STATUS OF DETECTION OF MINIMAL

RESIDUAL DISEASE (MRD) IN ACUTE

LYMPHOBLASTIC LEUKEMIAS

DEPT OF MOL ONCOLOGY

CANCER INSTITUTE (WIA)

ADYAR, CHENNAI - 600 020

INTRODUCTION

o ALL MOST COMMON PEDIATRIC MALIGNANCY REGISTERED AT CANCER INSTITUTE (WIA)

o 30-40% T-ALL- WESTERN STUDIES – 85% B-ALL AND 15% T-ALL

• MCP 841 –80-90 % ACHIEVE CR BUT 30-40% RELAPSE

o PERSISTENCE OF LOW NUMBERS OF RESIDUAL LEUKEMIC CELLS – NOT DETECTABLE BY CONVENTIONAL CYTOMORPHOLOGICAL METHODS (1-5%)

o 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?

o HELP ANTEDATE RELAPSE.

o THERAPY STRATIFICATION

o RISK STRATIFY PATIENTS

o ASSESS RESPONSE TO TREATMENT

o INTRODUCTION OF NEWER FORMS OF

BIOLOGICAL THERAPY WHEN TUMOUR

LOAD IS LOW

o EVALUATION AS A PROGNOSTIC MARKER

SENSITIVITY OF THE TECHNIQUES IN DETECTION OF MRD

NO. TECHNIQUE SENSITIVITY

1. MORPHOLOGY 1- 5% 2. CELL CULTURE SYSTEM 10-1 – 10-3

3. CYTOGENETICS 10-1 – 10-3

FISH 10-3

DUAL COLOR / TRIPLE COLOR INTERPHASE FISH

10-4

4 IMMUNOPHENOTYPE BY FLOW CYTOMETERY 10-3

MULTIPLE PARAMETER FLOW CYTOMETERY

10-4 – 10-5

5 SOUTHERN BLOT 1 – 5% 6. PCR 10-3 – 10—4

RT – PCR 10-4 - 10-5

REAL TIME QPCR 10-6

MARKERS USED FOR MRD IN ALL

o PCR ANALYSIS OF CLONE SPECIFIC JUNCTIONAL REGIONS OF TCR AND GENE REARRANGEMENTS

o PCR ANALYSIS OF BREAKPOINT FUSION TRANSCRIPTS OF LEUKEMIA SPECIFIC CHROMOSOMAL ABERRATIONS (BCR-ABL, TEL-AML,E2A-PBX, MLL-AF4. TAL-1 DELETION)

o MULTI PARAMETER FLOW CYTOMETRY

o QUALITATIVE AND QUANTITATIVE

TCR AND GENE REARRANGEMNTS

V D J 1

V1-J 1

DIVERSITY OF TCR BY T CELL DIFFERENTIATION-CORTICAL THYMOCYTES--V-D-J RECOMBINATION

V J1

V 1-J1

T-ALL ARREST IN DIFFERENTIATION

CLONAL PROLIFERATION OF ARRESTED CELL

EACH CELL IN CLONE --IDENTICAL JUNCTIONAL SEQUENCE

JUNCTIONAL REGION JUNCTIONAL REGIONRearranged

Germline

TCR AND ARE GOOD MARKERS FOR MRD – PCR

o 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.

o DEVISE PATIENT SPECIFIC PRIMERS/PROBES(ASO) o SOMATIC MUTATIONS NOT REPORTED IN REARRANGED TCR GENES

o 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

o FALSE POSITIVE

o BACKGROUND AMPLIFICATION OF SIMILAR

REARRANGEMENTS IN POLYCLONAL REACTIVE T

LYMPHOCYTES / NORMAL LYMPHOCYTES

o HETERO DUPLEX ANALYSIS – SIMPLE, FAST

CHEAP, RELIABLE METHOD TO CONFIRM CLONALITY

DETECTION OF MRD

DETECTION OF MINIMAL RESIDUAL DISEASE

INSTITUTE EXPERIENCE

o GENOMIC DNA -NORMAL & LEUKEMIC CELLS

o QUANTITATION -DIAGNOSIS , REMISSION, NORMAL

o ( SPEC)

o PCR AMPLIFICATION OF ABL, TCR AND TCR AT

PRESENTATION

o HETERODUPLEX ANALYSIS—PAGE

o HD BAND CUT ,ELUTED ,PCR REAMPLIFIED AND

o SEQUENCED TO DESIGN ASO (ALLELE SPECIFIC OLIGO)

o 50 CASES OF T-ALL STUDIED AT PRESENTATION o PCR–CLONALITY CONFIRMED BY HD ANALYSIS

o 24 CASES WERE AVAILABLE FOR FOLLOW-UP STUDIES

o DURATION OF FOLLOW-UP FROM 6 TO 72 MONTHS

o V1-J11.3/2.3 62.5% o V1 - J1 64%

o 2 V CLONAL MARKERS 17.5%

o V-J1 AND V1-J11.3/2.3 46%

DETECTION OF MINIMAL RESIDUAL DISEASE IN T-ALL -PCR-HDA

MRD-PCR FOLLOW UP – REMISSION / RELAPSE

ALL PATIENTS WERE PCR +VE/HD +VE AT END OF INDUCTION THERAPY (3 MOS) -MCP 841 o 6 PATIENTS IN CR BUT REVEALED CONTINUOUS PCR +VE/ HD +VE RELAPSED AND DIED

o COMBINATION OF PCR PRODUCTS AT PRESENTATION AND RELAPSE - SAME HD PATTERN - IDENTICAL CLONALITY

o 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

hetero

homo

QUANTITATION OF MRD

QUANTITATION OF MRD o DETECT AND ACCURATELY ASSESS THE VOLUME OF PERSISTENT SUB -CLINICAL DISEASE -LEVELS AND DYNAMICS OF MRD o 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

o SYBER GREEN 1 -BINDS TO DOUBLE STRANDED DNA

o HYBRIDISATION PROBE – DONOR AND ACCEPTOR FLUROCHROMES-FRET

o HYDROLYSIS PROBE -TAQMAN PROBE- 5’-3’ NUCLEASE ACTIVITY OF TAQ POLYMERASE

3 5

R Q

Fl quenched Fl emitted

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-J1 –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 ALLWHEN 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

1 AT END OF INDUCTION 1-RESPONSE TO TREATMENT

2 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

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)