREST IN DIFFERENTIATION CLONAL PROLIFERATION OF ARRESTED CELL EACH CELL IN CLONE --IDENTICAL JUNCTIONAL SEQUENCE

7. TCRgANDd ARE GOOD MARKERS FOR MRD – PCR • LIMITED GERMLINE AND COMBINATORIAL DIVERSITY • OF TCR  AND  GENES BUT EXTENSIVE JUNCTIONAL • REGION DIVERSITY (LEUKEMIA SPECIFIC DNA • FINGERPRINT) - DIFFERENT IN EACH LYMPHOCYTE • AND EACH LYMPHOID LEUKEMIA. • DEVISE PATIENT SPECIFIC PRIMERS/PROBES(ASO) • SOMATIC MUTATIONS NOT REPORTED IN • REARRANGED TCR GENES • IN 95% OF T-ALL, REARRANGED TCR  AND  • JUNCTIONAL REGIONS OR BOTH ARE USED AS • TARGETS FOR MRD-PCR

8. PITFALLS IN THE USE OF JUNCTIONAL REGIONS AS MRD PCR TARGETS • FALSE POSITIVE • BACKGROUND AMPLIFICATION OF SIMILAR • REARRANGEMENTS IN POLYCLONAL REACTIVE T • LYMPHOCYTES / NORMAL LYMPHOCYTES • HETERO DUPLEX ANALYSIS – SIMPLE, FAST • CHEAP, RELIABLE METHOD TO CONFIRM • CLONALITY

9. DETECTION OF MRD

10. DETECTION OF MINIMAL RESIDUAL DISEASE • INSTITUTE EXPERIENCE • GENOMIC DNA -NORMAL & LEUKEMIC CELLS • QUANTITATION -DIAGNOSIS , REMISSION, NORMAL • ( SPEC) • PCR AMPLIFICATION OF ABL, TCR AND TCR  AT • PRESENTATION • HETERODUPLEX ANALYSIS—PAGE • HD BAND CUT ,ELUTED ,PCR REAMPLIFIED AND • SEQUENCED TO DESIGN ASO (ALLELE SPECIFIC OLIGO)

11. DETECTION OF MINIMAL RESIDUAL DISEASE IN T-ALL -PCR-HDA • 50 CASES OF T-ALL STUDIED AT PRESENTATION • PCR–CLONALITY CONFIRMED BY HD ANALYSIS • 24 CASES WERE AVAILABLE FORFOLLOW-UP STUDIES • DURATION OF FOLLOW-UP FROM 6 TO 72 MONTHS • V1-J11.3/2.3 62.5% • V1 - J1 64% • 2 V CLONAL MARKERS 17.5% • V-J1 AND V1-J11.3/2.3 46%

12. MRD-PCR FOLLOW UP – REMISSION / RELAPSE • ALL PATIENTS WERE PCR +VE/HD +VE AT END • OF INDUCTION THERAPY (3 MOS) -MCP 841 • 6 PATIENTS IN CR BUT REVEALED CONTINUOUS • PCR +VE/ HD +VE RELAPSED AND DIED • COMBINATION OF PCR PRODUCTS AT • PRESENTATION AND RELAPSE - SAME HD • PATTERN - IDENTICAL CLONALITY • ALL PATIENTS IN LONG TERM CR WERE HD –VE • IN 8-12 MONTH REMISSION SAMPLES AND • CONTINUED TO BE PCR –VE/HD -VE Leukemia Research 2002 Vol 26, 335-43

13. RESULTS - MRD IN T-ALL-MCP 841 LEUKEMIA rESEARCH

14. HETERODUPLEX ANALYSIS hetero homo homo

15. QUANTITATION OF MRD

16. QUANTITATION OF MRD • DETECT AND ACCURATELY ASSESS THE • VOLUME OF PERSISTENT SUB -CLINICAL • DISEASE -LEVELS AND DYNAMICS OF MRD • DEFINE THE EXTENT OF REDUCTION IN • TUMOR VOLUME REQUIRED TO PREVENT • RELAPSE AND ENSURE LONG TERM DISEASE • FREE SURVIVAL • COMPETITIVE PCR • LIMITING DILUTION • REALTIME PCR LABORIOUS, MORE AMOUNT OF DNA , RISK OF CONTAMINATION

17. QUANTITATION OF MRD – REAL TIME Q-PCR • AFFORDS BOTH AMPLIFICATION AND ACCURATE QUANTIFICATION DURING EXPONENTIAL PHASE OF INITIAL TARGETS - SHORT TIME • FLOURESCENCE IS MONITORED AND THE CROSSING POINT/THRESHOLD CORRELATES TO AMOUNT OF INITIAL COPIES OF TARGET • NO NEED FOR GELS, RADIOACTIVITY AND POST -PCR MANIPULATION • DETERMINATION OF LARGE DYNAMIC RANGE OF STARTING TARGET MOLECULE DETERMINATION

18. REAL TIME PCR TECHNIQUES • SYBER GREEN 1 -BINDS TO DOUBLE STRANDED DNA • HYBRIDISATION PROBE – DONOR AND ACCEPTOR FLUROCHROMES-FRET • HYDROLYSIS PROBE -TAQMAN PROBE- 5’-3’ NUCLEASE ACTIVITY OF TAQ POLYMERASE Q R 3 5 Fl emitted Fl quenched

19. Real time PCR-Amplificaton plot and Standard curve -

20. REAL TIME PCR -QUANTITATION OF MRD 1 ASO- PCR - SPECIFICITY AND SENSITIVITY ( 1 IN 10-5 ) 2 ASO-PCR NORMAL DNA-NON SPECIFIC AMPLIFICATION 3 STANDARD CURVE PCR WITH KNOWN INTERNAL CONTROL-(RNASE P)(50 ng--50 pg) QUANTITATE SAMPLES 4 STANDARD CURVE PCR WITH ASO-J1 –SERIAL DILUTION OF PRESENTATION LEUKEMIC DNA (50ng--5pg) IN 500ng OF NORMAL DNA. 5 REMISSION SAMPLE QUANTITATED USING ABOVE STD CURVE

21. WHEN AND HOW OFTEN SHOULD MRD BE MONITORED • SINGLE TIME POINT ANALYSIS IS INADEQUATE • AT LEAST 2 SERIAL MEASUREMENTS ARE NEEDED DURING EARLY MONTHS OF TREATMENT • AT END OF INDUCTION 1-RESPONSE TO TREATMENT • AT START OF CONSOLIDATION-RISK OF RELAPSE IS PROPORTIONAL TO MRD LEVELS-POWERFUL PROG NOSTIC MARKER • LOW RISK  10-3 INTERMEDIATE RISK 10-3 HIGH RISK  10-2 • SLOWER KINETICS OF CLEARANCE IN T-ALL COMPARED TO PRE-B -ALL PROGNOSTIC VALUE OF MRD IN ALL

22. FUTURE STUDIES MICROARRAYS

23. THIS STUDY WAS FUNDED BY THE NCI GRANT FRA No N427-645 AND THE DEPARTMENT OF SCIENCE AND TECHNOLOGY, GOVT OF INDIA THANKS TO Dr T RAJKUMAR, SCIENTIFIC DIRECTOR MR SUDHAKAR, SRF IN THE DEPT DR RAJALEKSHMY, HEMATOPATHOLOGIST MISS MEENA , GRADUATE TECHNICIAN DR T G SAGAR ,DR ANITHA & DR S G RAMANAN DR V SHANTA, CHAIRMAN , CANCER INSTITUTE(WIA)