Bcr-Abl Kinase Domain Mutations

Martin Martin MMüüllerller

Primary resistance Relapse/ progression

Early chronic phase

Late chronic phase

Accelerated phase

(600 mg/d)

Blastic phase

(600 mg/d)

0

25

50

75

100

4 7

20

4

24

60 66

93

Frequency of imatinib resistance within 3 years

Perc

ent

Hochhaus and La Rosée Leukemia. 2004

Observations associated with resistance

Genomic amplification / overexpression BCR-ABL Mutations Clonal evolution / aneuploidy Pharmacologic mechanisms Association with AGP-levels? Low MRP-1 levels independent prognostic factor (Lange et al.) Decrease of intracellular imatinib-levels in PGP+ patients (Illmer et al.)

BCR-ABL mutations (%) Clonal evolution (%) Combination (%)

10/20 (50)

15/29 (52)

5/17 (29)

13/21 (62)

8/16 (50)

2/9 (22)

10/33 (30)

16/22 (73)

4/17 (24)

33/74 (45)

39/67 (58)

11/43 (26)

Patients With Hematologic Resistance/Relapse

Chronic phase (n=35)

Accelerated phase (n=33)

Blastic phase (n=66)

All (n=134)

Lahaye et al. Cancer. 2005

Molecular - cytogenetic causes of resistance

M24

4V

G25

0E

Y253

F/H

E255

K/V

F311

L

T315

I

F317

L

M35

1T

E355

G

F359

V

V379

I

L387

M

H39

6R/P

BiochemicalCellular

Mutant BCR-ABL have increased IC50 values for imatinib mesylate

wt

5

10

15

20

Fold

incr

ease

in IC

50

Corbin et al. Blood. 2003

Resistance to imatinib: 31 BCR-ABL mutations

in 25 amino acids (n=126)

P-Loop Kinase domain A-Loop

M244V

Q252H Y253F/H

E255K/V

T315I M351T E355G

L387F/M

H396R

L248V F317L

E279K F359C/V A397P

T277A D267G

V379I

G250E L324Q

F311L

M388L

A344V A350V

E450G/K E459K

Polymorphisms: K247R T315T E499E

Survival after imatinib failure Initial Australian experience

Branford et al. Blood. 2003.

P-Loop: Highly conserved area

250 252 253 255

G G G Q Y G E V

E H F K

R H V

15 0 5 10 0

20

40

60

80

100

% S

urvi

ving

Months since mutation detected

p=0.0024

n=27

P-loop, n=13

Non P-loop, n=14

Survival after imatinib failure Current experience

0 1 2 3 4 0

25

50

75

100

T315I, n=14

Others, n=53

P-Loop, n=38 p=0.031 for T315I

Prob

abili

ty o

f sur

viva

l (%

)

Years

n=105

Imatinib 400-600 mg/d

Hydroxyurea

0 1 2 3

0

25

50

75

100

% M

utat

ed B

CR

-AB

L

Y253H, 100%

Years after start of imatinib mesylate

CML, chronic phase, IFN-resistant

Significance of P-loop mutations in CML

• 89 imatinib resistant pts in 5 French centers; 26 with P-loop mutations, 18 with T315I, 50 with other mutations → significantly worse survival from diagnosis

for P-loop or T315I mutations (p=0.014)

• 159 pts from MDACC: 52 mutations in 49 pts; 19 P-loop mutations median F/U 6 mo: 1/19 P-loop vs 4/30 non P-loop died; 15 mo surv 95% vs 83%

Nicolini. Leukemia. 2006 Apr 27 [Epub ahead of print] Jabbour. Blood 104; 288a, abst 1007, 2004

Indications for mutation screening

hematologic resistance / relapse

cytogenetic resistance / relapse

5-10-fold increase of BCR-ABL load

prior to therapy with alternative kinase inhibitors (nilotinib, dasatinib)

3-monthly intervals under therapy with nilotinib and dasatinib

Methods to detect and quantify BCR-ABL mutations

Specificity Sensitivity Sequencing unspecific 10-20% Restriction digest analysis specific ~2-5% D-HPLC unspecific 0.1-10% Allele specific PCR specific 0.01% Sequencing of clones unspecific 1-5%

D-HPLC: nested RT-PCR

Fragment A Fragment A: : BCR BCR ((ExonExon b b2 2 / / ee11) ) –– ABL ABL ((ExonExon 10/1110/11))

BCRBCR ABL ABL KinaseKinase Domain Domain ((AA AA 235235--509509))

B B ((AA AA 207207--324324))

C C ((AA AA 279279--414414))

D D ((AA AA 382382--517517))

11stst stepstep

22ndnd stepstep

50%

10%

1%

0.1%

normal BaF3BCR-ABL

100%

*

*

*

*

*

mutant BaF3T315I *

Sensitivity of D-HPLC

0.1-1%

Ba/F3 BCR-ABL E255K

M351T M244V G250E Q252H Q252R Y253F Y253H E255V F317L E355G F359V H396R F486S

T315I

0

0.2

0.4

0.6

0.8

1

1.2

0 1 5 10 50 100

Rel

ativ

e gr

owth

afte

r 48

ho

urs

of d

rug

expo

sure

Concentration of Dasatinib (nM)

Parental Ba/F3 cells

“wt” BCR-ABL

M351T

E255K

0 1 5 10 50 100

“wt” BCR-ABL

M351T

T315I

Dasatinib inhibits growth of 14/15 imatinib-resistant BCR-ABL-expressing Ba/F3 cell lines in vitro

Shah et al., Science, 2004

Conclusions • Frequency of imatinib resistance depends on the stage of CML.

• The major causes of resistance are BCR-ABL mutations and clonal evolution.

• The impact of early detection of mutated clones by sensitive assays requires prospective evaluation.

• Certain BCR-ABL mutations might especially impair prognosis, in particular on continuous imatinib therapy.

• Early elucidation of imminent resistance to kinase inhibitors might contribute to individualized therapy according to molecular data.