1. Acute lymphoblastic leukemia in children Dragana Janić University Chldrens Hospital, Belgrade, Serbia

2. EPIDEMIOLOGY OF ALL IN CHILDREN Malignancie in children – 1% of all malignancies ALL 1/3 of all - 40/1 million children/y Slightly more frequent in males, except for infants (females > males) Peak incidence 2-5 years 85% cure rate vs none some 60y ago

3. LEUKEMOGENESIS - E LEUKEMOGENESIS - Environmental factors Ionizing radiation –Risk of ALL increased to 1:60 during 12 years after nuclear bomb explosion –Intrauterine exposure to X-rays (historical data) for diagnostics increases relative risk of ALL to 1.5 Role of infection –Protective at an early age –Increasing the incidence when populations mix

4. LEUKEMOGENESIS - LEUKEMOGENESIS - Genetic factors Congenital gene aberrations –Inherited syndromes (Down, neurofibromatosis type I. Bloom, ataxia telangiectasia, Schwachman) –Family clustering - Li Fraumeni sy: TP53 Acquired gene aberrations: detected in up to 50% by cytogenetics and far more with molecular genetics –Hypodyploidy <44 chr –High hyperdyploidy 51-65 chr –BCR-ABL t(9;22), ETV6/RUNX1 t(12;21), MLL 11q 23 mostly resulting from t(4;11) –iAMP 21 –Oher reccurent abnormalities

5. FREQUENCY OF SPECIFIC GENE ALTERATIONS IN ALL Pui et al. NEJM. 2004 10% 2% 7% 3% 25% 53% ADULTDECA 25% 3% 5% 8% 22% 37% Hyperdiploidy TEL/AML1 MLL/AF4 BCR/ABL E2A/PBX1 Other CHILDREN

6. Acquired gene aberrations in explaining ALL Prenatal origin of leukemogenic event & theory of placental metastasisPrenatal origin of leukemogenic event & theory of placental metastasis “Two-hit theory” (two leukemogenic events)“Two-hit theory” (two leukemogenic events)

7. Prenatal origin of leukemogenic event & theory of placental metastasis 58% of identical twins share the placenta, monochorionic, blood vessel anastomosis. Clarkson BDClarkson BD. Boyse EA Possible explanation of the high concordance for acute leukaemia in monozygotic twins. Lancet. 1971 Apr 3;1(7701):699-701.Boyse EA 1617 NN: De Wikkelkinderen (The Swaddled Twins). The Muiderslot castle near Amsterdam

8. “Two-hit” theory first mutation first mutation second mutation second mutation External factors External factors in utero postnatal Monozygotic twins Guthrie test cards Greaves MF. et al. Leukemia in twins: lessons in natural history Blood. 1 October 2003. Vol. 102. No. 7. pp. 2321-2333 ALL

9. Retrospective analysis of Guthrie cards for fusion genes Guthrie catds from the 1950s used for PKU The same mutation found on card as in leukemia patient (TEL-AML1) Prenatal initiation – first hit Latency 3 years Not all leukemias are initiated in utero - t(1;19), negative on cards

10. Second hit Different age in other twin with leukemia (latency 3 years) 1% of healthy children bear TEL-AML1, never to develop leukemia

11. DIAGNOSIS History Physical examination Lab

12. History Short duration Fatigue Fever Bone and joint pain Hemorrhagic syndrome

13. ALL common presentation Findings % pts Hepatosplenomegaly 70 Splenomegaly 65 Fever 60 Bleeding 50 Lymphadenopathy 50 Pain/swelling bone/joint 25 Lab % pts WBC (mm 3 )  10 000 55 10 000 – 49 000 30  50 000 15 Hgb (g/dL)  70 45 70 – 110 45  110 10 PLT (mm 3 )  20 000 30 20 000 – 99 000 50  100 000 20

14. ALL uncommon presentation CNS - 5% Increased preassure Sight disturbances Paralysis of cranial nerves Vertigo Hearing disturbances Cerebelar disturbances Hyperpnea Less than 1% BM aplasia Hypereosinophilia Hypercalcemia Cyclic neutropenia SLE Renal failure IHA

15. The most common presentations of ALL 1.B cell precursor ALL 2.T-ALL (15% of ALL) 3.ALL in infants and small children

16. B CELL PRECURSOR ALL Common ALL, CALLA+, CD10+ Most common IPH Age 2-5 yrs Best prognosis Up to 5% may be BCR-ABL+

17. T-ALL Older age Male predominance Mediastinal mass, hyperleukocytosis, CNS affection

18. INFANT ALL Hyperleukocytosis, organomegaly, CNS Early pre-B cells, coexpression of myeloid antigens Structural alterations in 11q23 (MLL gene), most commonly t(4;11)

19. Lab analysis Complete blood count Bone marrow –morphology –cytochemistry –immunophenotype –bone marrow cytogenetics –molecular genetics

20. Bone marrow morphology % of leukoblasts FAB morphology classification L1 L2 L3

21. Bone marrow cytochemistry Specific dyes –PAS –Sudan black –Myeloperoxidase... PAS+ Sudan BlackMyeloperoxidase

22. Bone marrow immunophenotype Classification T (15%) (CD3, CD2, CD5 and CD7) –Early (CD34, CD7, CD5, CD2, cyCD3, TdT) –Medium (CD4 and CD8, CD3) –Late (either CD4 or CD8) B –Pre-pre-B-ALL (CD10, CD19, CD24, CD34, HLA-DR, cyCD22, CD79a, CD79b and nu-TdT) –Pre-B-ALL (CD19, CD20, HLA-DR, CD10 in 90% (CALLA- Common ALL antigen) Mature B ALL (1-2%) (IgM, CD20, CD19, HLA-DR, CD21)

23. Cytogenetics and molecular genetics Numeric and structural aberrations Methods: 2. FISH 3. PCR 4. micro array 1. karyotype

24. Case reviews

25. Case №1 Boy aged 7 Presented with: –Fever –Lymphadenopathy –Splenomegaly of short duration CBC showed WBC↑ 20.000/mm3, moderate anemia and thrombocytopenia

27. Case №1 Differential diagnosis –Infectious mononucleosis –Malignancy? Diagnostic work-up –CBC with peripheral smear –BM aspiration –Immunophenotype Dg: T - ALL

28. Case №2 6 year old girl Presented with: –Bone and joint pain with a limp –Knee swelling –Thrombocytopenia

30. Case №2 Diagnostic work-up: –CBC with peripheral smear revealed Plt↓ –Careful re-examination revealed splenomegaly Dg: B cell precursor ALL

31. Case №3 4 year old girl Presented with: –Fever –Severe aphthous stomatitis –“Strange” lesions on the skin –Pancytopenia –Hepatosplenomegaly

33. Case №3 CBC with peripheral smear showed pancytopenia with scarce leukoblasts BM aspiration with immunophenotyping showed B-cell precursor ALL Skin biopsy: atypical form of pyoderma gangrenosum (paraneoplastic neutrophilic dermatosis – very rare in children!)

34. Case №4 3 year old boy Presented with: –Leucocytosis –Anemia –Thrombocytopenia –Massive hepatosplenomegaly –Scrotal swelling

36. Case №4 Diagnostic work-up: –BM aspiration with immunophenotype –Abdominal and testicular US Dg: B cell precursor ALL with testicular infiltration

37. Special therapy for subtypes? IV Balkan Hematology Days, Sept. 2009, Sofia From: Acute lymphoblastic leukaemia. Pui CH, Robison LL, Look AT. Lancet 2008.

38. PROGNOSTIC FACTORS Gene alterations Age, WBC, Tx response Imnunophenotype: pre-B and T

40. I-BFM SG

41. Chemotherapy-treatment protocol

42. General principles of ALL therapy Induction CNS-directed therapy and consolidation Reinduction Maintenance

43. General principles of ALL therapy: Induction 98% - morphological remission and restauration of hematopoesis 4-6w of 4 sistemic drugs (glucocorticoids, vincristine, anthracycline and L-asparaginase) + IT therapy with MTX or MTX, hydrocortisone and cytarabine

44. General principles of ALL therapy: CNS-directed therapy and consolidation Prevention of CNS relapses High-dose MTX and 6-MP + IT Th T ALL with high WBC, high-risk pts - 12Gy CNS involvement -18Gy

45. General principles of ALL therapy: Reinduction or delayed intensification Introduction of reinduction therapy brought the major improvement in EFS rates Therapy repeats the elements of induction

46. General principles of ALL therapy: Maintenance Oral daily 6-MP and weekly MTX for total therapy duration of 2 to 3 years Adjusting the doses of the drugs in order to keep the WBC count between 1500 and 3000/mm3, which requires frequent check- ups because of high inter-individual variability

47. Selected randomized studies comparing Pred with Dexa in the treatment of childhood ALL TrialYearsOutcome CALGB 71111971-1974 Isolated CNS relapse rates: Pred 25.5% Dexa 14.3% CCG 19221993-1995 EFS at 6 y: Pred 77% Dexa 85% UK MRC ALL97 and ALL97/99 1997-2002 EFS at 5y: Pred 76% Dexa 84% Stanulla M, Schrappe M. Treatment of childhood acute lymphoblastic leukemia. Semin Hematol. 2009;46(1):52-63

48. Randomized trials of 6-MP and 6- TG for childhood ALL TrialYearsOutcomeToxicity COALL-921992-1997 EFS at 6.6y: 6-MP 79% 6-TG 78% 6-TG: prolonged myelosuppression with marked thrombocytopenia UK MRC ALL97 and ALL97/99 1997-2002 EFS at 6.6y: 6-MP 81% 6-TG 80% 6-TG: non-fatal hepatotoxicity with features of VOD at 11% pts CCG-19521996-2000 EFS at 5y: 6-MP 77% 6-TG 85% 6-TG: reversible VOD at 20% pts; portal hypertension as a late effect Stanulla M, Schrappe M. Treatment of childhood acute lymphoblastic leukemia. Semin Hematol. 2009;46(1):52-63

50. Choosing treatment protocol - Serbian experience In 2002, all Serbian centers treating pediatric ALL achieved consent to participate in the randomized study of ALL treatment Concerning our long-term experience in providing treatment according to BFM- based protocols, we participated in ALL IC BFM 2002 protocol and became members of I-BFM-SG

51. Janic et al. Rezultati lečenja dece obolele od akutne limfoblastne leukemije po modifikovanom BFM protokolu. Srp arh celok lek 2004; 132:17-22

52. SCG National Group for ALL IC-BFM 2002 Dragana Janić, National Coordinator Intensive chemotherapy for childhood acute lymphoblastic leukemia: results of the randomized intercontinental trial ALL IC-BFM 2002. Stary J, Zimmermann M, Campbell M, Castillo L, Dibar E, Donska S, Gonzalez A, Izraeli S, Janic D, Jazbec J, Konja J, Kaiserova E, Kowalczyk J, Kovacs G, Li CK, Magyarosy E, Popa A, Stark B, Jabali Y, Trka J, Hrusak O, Riehm H, Masera G, Schrappe M. J Clin Oncol. 2014 Jan 20;32(3):174-84 EXPERIENCE IN PROTOCOL ADMINISTRATION

53. Novi Sad Belgrade Banja Luka Niš Serbia Adriatic sea Bosnia and Herzegovina Croatia Hungary Romania Bulgaria FYR Macedonia Albania Immunophenotyping Cytogenetics Immunophenotyping BMT Molecular genetics Podgorica Montenegro

54. NEGATIVES HEAVY WORK LOAD!

55. Conclusions ALL displays an enormous biological variety which is not yet fully elucidated Great expectations that molecular genetic methods will enable us to recognize new risk factors related to biology of the disease Being involved in one of the large international studies is probably the best way toward optimizing the treatment of each individual patient

56. Supportive treatment Blood component transfusion Infection prophylaxis and treatment Psychosocial Nutrition Various disease and treatment related complications