Myeloprolipherative diseases (MPD)

Chronic myeloid leukemia(CML)

Polycythaemia Vera (PV)

Essential thrombocythaemia (ET)

Primary myelofibrosis (MF)

Common signes: proliferation 1-3 cell lines with excess in peripheral blood, extramedullary haemopoiesis, bone marrow fibrosis

CML- historical milestones

1845: John Hughes Bennet (Edinburgh)

Rudolf Virchow (Berlín)

„Unusual“ cases of splenomegaly and blood „suppuration“ resulting in death 1872: Ernst Neumann: primary site is bone marrow

1882 LANCET: XXX Response of patient with splenomegaly to arsenic treatment

1973: BLOOD: J. Rowley: Ph chromosome… BCR-ABL 1992: FASEB Journal: A. Levitzki: tyrohosthins – small molecules to inhibit

ABL (future Tyrosine kinase inhibitors)

Scotish physicianWriter

Military awards & aristocratic state from Burs wars

Driver´s licence No. 1 UK

First downhill ski teacher in UK

Planned to build tunnel under the la Manche channel

Died 1930

…the findings suggest a causal relationshipbetween the chromosome abnormality observed and chronic granulocytic leukemia…

A minute chromosome in human granulocytic leukemia. Science 132, 1960, 1497.P.C. Nowell, D.A. Hungerford, University of Pennsylvania in Philadelphia

19196060

The ‘Philadelphia chromosome’

Philadelphia chromosome described in 1960

>95% of CML patients have the Philadelphia chromosome

Genetic fault, BCR-ABL, described in mid 80s

In previous studies:

The less Philadelphia chromosomes there are in the bone marrow - the longer you live

BCR/ABLdual fusion

t(9;22)(q34;q11)

9 der(9) 22 der(22)

ABL9q34

BCR22q11

BCR

ABL

ABL

BCR

FFRGTypical abnormal

9 9 22 22

ABL9q34

BCR22q11

BCR22q11

ABL9q34

RRGGNormal signal pattern

FISHFluorescent In Situ Hybridisation

Chronic myeloid leukaemia (CML)

Definition: myeloprolipherative clonal disease derived from a stem cell, bcr/abl rearrangement involved

Epidemiology: incidence 1/100 000

Etiology: unknown

Risk factors: radiation

Pathophysiology: gene rearrangment bcr, abl bcr/abl – tyrosinkinase. Cytogenetic marker: t(9;22) = Ph chromosome.

Course of the disease

Chronic phase: 50% patients diagnosed incidentialy: sweating, weight loss, pruritus, left hypochondrium pressure

Acceleration phase: symptoms stressed, treatment with decreased effect (need for increase effect)

Blastic phase: anaemic syndrome, infection, haemorhagia, left hypochondrial tension – splenomegaly

Diagnosis of CMLBlood counts:

Chronic phase: leukocytosis with left shift to myeloblasts

Acceleration phase: Number of blasts in peripheral blood or bone marrow: 10-30%, eosinophils and basophils: > 20%

Blastic phase: blasts >30%, blasts and promyelocytes > 50%

Bone marrow cytology: rich BM with majority of myelocytes (chronic phase)

Bone marrow - histology: different degree of fibrosis

Other: erythrocytosis, thrombocytosis, splenomegaly, hepatomegaly low ALP index in leukocytes, uric acid

CYTOGENETICS: Ph chromosome,

MOL. GENETICS: bcr/abl +

Treatment of CML

• Risk assessment according to Hasford´s index

• IMATINIB MESYLATE (GLIVEC): treatment of choice for 1.line in patients with low H. index

• Young patients (pod 45 let) with higher Hasford´s index and available HLA donnor: alogenneic stem cell transplantation

• Patients reafractory to GLIVEC or in relaps: allogeneic stem cell transplantation or new thyrosine kinase generation (dasatinib, nilotinib)

• Alternative and obsolete: Interferon-, hydroxyurea, busulfan

Imatinib mesylate (Glivec®)

• first specific inhibitor of tyrosinkinase bcr/abl (TKI)

• mode of action: occupies binding site ATP

• standard dose: 400mg/D p.o.

• 90,3% of patients survive after 54 months of therapy

Tolerance: very good

Side effects: swellings, rush, neutropenia

New TK inhibitors: dasatinib (SPRYCEL®), nilotinib (TASIGNA®)

CHRMCyRCCyR

% re

spon

ding

0

10

20

30

40

50

60

70

80

90

100

Months since randomization to Imatinib0 6 12 18 24 30 36 42 48 54 60 66

C u m u la tiv e B e s t R e s p o n s e a t 1 2 a n d6 0 m o n th s o n F irs t-lin e Im a tin ib

9 6 %

8 5 %

6 9 %

9 8 %9 2 %8 7 %

8 0 %8 4 %

Survival without AP/BC

Event-free Survival% w

ithou

t eve

nt

0

10

20

30

40

50

60

70

80

90

100

Months since randomization0 6 12 18 24 30 36 42 48 54 60 66

E v e n t-fre e S u rv iv a l a n d S u rv iv a lW ith o u t A P /B C o n F irs t-lin e Im a tin ib

A c tu a l E v e n ts6 .3 % A P /B C (n = 3 5 )5 .1 % lo s s o f M C yR (n = 2 8 )2 .5 % lo s s o f C H R (n = 1 4 )1 .6 % C M L -u n re la te d d e a th s (n = 9 )

8 3 %

(9 0 -9 6 )

(8 0 -8 7 )

E s tim a te d ra te a t 6 0 m o n th s (w ith 9 5 % C I)

9 3 %

S u rv iv a l W ith o u t A P /B C b y S o k a l G ro u p in P a tie n ts w ith C C y R o n F irs t- lin e Im a tin ib

n = 1 7 9 9 9 % n = 9 1 9 5 %n = 4 9 9 5 %

E s tim a te d ra te a t 6 0 m o n th s

p = 0 .1 6 p = 0 .0 9

p = 0 .2 0 0 (o v e ra ll)

Low riskIntermediate riskHigh risk

% w

ithou

t PD

to A

P/B

C

0

10

20

30

40

50

60

70

80

90

100

Months since randomization0 6 12 18 24 30 36 42 48 54 60 66

IRIS study – complete cytogenetic response

25%

9% 3%1%

12%14%

51%

63%69%

76%

p<0.001

Months after randomization

CML- terms definition Complete hematological response (CHR)

No cytogenetic response: >95% Ph+ Minimal cytogenetic response: 66-95% Ph+ Minor cytogenetic response: 36-65% Ph+ Parcial cytogenetic response (PCyR): 1-35% Ph+ Complete cytogenetic response(CCyR): 0% Ph+ MAJOR cytog. Resp.: CCyR + PCyR

MAJOR cyt. resp. : <0,1% RQ-PCR CMR: RQ-PCR neg.

Aim - imatinib tratment

Complete hematol. remision: 3m

Parcial cytogenet. remision: 6m

Complete cytogenet. remision: 12m

Major molecular response: 18m

Treatment failure in CML• Hematologic response

• Cytogenetic response

• Molecular response

• Rezistent mutants

No hematol. Response in 3m, loss of CHR any time

No cytogenet. Response in 6 m MCyR not completed in 12mCCyR not completed in 18mLoss of CCyRany time

NA

Detection of imatinib-rezistant variant of BCR/ABL any time

IMATINIB: treatment failure or suboptimal response

Primar rezistance Patients seldomly don´t reach CHR But : 20-25% do not reach CCyR

Secundary rezistance 20-25% pacients, who reach CHR and CCyR

but do not withold Molecular response? (>0,1%)?

Reasons for resinstance to (TKI) Farmakokinetics: biol. availability and blood levels differs

inter-individually

In future :dose according to blood levels

•Reasons for rezistence due to the clone1.Mutation Bcr-Abl2.Increased expression of Bcr-Abl3. increased expression ofMDR1 ( P-glykoprotein)4.Src family kinase activation5.„sleeping“ CML stem cells

BCR-ABL mutation

Highly resistent to imatinib, nilotinib, dasatainib: T315I

Mutation relatively resistant to nilotinib :

E255/KV

Mutation relatively rezistant to dasatinib:

F317L/I

Approach in case of imatinib rezistance

Not reaching or loss of CHR and/or CCyR

Not reaching major molecular response (?)

Imatinib 600-800mg/D Dasatinib

Nilotinib

Allogeneic HSCT

Acceleration phase and blastic phase AP CML: blasts 10-19%, basophilia ≥20%,

↑ splenomeglia, constitution sy, thrombocytosis + leukocytosis.

BP CML blasts ≥20%, chloroma

Allogeneic SCT IF- α, hydroxyurea, ARA-C

Dasatinib, Nilotinib

Nilotinib (Tasigna®) Molecule obtained by crystalization of imatinib 30-50x more active inhibition of Bcr-Abl and

most variants excepta T315I Based on results of studies phase II – indication

in patients with CP and AP CML resistant to imatinib (or intolerance to imatinib)

Minimal toxicity: hematologic toxicity, QT interval prolongation.

NilotinibNilotinib

Lipofile interactions instead of hydrogen bridges => smaller sensitivity towards

point mutations Bcr-Abl

More effective bonding to ATB binding site

ImatinibImatinib

ATP binding site

Hydrogen bridges with certain amino acids

Structure of nilotinib - optimaliztion of bonding and inactivation of ABL kinase domaine

Dasatinib (Sprycel®)

„multi target“ kinase inhibitor (Bcr-Abl, SRC, TEC, PDGFR, kit etc.)

cca 100x more active then imatinib , but inactive in T315I

Inactive in F317L variant Unable to eliminate sleeping CML stem cells

(CD34+CD38-) Toxic profile: hematological toxicity, fluidothorax

Bosutinib (SKI-606, Wyeth)

Effective in resistant variants: Y253F, E255K, atd.

Ineffective in T315I Ineffective inc-kit a PDGFR Very promising toxic profile (GIT,

hematological toxicity–only in advanced CML)

Other medicaments in CML treatment

heat shock proteinu 90 inhibitors Arsenic trioxide (Trisenox) Homoharringtonine Histondeacetylase inhibitors Farnesyltransferase inhibitors (RAS) Etc.

Polycythaemia vera (PV)Incidence: 0,5 – 0,8/100 000

Median: 60 years

Etiology: unknown in most cases (some: ionizing radiation)

Definition: myeloprolipherative clonal disease derived from a stem cell with major erythrocyte prolipheration

Patophysiology: > 25% erythrocyte mass, erythrocytosis, hyperviscosity, hyperuricaemia, trend to vessel complications

PV – diagnosis1. Exclude secondary and relative erythrocytosis

(ChOBPD, EPO doping, plasma depletion, etc.)

2. Exclude other MPDs (bcr/abl),endo EPO level

If JAK-2 negative, than

1. Erythrocyte mass > 25% above normal, practically: hematocrit in females > 0,56, in males: > 0,60.

2. Bone marrow: cytology only supportive, histology: degree of fibrosis + erythropoiesis prolipheration

3. BM cultivation in EPO presence, EPO level

JAK-2 mutation +

PV - treatment

1. Blood draw ( 300-500 cc (1x in 6 – 8 weeks). Aim: HCT < 0,45

2. If venepunction is not effective: Litalir (hydroxyurea) in older patients

3. Interferon alfa in younger patients and in women of fertile age

4. Anagrelide (Tromboreduktin)

All patients: antiaggregation therapy (Acetylsalic acid) if not contraindicated

Essential thrombocythaemia (ET)

Incidence: 0,1/100000

In majority clonal disease with accelerated proliferation of megakaryocyte line

Peripheral blood count: PLT > 600 x109/l, slight leukocytosis with a left shift.

Blood counts: 1. Bleeding (PLT > 1500 x109/l), 2. Thrombosis (PLT < 1500 x109/l). Thrombosis in atypical sites: v. jugularis, v. portae etc.

Dif. diagnosis: iron deficiency, reactive thrombocytosis (chron. Inflammatory diseases), tumors.

Treatment of ET

Risk factors: younger patients(< 60 let) without thrombosis or haemorrhagia in history: no therapy

Other:

1. Hydroxyurea in men, women after fertile age

2. Interferon - (3MU 3 x in a week) in women in fertile age

3. Anagrelide

Supportive treatment: Anopyrine, thrombocytapheresis.