Children ALL MRD study according to protocol of D. Campana

Children ALL MRD study according to protocol of D. Campana L.Yu. Grivtsova Laboratory of Haematopoiesis Immunology State N.N.Blokhin Russian Cancer Research Center affiliated to the Russian Academy of Medical Sciences, Russian Federation

Risk group stratification Age Leukocyte count Immunophenotype of blasts genetic abberation (BCR/ABL, MLL/AF 4) Therapy induction efficacy (prednisolone response) Leukemic cytoreduction (d 15-th induction chemotherapy)

ALL-IC-BFM 2002 (National coordinator from Russia Professor A.V.Popa)

Morphological detection of residual disease 7-th & 15-th days therapy of induction Miller et. al. 1989 Steinherz et al 1996 Sandlund et al 2002

Flow cytometry MRD detection Blasts have aberrant immunophenotype G. Janossy et.al. J. of Immunology, 1979, Blood, 1980 M.F. Greaves et. al. Cancer Research, 1981, Blood, 1983

BFM-AIEOP Examples of deranged patterns of antigen expression in BCP-ALL with respect to normal differentiation normal frequently involved antigens: CD10, CD11a, CD19, CD20, CD34, CD44, CD45, CD58 M. N. Dworzak et. al. 1999 CCRI St. Anna Kinderspital, Vienna, Austria

A leukemic preb- C-ALL physiologic 34 + BCP 34 - BCP global B leukemic preb- C-ALL physiologic BCP1 BCP2 BCP3 BFM-AIEOP 10000 1000 65 % overexpression 10000 1000 MFI MFI 100 Deranged patterns of antigen expression in BCP-ALL 100 10 CD10 10 1 58 % underexpression CD45 RA with respect to normal differentiation C 1000 leukemic preb- C-ALL physiologic BCP1 BCP2 BCP3 D 10000 leukemic preb- C-ALL physiologic BCP1 BCP2 BCP3 M. N. Dworzak et. al. 1999 CCRI St. Anna Kinderspital, Vienna, Austria 100 MFI 10 1 33 % underexpression CD11a 1000 MFI 100 10 1 7 % underexpression CD 44

BFM-AIEOP normal Examples of deranged patterns of antigen expression in BCP-ALL with respect to normal differentiation frequently involved antigens: CD10, CD11a, CD19, CD20, CD34, CD38, CD44, CD45, CD58 M. N. Dworzak et. al. 1999 CCRI St. Anna Kinderspital, Vienna, Austria

Comparison of established MRD-MoAb panels St. Jude CRH AIEOP-BFM Biomed MyM cym 22 10 34 19 58 10 34 19 45 10 34 19 38 10 34 19 13 10 34 19 33 10 34 19 15 10 34 19 65 10 34 19 66c 10 34 19 56 10 34 19 7.1 10 34 19 TdT 10 34 19 Cµ 10 34 19 WT1 10 34 19 20 10 34 19 58 10 34 19 10 34 45 19 10 11a 45 19 (10 38 45 19) 10+20 38 34 19 15 34 45 19 65 34 45 19 limited panel!! triple-backbone! TdT 10 19 19 34 45 10 20 19 34 22 45 (19) 34 38 19 10 13 19 33 15 65 CD diversity: n= 17 n= 10 n= 12

Mini Mini study results (Prague -2007) BFM-AIEOP Patients: Czechia, Croatia, Hong Kong, Hungary, Israel Syto16/CD19/CD45 CD20/CD10/CD19/CD34 CD58/CD10/CD19/CD34 CD10/CD66c/CD19/CD45 standard subpopulations evaluated regardless initial immunophenotype normal immature cells with the same immunophenotype typical rebound regeneration after chemotherapy

Mini Mini study results (Prague -2007) BFM-AIEOP Live gate strategy (syto 16 all nucleated cells)

Mini Mini study results (Prague -2007) BFM-AIEOP cells expressing aberrant molecules CD20/CD10/CD19/CD34 CD58/CD10/CD19/CD34 CD10/CD66c/CD19/CD45

Mini Mini study results (Prague -2007) BFM-AIEOP BCP ALL: CD20-10-19-34

Mini Mini study results (Prague -2007) BFM-AIEOP BCP ALL CD10-66c-19-45

Mini Mini study results (Prague -2007) BFM-AIEOP

Mini Mini study results (Prague -2007) BFM-AIEOP Waiting for the antibodies PC5 DAKO PE BD

Mini Mini study results (Prague -2007) BFM-AIEOP 4-color FC - four to one tube CD19 PC5 to PC7 planned change as PC7 became available other changes (Coulter based system) CD66c conjugated with FITC changes in SYTO16 combinations (SYTO16-CD10-CD19-CD45) SYTO16 combination not measured extra templates needed SR: FCM-MRD d15 < 0.1% NC MR: FCM-MRD d15 >=0.1% NC, HR: FCM-MRD d15 > 10% NC.

Coustan-Smith E. et al. Blood, 2006 BM normal lymphoid progenitors CD10+CD19+CD34+ are exquisitely sensitive to corticosteroids and other antileukemic drugs In patients with B-lineage ALL cells with this phenotype detected early in treatment shoud be leukemic rather than normal

Coustan-Smith E. et al. Blood, 2006 Three-color FC (BCP ALL) T-Lineage ALL after 2 weeks of remission induction therapy (day +19) CD19+CD34+ and/or CD19+CD10+ undetecteble or represented less than 0,01% of BM MNC

Coustan-Smith E. et al. Blood, 2006 Three-color FC (BCP ALL 380 pts) FSC vs SSC 500 000 events (cells) CD19 gating CD10 CD34 PerCP- PE - FITC SR IR HR 0,01- <0,1%% 0,1 - < 1,0%% 1,0% and more MNC MNC MNC

Coustan-Smith E. et al. Blood, 2006 Three-color FC (BCP ALL)

Coustan-Smith E. et al. Blood, 2006 Three-color FC (BCP ALL)

Coustan-Smith E. et al. Blood, 2006 Three-color FC (BCP ALL)

Study design Started - Spring 2007 National coordinator from Russia Professor A.V.Popa 1. Flow cytometric assay according to Coustan-Smith E. et al. Blood, 2006 2. Measurement of blast percent (morphology) and CD10+CD19+ as well as CD34+CD19+ MNC (MNC were identified according to FSC/SSC) Day 15 of induction therapy Day 33 of induction therapy

Study design 21 pts Pre-pre-B ALL day + 15 21 pts day + 33 18 pts Morphology FC IgG1 PE IgG1FITC- CD19PE-cy5 Typical morphology of lymphoblast L1-L2 CD10PE- CD34 FITC CD19PE-cy5 or CD34PE- CD10 FITC CD19PE-cy5

Day +15 group I (morphology CR, blasts 0,0-1,0) n pts % blasts of morfology %CD19+CD10+ of MNC %CD19+CD34+ of MNC 1 Kalant. 0,6 0,39 0,33 2 Lob. 1,0 1,49 1,0 3 Min. 0,5 0,04 0,0 4 Lin. 0,0 0,01 0,57 5 Redzh. 0,0 0,54 <0,01 6 Les. 0,7 0,07 0 7 Grig. 0,0 0,0 0,0 8 Karp. 0,0 0,04 0,14 9 Gadz 0,2 0,01 1,7 10 Shaik. 0,2 0,0 0,0 Only 2 cases of complete morphological and FC remission (<0,01% MNC CD19+CD10+CD34+)

EXAMPLE MRD in BCP ALL Complete morphological (0% blasts) and FC remission (<0,01% MNC CD19+CD10+CD34+) A C D CD10 PE CD34 FITC B Patient G Day 15 Morphology 0,0% blasts FC 0,0% MNC (CD19+CD10+& CD19+CD34+)

EXAMPLE, MRD in BCP ALL Patient R, day 15 Morphologically 0% blasts Flow MRD <1,0% CD10+CD19+ & CD34+CD19+ MNC A B C D CD19+CD10+ 0,74% of MNC CD19+CD34+ 0,04% of MNC

Day +15 group II (morphologically blasts 1,1-5,0) n pts % blasts of morfology %CD19+CD10+ of MNC %CD19+CD34+ of MNC 1 Zhul. 3,4 3,3 1,1 2 Ilnits. 3,2 0,12 0,05 3 Vor. 2,0 0,08 0,06 4 Tash. 1,5 1,46 0,16 5 Saf. 2,4 0,06 0 6 Zhuik. 1,5 0 0 7 Siv 4,5 0,43 0,05 In 1 case (blasts 1%) FC not detected of MRD(0% MNC CD19+CD34+CD10+) In 6 cases flow cytometrically MRD (0,06 3,3%) MNC

EXAMPLE MRD in BCP ALL (intermediate group) Patient Zuik., day 15 Morphologically 1,5% blasts A B CD19+CD10+ CD19+CD34+ FC MRD 0,0% CD10+CD19+ & CD34+CD19+ MNC

EXAMPLE MRD in B-lineage ALL (intermediate group) Patient T., BCP All Day 15 Morphology - Blasts 1,5% FC MRD CD19+CD10+ CD19+CD34+ 1,46% MNC 0,16% MNC A B CD19+CD10+ CD19+CD34+

Day +15 group III (morphological blasts >5,0%) n pts % blasts of morphology %CD19+CD10+ of MNC %CD19+CD34+ of MNC 1 Bulig. 60,8 58,9 52,2 2 Mustaf. 8,2 2,85 1,15 3 Khleb. 17,5 28,7 20,4 4 Kisel. 16,0 20,8 0,01 In all these cases it was a prominent percent of CD10+CD19+ & CD34+CD19+ MNC CD19+CD10+ 28,7% of MNC CD19+CD34+ 14,17% of MNC Morphologycally 17,5% blasts

FC MRD Day +33 18 pts Mean Median variabl n Morphology blasts CD19+CD10+ MNC CD19+CD10+ MNC 1,2+/-0,35 0,8 0,0-5,8 18 2,06+/-2,01 0,02 0,0-36,4 18 0,87+/-0,84 0,015 0,0-15,3 18

FC MRD Day +33 18 pts Pts Blasts morfjlogycally (%%) CD19+CD10+ (%% MNC) CD19+CD34+ (%% MNC) Zhul. 3,6 0,04 0,0 Il. 1,4 0,09 0,03 Bu. 5,8 36,4 15,3 Kal. 0 0,07 0,0 Lo. 0,9 0,36 0,22 V. 0,0 0,01 0,0 Mu. 1,5 0,04 0,0 Khl. 2,2 0,09 0,12 Mi. 0 0,02 0,02 T. 1,2 0,01 0,02 G. 0,8 0,06 0 Kar. 2,6 0,02 0,05 Sha. 0,2 0,0 0,01 S. 0 0,0 0,0 R. 0,4 0,01 0,0 Le. 2,6 0,01 0 Zhui. 0 0 0 Ga 0,4 0 0

Summary, conclusions Day 15 Morphologically remission was noted in 17 of 21 pts (blasts not more than 5%) In group (10pts) where morphologycally blasts not more than 1,0%, flow cytometrically MRD was not noted in 2 cases. In cases (11 pts), where morphologically there were more than 1% of blasts in BM, flow cytometry confirmed MRD in 10 cases. Only in 1 pt with 1,5% blasts morphologically, FC not detect MRD (CD19+CD34+CD10+ MNC). So, in 3 from 17 cases with morphological remission (blasts less then 5%) immunologically MRD was not identified. These pts are possible candidates for reduction of intensity of induction therapy. Day 33 Morphologically less then 1% blasts were identified in 10 of 18 pts. In these cases flow cytometrically MRD was not noted in 6 cases (0,01 and less CD19+CD10+&CD19+CD34+ MNC cells. In 7 of 8 cases, where morphologically there were more than 1% of blasts in BM, flow cytometry confirmed MRD. Only in 1 pts (2,6% blasts morphologically) FC did not detect CD19+CD34+CD10+ MNC.

Children ALL MRD study State N.N..Blokhin Russian Cancer Research Center affiliated to the Russian Academy of Medical Sciences, Russian Federation Отделение химиотерапии гемобластозов НИИ ДО и Г ГУ РОНЦ им. Н.Н. Блохина РАМН Рук. Профессор А.В. Попа Лаборатория иммунологии гемопоэза Централизованный клиниколабораторный отдел НИИ КО ГУ РОНЦ им. Н.Н. Блохина РАМН Рук. Профессор Н.Н. Тупицын Лаборатория гемоцитологии НИИ ДО и Г ГУ РОНЦ им. Н.Н. Блохина РАМН Рук. Профессор И.И. Матвеева M. Г. Божьева Л.Ю. Гривцова И.Н. Серебрякова Н.А. Купрышина

Thanks a lot of attention!!!