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

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 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

6. 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

7. 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

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 FOR FOLLOW-UP STUDIES
DURATION OF FOLLOW-UP FROM 6 TO 72 MONTHS
V1-J11.3/ %
V1 - J %
2 V CLONAL MARKERS %
V-J1 AND V1-J11.3/ %

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,

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

14. HETERODUPLEX ANALYSIS
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. 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  INTERMEDIATE RISK HIGH RISK  10-2
SLOWER KINETICS OF CLEARANCE IN T-ALL COMPARED TO PRE-B -ALL

22. FUTURE STUDIES
MICROARRAYS

23. 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)