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

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)

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

SENSITIVITY OF THE TECHNIQUES IN DETECTION OF MRD

MARKERS USED FOR MRD IN 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

TCR g AND d 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

TCR g AND d 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

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

DETECTION OF MRD

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)

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 FOR FOLLOW-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%

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

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

HETERODUPLEX ANALYSIS homo homo

QUANTITATION OF MRD

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

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

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

Real time PCR-Amplificaton plot and Standard curve -

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

PROGNOSTIC VALUE OF MRD IN ALL 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

FUTURE STUDIES MICROARRAYS

THANKS TO 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)