Acute leukaemias: pathogenesis, diagnostics and treatment

1
Acute leukaemiaspathogenesis, diagnostics and
treatment

• Tomas Kozak
• Department of Internal Medicine and Haematology,
• 3rd Faculty of Medicine, Charles University in
  Prague
• Pregraduate lecture, Summer semester 2013

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• Acute myelogenous leukemia
• (AML)

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AML basic characteristics

• Complex CLONAL disease with considerable
  phenotypical and genotypical heterogeneity
• Proliferation and accumulation of immature
  haematopoietic cells in the bone marrow and blood
• Evalutaion and prognosis hase changed over 20
  years, however, still most patients will die of
  the disease or of the therapy.

Challenge for molecularly targeted therapy
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AML - epidemiology

• 1,2 of all cancer deaths
• Median age at diagnosis 60-65 years
• AML 60-90 of adult leukemias in adults
• Incidence by age
• 50 years 3,5/100 000
• 70 years 15/100 000
• 80 years 35/100 000

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AML - etiology

• No firm etiology factor known at this point
• Predispositions
• Inherited (genetic) family region, well
  difined cosntitutional aberation and mutation
  sydromes (Downs, 8, Blooms, LiFraumenis,
  Fanconis, etc.). Special regard monoz. twins.
• Environmental exposure Ionizing radiation,
  benzene (gas products), smoking? Magnetic field?
• Therapy related leukemia Hodgkins, breast
  cancer, etc. Alkylating
  agents chromosome 5, 7 etc.
  Inhibitors TP II 11q23
  (MLL associated aberations)
• Antecendent hem. disorders MDS, PV, ET, PMF,PNH
• 
  

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Pathogenesis of AML

• Inappropriate proliferation
• Differentiation blockade
• Escape from programmed cell death
• Self renewal
• Loss of cell cycle control
• Genomic instability
• Leukemia cell dissemination

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The leukemic stem cell

• only few leukemic blasts have capacity of
• self renew
• from animal studies
• one leukemia stem cell in 105 leukemia
• cells
• phenotype of leukemia SC CD34CD38-

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The leukemic stem cell
HSC
Leukemia oncogene
(MLL)
Second hit (FLT3 mutation)
Myeloid progenitor
granulocyte
AML
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Self renewal capacity
1.
Nucleophosmin (NPM) protein
CD 34
Stem cell phenotype continuation
FLT3-ITD
Wnt/?-catenin
? catenin
2.
RUNX1-MTG8, PML-RAR?
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Differentiation blockade

• Transcription factors
• In AML commonly disrupted by chromosomal
  translocation
• or point mutations.

Chr. translocation
Negative forms of CBF complex
1. Core binding factor complex RUNX1 (AML1) and
CBF? t(821) RUNX1/MTG8 Inv 16.CBF?/MYH1 2.
t(1517)..PML/RAR?
STOP in differantiation Self renewal capacity
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Inappropriate proliferation
Crucial role Signaling Molecules
Receptor tyrosine kinase
Intracellular thyrosine kinase
FLT3 tyrosine kinase expressed almost in all AML
Internal tandem duplication (ITD) or mutation
within activation loop of FLT3 (30 of AML)
Constitutive activation of FLT3
Other tyrosine kinases involved c-KIT, JAK-2
(V617F), etc.
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Escape from programmed cell death
1.
Proliferation
Phosphatidylinositol 3-kinase (PI-3 kinase)
TK activation
BCL-2
Survival
2.
RUNX1-MTG8 (t(821)
p53 stability
p14ARF
NPM (nucleophosmin) protein mutation
30 AML
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The role of RAS family
RTK
Mutated NRAS, KRAS, HRAS
RAS
Constitutive proliferative signal
NRAS mutated 10 20 AML KRAS mutated 5 - 15
AML
RTK Receptor Tyrosine Kinase
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MLL rearrangements
Fusion proteins involving PML-RAR? or RUNX1/MTG8
negative proteins Fusion proteins involving MLL
both activators negative proteins
Hox gene activation
p53 activation prevention
Self renewal capacity
genomic instability
Frequent MLL involvement MLL-AF10, MLL-CBP
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Cooperating mutations
Proliferative survival advantage
Self renewal properties and impaired
hematopoietic differentiation and
BCR/ABL, TEL/PDGFR?, N-RAS and K-RAS mutations,
c-KIT mutations
RUNX1/MTG-8, PML/RAR?, MLL rearrangements
AML
FLT3 inhibitors Prenylation inhibitors
ATRA, ? HDAC inhibitors
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Other mechanisms
Loss of cell cycle control
p53 dysfunction p15, p16 cyclin dependent
kinase gene methylation
Genomic instability
Evolution of leukemic clone accumulation of
defects in oncogene and tumor supressor pathways
p53 subversion Fusion transcription factors
repress genes associated with DNA repair
Leukemia cell dissemination
TNF stimulates endothelium
Adhesion egress through vessels
selectin, cadherin, integrin
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Classification of AML - FAB

• M0 AML, minimal differentiation
• M1 AML, without maturation (15 20)
• M2 AML, with maturation (25 30)
• M3 acute promyelocytic leukemia (M3v
  hypogranular variant)
• M4 acute myelomonocytic leukemia (M4eo variant)
  (20-30)
• M5a acute monoblastic leukemia, poorly
  differentiated
• M5b acute monocytic leukemia, differentiated
• M6 acute erythroleukemia
• M7 acute megakaryoblastic leukemia

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Classification of AML - FAB

• M0 AML, minimal differentiation
• M1 AML, without maturation (15 20)
• M2 AML, with maturation (25 30)
• M3 acute promyelocytic leukemia (M3v
  hypogranular variant)
• M4 acute myelomonocytic leukemia (M4eo variant)
  (20-30)
• M5a acute monoblastic leukemia, poorly
  differentiated
• M5b acute monocytic leukemia, differentiated
• M6 acute erythroleukemia
• M7 acute megakaryoblastic leukemia

OBSOLETE? Better traditional
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AML, WHO classification

• AML with recurrent genetic abnormalities

AML with t(821) (RUNX1/MTG-8) AML with abnormal
BM eosinofils Inv (16) or t(1616)
(CBF?/MYH11) APL with t(1517) (PML/RAR?) and
variants AML with 11q23 (MLL) abnormalities

• AML with multilineage dysplasia

• AML and myelodysplastic syndromes, therapy
  related

without maturation

• AML minimally differentiated
• Acute basophilic leukemia

with maturation
myelomonocytic
etc.

• Acute panmyelosis with myelofibrosis

• Myeloid sarcoma
• AML not otherwise categorized
• Acute leukemia of ambigous lineage
  (undifferentiated, bilineal, biphenotypical)

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AML M1
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AML M2
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AML M2
Auers rod
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APL (AML- M3 promyelocytic)
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APL (AML- M3 promyelocytic)
Auers rods
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AML M3v
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AML M5
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AML M6
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Diagnosis of AML - symptoms

• general symptoms brief infection-like
  illness, fatigue, malaise, progressive skin
  infection, anorexia, diffuse bone tenderness
  (25)
• symptoms from pancytopenia (anemic syndrom,
  infections, easy bruises, bleeding, etc.)
• Physical examinantion pallor, sites of infection
  especially in mouth (oozing from gums) and ENT
  region, skin petechiae.

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Diagnosis of AML, special consideration

• Splenomegaly rare
• Lymphadenopathy rare
• CNS involvement 5-7
• Chloromas (myeloblastic sarcomas) M1, M2
• Skin involvement 10
• Metabolic and electrolyte derrangement

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Making the diagnosis of AML
Peripheral blood finding leucocytosis with
blasts anemia, thrombocytopenia in 90 10
pancytopenia (or normal leukocyte count with
blasts)
BM aspiration ( biopsy if dry tap)
Morphology (Giemsa) cytochemistry
cytogenetics
Molecular cytogenetics (FISH)
FACS
Molecular biology
FLT3
PML/RAR? !!
Other RUNX1/MTG8
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AML risk assesment
Risk Chromosomal aberation Prognosis
Low t(1517), inv(16), t(1616), t(821) CR 90 OS in 5 years65
Standard Normal karyotype or aberation non standard, non- high risk CR 80 OS in 5 years 30-40
High -7, -5, -5q, abnormity 3q, abnormity 11q23 and complex karyotyp ( 3 cytogenetic aberations) CR 60 OS in 5 years 10
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Treatment of AML
General strategy and stratification
AML ? 65 years
AML gt 65 years
Acute promyelocytic leukemia (APL)
Intensive chemo with curative intent - BMT
ATRA chemotherapy
Palliative approach
Special consideration supportive care
Overal survival (OS) in AML 40 in 5 years
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Acute promyelocytic leukemia
Low risk Leuko ? 10 x109/l PLT ? 40 x109/l
Interm. risk Leuko ? 10 x109/l PLT 40 x109/l
High risk Leuko 10 x109/l
Common induction ATRA 45mg/m2 Idarubicin
3x consolidation with idaribucin, mitoxantrone
3x consolidation with idaribucin, mitoxantrone
ATRA
3x consolidation with idaribucin, mitoxantrone
ATRA
PML/RAR? neg
PML/RAR? pos
BMT
2 years maintenance ATRA 6-MP MTX
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AML, non-APL, treatment, age ? 60
Induction Idarubicin ID ARA-C or Daunorubicin
ARA-C
Low risk cytogenetics low risk 1. CR
(FLT3/ITD neg)
IM risk cytogenetics normal 1. CR (FLT3/ITD
neg)
High risk risk cytogenetics or no 1. CR (FLT/ITD
)
Consolidation HD ARA-C Mitoxantrone
3-4 consolidation treatment with HD ARA-C
Allogeneic BMT
3 consolidation treatment with HD ARA-C
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Acute lymphoblastic leukemia
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Epidemiology, etiology of ALL
Incidence 1,1 1,4/100 000 Double peak
incidence 4 years and 50-80 Etiology not
known Risks Inherited chromosomal aberrations
21, Fanconis anemia, Blooms sy, etc. External
factors alkylating agents, inhibitors of
topoisomerase II (etoposide), organic dissolvents
EBV/plasmodium malariae mature ALL (Burkitts
lymphoma) HTLV I adult T leukemia/lymphoma
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Patophysiology of ALL
ALL is derived from morphologically immature
precursors of lymphocytes Primarily involves
bone marrow Infiltrates lymph nodes, spleen,
liver, testicules, other parenchymatous orgáns,
CNS. T- ALL involves more frequently mediastinal
lymph nodes B- ALL spleen, liver Cytogenetics
t(9,22) Ph chromosome, other t(411),
t(821), t(28), t(814)
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ALL classification

• 
  FAB
• L1 small uniform blasts, narrow belt of
  cytoplasma (T-ALL, pediatric ALL)
• L2 heterogenous blasts, often irregular nucleus
• L3 large uniform blasts, often vacuolised
  cytoplasm, somtime Burkitts like

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ALL classification

• 
  FAB
• L1 small uniform blasts, narrow belt of
  cytoplasma (T-ALL, pediatric ALL)
• L2 heterogenous blasts, often irregular nucleus
• L3 large uniform blasts, often vacuolised
  cytoplasm, somtime Burkitts like

Today obsolete, does not have influence on the
management of ALL
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ALL clinical course

• Symptoms from bone marrow failure and organ
  infiltration dominate
• fatigue, weakness, breathlessness,
  tinnitus,colapses.. anemic sy
• Fever, chills. Infections in neutropenia
• Bleeding mucosal, nasal, hematomas,
• Usualy no serious coagulation lab. abnormalities
• Other
  symptoms
• Hyperviscosity
• Lymphadenopathy, splenomegaly
• Relativly frequent CNS infiltration

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ALL making diagnosis

• Blood counts leukocytosis blasts gt20,
  leukopenia, anemia, thrombocytopenia.
• Bone marrow smear blasts gt20, PAS positivity
• Flow cytometry CD19, and/or CD79a and/or
  CD22, or CD3, CD33-,
• Molecular biology bcr/abl, MLL-AF4
• Cytogenetics t(922), t(411), polyploidy, etc.
• RTG PS (CT) mediastinal involvement
• lumbar punction (MRI)

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ALL prognostic groups
Standard risk leuko lt 30x109/l, pro- or pre-B
ALL, leuko lt 100x109/l if T-ALL. No Ph
chromosome, nor t(411), complete remmission in 3
weeks after start of chemotherapy High risk
t(411) or MLL-AF4 positivity Very high risk
t(922) or bcr/abl positivity Ph chromosome
t(922)(q34q11)
Fusion protein bcr/abl
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ALL example of treatment (CALGB)
Induction therapy Daunorubicine,
cyclophosphamide, Vincristine, L- asparaginasae
Prednisone (Dexametason) Consolidation therapy
daunorubicine, higher dose of cyklophosphamide
(T-ALL), ARA-C, Intensification methotrexate,
ARA-C CNS LP 3 methotrexate i.t., or MTX,
HCT, ARA-C. Radioterapy of CNS 24 Gy Maintenance
th 6-mercaptopurin (6-MP) methotrexate Total
therapy 24 MONTHS
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ALL treatment example Hyper C VAD HD
MTX/ARA-C
Odd cycles (1,3,5,7) Cyklophosphamide 300mg/m2
i.v. á 12 h, together 6 dosis
MESNA Vincristine 2mg i.v. day 4 a
11 Doxorubicine 50mg/m2 24 h infusion day
4 Dexamethasone 40mg i.v. D 1-4, D 11-14 G-CSF
10ug/kg s.c. 24 hours after the completion of the
therapy daily untill ANC gt 1,0 x109/l
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ALL treatment example Hyper C VAD HD
MTX/ARA-C
Even cycles (2,4,6,8) Methotrexate 1g/m2 24 hod
infuze Den 1 ARA-C 3g/m2 i.v. á 12 hod den 2 a 3
(celkem 4 dávky) G-CSF 10ug/kg s.c. 24 hours
after the completion of the therapy daily untill
ANC gt 1,0 x10(9)/l
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Imatinib mesylate (Glivec)

• first specific TKI (TyrosineKnase Inhibitor) of
  bcr/abl
• mechanism occupation of the binding site for
  ATP
• first significant breakpoint in the CML
  management
• 1. 90,3 alive after 54 months on imatinib
• 2. Tolerance better than that of interferon ?
  ARA-C or
• allogenneic stem cell transplantation
  (previous standard
• treatment of chronic phase of CML)

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Imatinib mesylate toxicity and adverse events

• Not serious in general in monotherapy
• mostly in 1. year of the therapy
• 3. 4. grade of toxicity
• hematologic neutropenia (16,7 ),
  thrombocytopenia (9,8 ), anemia (4,7 )
• liver toxicity liver tests elevation 5,2
• Other (16,5 ) edema, fluid retention,
  parestesia, skin rash and induration

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Ph ALL results of treatment without imatinib

• 3-5 children and 20-30 adults with ALL
• Remmission after induction therapy až 80
• Relaps 85
• 5 year survival lt 10 adults (25-30 children)
• longer EFS in adults after HSCT 28-40
• Relaps rate after HSCT up to 50
• Mortality after alloHSCT up to 30
• Výsledky lécby Ph (bcr/abl) ALL jsou stále
  neuspokojivé.

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Imatinib in the treatment of ALL
Imatinib In vitro synergy antracyclins,
vincristine anad ARA-C Imatinib monotherapy in
relapsing PhALL response , but short Imatinib
monotherapy in bcr/abl reoccurence after allo
HSCT good response
The highest effectiveness may be expected in the
combination therapy as part of induction or
consolidation treatment
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Imatinib in combination with Hyper C-VAD
HDMTX/ARA-C
Scheme D1-D14 Imatinib 400mg p.o. of each
cycle Odd cycles Hyper C-VAD MESNA Even
cycles HD mehtotrexate D1 Leukovorin HD ARA-C
D2,3 G-CSF 24hod after the end of the cycle
10ug/kg s.c.
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Imatinib in Ph ALL, conclusion

• Imatinib in combination with chemoterapy results
  induces high remission rate and cca 70 patients
  achieve molecular remission.
• Promissing strategy but need for further
  confirmation (higher toxicity in older patients,
  etc.) .
• Indication for allogenneic HSCT in 1. remmission
  if suitable donnor available remains.