CML Treatment Failure:

More Threatening Than

It Appears

Mutation Testing

Content

• Mutations and treatment failure

• Single mutations

• Compound mutations

• Mutation testing

• Guideline recommendations

• Summary

2

Mutations and

Treatment Failure

BCR-ABL1 mutation can confer TKI resistance and

is a mechanism of treatment failure in CML

4

1. Soverini S, et al. Clin Cancer Res. 2006;12:7374-7379. 2..Cortes J, et al. Blood. 2007;109:3207-3213. 3. Bixby D, et al. Leukemia. 2011;25:7-22. 4. Bowlin SJ, et al. ASCPT 2012. 5. Quintás-Cardama A, et al. Cancer Control. 2009;16:122-131. 6. Valent P. Biologics. 2007; 1:433-438. 7. Corbin AS, et al. J Clin Invest. 2011;12:396-409.

Resistance

BCR-ABL1 Mutation

(~40%)1,2

Activation of SRC family kinases3

Drug binding, inadequate drug concentration, or

epigenetic modifications3

Drug-drug interactions4

Increased drug efflux or influx3,5

Inability of TKIs to eradicate mutant

stem cells or cross the blood-brain barrier6,7

BCR-ABL1 gene amplification3

BCR-ABL1 independent

BCR-ABL1 dependent

Others

(~60%)

How do BCR-ABL1 mutations emerge?

5

Sensitive

to TKI

Resistant

to TKI

Treat with TKI

Sensitive

to TKI

Resistant

to TKI

1. Soverini S, et al. Blood. 2011;118:1208-1215. 2. Sierra JR, et al. Molec Cancer. 2010;9:75. 3. Bauer RC, et al. Clin Cancer Res. 2013;19: 2962-2972.

• Mutations can occur in cancer cells where genetic instability is high and

accumulation of further abnormalities is likely1,2

• TKI therapy can select for BCR-ABL1 mutations in unstable cancer cells

– Small cell populations containing mutations may have a survival advantage

during TKI therapy and emerge later as the dominant clone1,2

– In an in vitro simulation, sequential use of TKIs increases the probability that

mutations will arise, and the sequence of TKIs used may influence the types

of mutations that emerge3

Mutation-related TKI resistance is

common in patients with CML

6 Ai J, et al. Ther Adv Hematol. 2014;4:107-120.

Up to one in five newly-diagnosed CP-CML patients who start first-line TKI

therapy will develop a BCR-ABL1 mutation

Physicians do not always test for mutations

according to guideline recommendations

7

• According to a 2010 survey of 507 physicians treating

patients with CML1*

• Many clinicians do not appreciate the role of mutation

analysis in the overall management of CML2

Nearly half would not

test for BCR-ABL1 KD

mutations in patients not

achieving MMR two years

after the initiation of

TKI therapy1

9% indicated that they

were unfamiliar with, never

ordered, or did not have

access to the test for

BCR-ABL1 KD mutations1

*Prospective United States-based, noninterventional, cross-sectional study conducted through an online survey in December 2010. 1. Kantarjian HM, et al. Clin Lymphoma Myeloma Leuk. 2013;13:48-54. 2. Ai J, Tiu RV. Ther Adv Hematol. 2014;5:107-120.

Single Mutations

Over 90 BCR-ABL1 point mutations have

been identified that affect sensitivity to certain TKIs

9

1242T M244V

L248V

G250E/R

Q252R/H

Y253F/H

E255K/V

D276G

T277A

E279K

V280A

V289A/I

F311L/I

T315I

F317L/V/I/C

Y320C

L324Q

F359V/I/C/L

D363Y

L364I

A365V

A366G

L370P

E373K

V371A

S417F/Y

I418S/V

A433T

S438C

E450K/G/A/V

E453G/K/V/Q

E459K/V/G/Q

M237V E258D

W261L

L273M

E275K/Q

E292V/Q

I293V

L298V V299L

Y342H

M343T

A344V

A350V

M351T

E355D/G/A

V379I

A380T

F382L

L384M

L387M/F/V

H396P/R/A

M388L

Y393C

A397P

M472I

P480L

F486S

E507G

P-loop SH3

contact SH2

contact A-loop

Data from patients resistant to first-generation TKI therapy, collated from 27 studies

published between 2001 and 2009.

Star indicates amino acid position reported to be directly involved in first-generation TKI binding via hydrogen bonds or

van der Waals interactions.

Soverini S, et al. Blood. 2011;118:1208-1215.

Spectrum and frequency of BCR-ABL1

KD mutations recovered after TKI therapy

10

Lighter color corresponds to the first amino acid change; darker color corresponds to the second amino acid change,

if applicable. Cortes J, et al. Blood. 2007;110:4005-4011.

Higher number of mutations were associated

with poor survival and 4 year EFS

11

• According to a single-center study of 207 patients with chronic

phase, accelerated phase, or blast phase CML who failed

first-line TKI, those with >1 BCR-ABL1 mutation exhibited

worse response rates and long-term outcomes with TKI

therapy compared with those with ≤1 BCR-ABL1 mutation.

Among patients with 0, 1, or >1 BCR-ABL1 mutation1:

– 4-year EFS were 56%, 49%, and 0%, respectively (P=0.02)

– Overall survival rates were 91%, 69%, and 75%,

respectively (P=0.13)

1. Quintas-Cardama A, et al. Haematologica. 2011;96(6):918-921.

NCCN Clinical Practice Guidelines In Oncology (NCCN

Guidelines®) treatment recommendations

based on BCR-ABL1 mutations

12

Mutation present 2L and subsequent therapy options

Y253H DAS, BOS

E255K/V DAS, BOS

F359V/C/I DAS, BOS

F317L/V/I/C NIL, BOS

V299L NIL

T315A NIL, BOS

T315I PON, OMA, HCT, or clinical trial

Adapted with permission from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for Chronic Myeloid Leukemia V.2. 2017. © 2017

National Comprehensive Cancer Network, Inc. All rights reserved. The NCCN Guidelines® and illustrations herein may not be reproduced in any form for

any purpose without the express written permission of NCCN. To view the most recent and complete version of the NCCN Guidelines, go online to

NCCN.org. The NCCN Guidelines are a work in progress that may be refined as often as new significant data becomes available. NCCN makes no

warranties of any kind whatsoever regarding their content, use or application and disclaims any responsibility for their application or use in any way.

ELN guidelines: Treatment recommendations

based on BCR-ABL1 mutations

13

In vitro sensitivity based on select BCR-ABL1 mutations*

Mutation present Second-generation TKI IC50, range (nM)

NIL DAS BOS PON

Y253H 450-1300 1.3-10 NA 6.2

E255K 118-566 5.6-13 394 14

E255V 430-725 6.3-11 230.1 36

F359V 91-175 2.2-2.7 38.6 10

F317L 39.2-91 7.4-18 100.7 1.1

F317V 350 NA NA 10

V299L 23.7 15.8-18 1086 NA

T315A NA 760 NA 1.6

T315I 697 to >10,000 137 to

>1000 1890 11

Baccarani M, et al. Blood. 2013;122:872-884.

*Per the ELN guidelines, one factor to take into consideration when considering switching TKIs is the presence and type of mutation.

The probability of TKI-resistant mutations may

increase as the line of therapy increases and CML

disease progresses

14 1. Bauer RC, et al. Clin Cancer Res. 2013;19:2962-2972. 2. Soverini S, et al. Blood. 2011;118:1208-1215. 3. Soverini S, et al. Blood. 2009;114: 2168-2171.

• Although patients may develop resistance to TKI therapy at any time,

the risk of developing additional mutations may increase as patients

move to later lines of TKI therapy and as the disease progresses1

Up to 80%

of patients with BP-CML

have mutations2

83% of first-generation

TKI-resistant patients who

relapsed while on a second-

or third-line TKI experienced

an emergence of newly

acquired BCR-ABL1

mutations3

Compound Mutations

Compound mutations

16

• Treatment with multiple TKIs may select for compound mutations (≥2 mutations in the same

BCR-ABL1 molecule) that confer resistance to multiple TKIs1,2

• In vitro data suggest that compound mutations can be highly resistant to certain TKIs3

BCR-ABL1 compound mutations1

1. Khorashad JS, et al. Blood. 2013;121:489-498. 2. Shah NP, et al. J Clin Invest. 2007;117:2562-2569. 3. O’Hare T, et al. Nat Rev Cancer. 2012;12:513-526.

CML cells

Sequential therapy may increase the

probability of compound mutations1

17 1. Shah et al. J Clin Invest. 2007;117:2562-2569.

Mutation Testing

A variety of mutational analysis

techniques exist

19

Abbreviations: AP, accelerated phase; BP, blast phase; CP, chronic phase.

Method Sensitivity Pros Cons

Direct (Sanger) 15-25%

• Mutation characterization

• Semiquantitative

• Bidirectional conformation of

mutations

• Least sensitive (but sensitive enough for general

use)

High Resolution

Melt (HRM)

analysis

5-10%

• High throughput

• Cost effective

• DNA Spiking not required

• No additional steps required

after the PCR step

• Ideal for large scale screening

• Unable to characterize the mutations

• Optimal function requires small amplicon size

which implies several (4-5) PCR reactions

Pyrosequencing 5%

• High sensitivity and specificity

• Quantitative

• Internal quality and negative

controls

• Short read length

• Prior mutations knowledge is required

• Labor intensive

Denaturing High

Pressure Liquid

Chromatography

(DHPLC)

1%

• High throughput

• Cost effective

• Good for large scale studies

• Unable to characterize the mutations

• Wild Type DNA Spiking required

• Prone for contamination

• Requires several PCR amplifications to obtain

amplicons of ideal size

• Occurrence of non-specific peaks making data

interpretation difficult

Allele-specific,

oligonucleotide

(ASO)-PCR

0.001-1%

• High sensitivity

• Quantitative

• Easy to perform

• Sensitivity could be compromised by closely

located mutations

• Requires prior knowledge of mutations

• False positives issue

• Labor intensive if screening for multiple mutations

Alikian M, et al. Am J Hematol. 2012; 87:298-304.

Sen

sit

ivit

y

Clinical conditions and the presence

of BCR-ABL1 mutations

20

Mutation analysis in 399 patients with CP-CML receiving first-line, first-generation TKI

Clinical condition Patients tested for

mutations, n Presence of ≥1 BCR-ABL1

mutations (%)

Failure 166 45 (27)

No CHR at 3 mo 16 3 (19)

No CyR at 6 mo 9 1 (11)

No PCyR at 12 mo 24 4 (17)

No CCyR at 18 mo 36 6 (17)

Loss CCyR 49 15 (31)

Loss CHR 32 16 (50)

Suboptimal 233 11 (5)

No CyR at 3 mo 15 1 (7)

No PCyR at 6 mo 20 1 (5)

No CCyR at 12 mo 51 5 (8)

No MMR at 18 mo 52 0 (0)

Loss MMR (but not CCyR) 95 4 (4)

Soverini S, et al. Blood. 2011;118:abstract 112.

Sanger sequencing is the most

extensively used technique

• Direct (Sanger) sequencing is currently the most extensively

used technique to detect BCR-ABL1 mutations1,2

– Least sensitive method, but sufficient for general use

– Several contract labs offer this service

• Sanger can be useful for predicting the best course of

treatment for TKI-resistant patients and for monitoring

resistant mutations in subsequent treatment settings2,3

21 1. Baccarani M, et al. Blood. 2013;122:872-884. 2. Alikian M, et al. Am J Hematol. 2012; 87:298-304. 3. Soverini S, et al. Blood. 2011;118:1208-1215.

Sanger sequencing may not detect all

mutations present

10

F359C

E255K

Y253H

E255V

F317L

F359V

T315I

Mutations detectable by Sanger

sequencing (n=169)

Low-level mutations detectable by mass-

spectrometry assay only (n=132)

Frequency of mutations (%)

4 2 6 0

22 Parker WT, et al. J Clin Oncol. 2011;29:4250-4259.

• Mutation testing performed

after failure with a first-

generation TKI prior to

treatment with second-

generation TKIs (n=220)

• Mutations that would

influence therapeutic

decisions after failure with

a first-generation TKI were

found in more patients with

mass spectrometry than

direct sequencing

(32% vs 23%; P=0.03)

8

Low-level mutations can influence failure-free survival

in CP-CML patients treated with second-generation TKIs

after failure with a first-generation TKI

23

Pro

babili

ty o

f fa

ilure

-fre

e s

urv

ival (

%)

0 2 4 6 8 10 12 14 16 18 20 0

20

40

60

80

100

Low-level mutations by

mass spectrometry (n=6)

Mutations by sequencing (n=9)

Months since start of second-line therapy

No mutations (n=38)

Parker WT, et al. J Clin Oncol. 2011;29:4250-4259.

NGS provides a higher level of sensitivity to detect

clinically relevant BCR-ABL1 mutations that are not

detected by Sanger sequencing1

24 1. Molecular MD. http://www.molecularmd.com/case-studies/next-generation-sequencing-ngs-of-bcr-abl1-kinase-domain. Accessed December 2015. 2. Baccarani M et al. ASCO. 2014:167-175. 3. Shuen A, Foulkes WD. Curr Onc. 2010;17(5):39-42.

• NGS performs ultra-deep sequencing (UDS) of the

BCR-ABL1 kinase domain2

– UDS provides increased sensitivity and dissects qualitatively

and quantitatively the clonal texture of the mutated BCR-ABL1

positive subpopulations2

• NGS is comprehensively selective for mutations that likely

contribute to disease pathogenesis3

– For example, NGS identifies potential driver mutations (genetic

alterations that provide the cell with a survival advantage),

while filtering out passengers (mutations that can be expected

to have no effect on cell survival)3

NGS detects more mutations than

Sanger sequencing

25

51 49

10

39

61

23

0

10

20

30

40

50

60

70

No mutations Mutations ≥2 mutations

Pro

po

rtio

n o

f p

ati

en

ts, %

Sanger

NGS

Baseline mutation status. 267 patients with CP-CML resistant/intolerant to at least 2 prior TKIs or with

the T315I mutation.

Deininger MW, et al. Blood. 2016;127:703-712.

Deep sequencing (DS) may detect mutations

earlier than conventional sequencing

26

Soverini S, et al. Blood. 2014;124: Abstract 815.

• 51 patients treated with second-generation TKIs for a median of 9

months following failure of a first-generation TKI acquired BCR-ABL1

mutations detected by conventional sequencing

– Previously collected samples were analyzed for mutations using DS

• In 23 patients (45%), DS identified mutations that may confer

resistance following failure of a first-generation TKI

– Median interval between detection by DS and conventional sequencing:

3 months (range, 1-9 months)

• Response status (as per ELN 2013 guideline) at the time of mutation

detection by DS was Optimal (n=1), Warning (n=13), Failure (n=4);

5 patients had mutations at baseline (i.e., after failure of a first-

generation TKI)

• Results suggest that mutation testing using DS may be useful for

patients with Warning during second-line therapy

Guideline Recommendations

Mutational analysis may provide additional

information for patients with inadequate response

28

• The presence of mutations remains a consideration when making treatment

decisions1

• Routine monitoring of BCR-ABL1 transcripts, in conjunction with cytogenetic

evaluation, provides important information about long-term disease control in

patients with CML2

• National Comprehensive Cancer Network® (NCCN®) and ELN guidelines do not

recommend a specific technique1,2

Recommendations on When to Perform Mutational Analysis

ELN1,3:

• In case of treatment failure or

progression to AP or BP

NCCN2:

• CP-CML

- Failure to reach response milestones

- Any sign of loss of response (defined as hematologic or

cytogenetic relapse)

- 1-log increase in BCR-ABL1 transcript levels and loss of

MMR

• Disease progression to AP or BP

IS, International Scale. 1. Baccarani M, et al. Blood. 2013;122: 872-884. 2. Referenced with permission from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for Chronic Myeloid Leukemia V.2.1017. © National Comprehensive Cancer Network, Inc. 2017 All rights reserved. Accessed January 20, 2017. To view the most recent and complete version of the guideline, go online to NCCN.org. 3. Soverini S, et al. Blood. 2011;118:1208-1215.

Analysis of mutations in CP-CML

• Mutations studies can help make treatment decisions in the

event of cytogenetic or hematologic relapse1,2

– Certain mutations confer resistance to certain TKIs1,2

• There is currently no role for mutation analysis at diagnosis

or in patients with adequate response to therapy1,2

29

1. Referenced with permission from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for Chronic Myeloid Leukemia V.2.1017. ©

National Comprehensive Cancer Network, Inc. 2017 All rights reserved. Accessed January 20, 2017. To view the most recent and complete version of

the guideline, go online to NCCN.org. 2. Soverini S, et al. Blood. 2011;118:1208-1215.

Treatment options for TKI-resistant CML

1. Ai et al. Ther Adv Hematol. 2014;5:107-120. 2. Baccarani M, et al. Blood. 2013;122:872-884. 3. Referenced with permission from the NCCN Clinical

Practice Guidelines in Oncology (NCCN Guidelines®) for Chronic Myeloid Leukemia V.2.1017. © National Comprehensive Cancer Network, Inc. 2017

All rights reserved. Accessed January 20, 2017. To view the most recent and complete version of the guideline, go online to NCCN.org. 4. Baccarani M,

et al. Ann Oncol. 2012;23(Suppl7):vii72-7. 30

• The results of mutational analysis is one of many factors

(eg, efficacy, safety, patient comorbidities, cost) in making

treatment decisions1

• For patients with TKI-resistant CML, potential treatment

options include2-4:

– Alternate TKI

– Protein synthesis inhibitors

– Allogeneic hematopoietic stem cell transplantation (HCT)

– Clinical trial

Summary

1. Soverini S, et al. Clin Cancer Res. 2006;12:7374-7379. 2..Cortes J, et al. Blood. 2007;109:3207-3213. 3. Bixby D, et al. Leukemia. 2011;25:7-22. 4. Bowlin SJ, et al. ASCPT 2012. 5. Quintás-Cardama A, et al. Cancer Control. 2009;16:122-131. 6. Valent P. Biologics. 2007; 1:433-438. 7. Corbin AS, et al. J Clin Invest. 2011;12:396-409. 8. Referenced with permission from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for Chronic Myeloid Leukemia V.2.1017. © National Comprehensive Cancer Network, Inc. 2017 All rights reserved. Accessed January 20, 2017. To view the most recent and complete version of the guideline, go online to NCCN.org. NCCN makes no warranties of any kind whatsoever regarding their content, use or application and disclaims any responsibility for their application or use in any way. 9. Kantarjian HM, et al. Clin Lymphoma Myeloma Leuk. 2013;13:48-54. 10. Ai J, Tiu RV. Ther Adv Hematol. 2014;5:107-120. 11. Baccarani M, et al. Blood. 2013;122: 872-884. 12. Soverini S, et al. Blood. 2011;118:1208-1215.

Summary

• Mutations in the BCR-ABL1 kinase domain may emerge at any time

during TKI therapy and confer treatment resistance1-7

• The type of mutation can help determine the most appropriate

subsequent therapy8

• Despite its recommendation in current treatment guidelines, mutational

analysis is not always performed in patients with suspected TKI

resistance8,9

• Mutational analysis, as recommended in current treatment guidelines,

should be considered a standard part of monitoring CML patients

treated with TKIs8,10-11

32

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