Role of TP53 abnormalities in CLL

Overview

Sarka Pospisilova

Department of Internal Medicine – Hematology and Oncology, University Hospital Brno, Czech Republic

CEITEC - Center of Molecular Medicine, Masaryk Univerzity, Brno, Czech Republic

Complex

Karyotype

Chromosomal Aberrations

(FISH) TP53 mutations

IGHV gene

mutational

status del(11) trisomy 12 del(13) del(17)

Initial diagnosis Optional Yes Yes Yes Yes Yes Yes

Disease progression

/ Before frontline

therapy

Yes Optional Optional Optional

Yes,

unless

detected before

Yes,

unless

detected before

Yes,

unless

performed

before

Relapse / Before

subsequent

therapies

Yes Optional Optional Optional

Yes,

unless

detected before

Yes,

unless

detected before

Yes,

unless

performed

before

Prognostic

significance Yes

Yes, mainly

in elderly Yes Yes Yes Yes Yes

Predictive

significance

Yes

(BcR

Inhibitors)

Yes To be

clarified Yes Yes Yes

Yes

(FCR, BcR

Inhibitors)

Genetic prognostic and predictive factors in CLL

Pospisilova & Doubek, 2017, EHA BOOK „EDUCATIONAL UPDATES IN HEMATOLOGY“, modified

Types of p53 inactivation in tumor cells

P53 protein in coded by TP53 gene

localised in 17p13.1 (2 alleles)

P53 binds DNA as a tetramer

1) Wild-type p53 (both alleles)

2) Deletion of 17p13.1 locus (+ wt allele)

3) Mutation of TP53 gene (1 or 2 alleles)

4) Deletion of 1st allele + mutation of the 2nd allele

Haploinsufficiency – product of one allele is not sufficient for p53 protein function

Dominant - negative effect – 1 mutant protein in the tetramer influences the function of wt protein

Different mutations have different impact on p53 behavior

Types of TP53 gene mutations in tumors

www-p53.iarc.fr

CLL exhibits a disease-specific p53 mutation profile

CLL

all tumors

175 220 281

del GA

www-p53.iarc.fr

Zenz et al., Leukemia 2010

Antioncogene TP53 = "the guardian of the genome"

Conserves genetic stability by preventing genome mutation

TP53 gene is located on the short arm of chromosome 17 (17p13.1)

Tumors with p53 dysfunction - bad prognosis and poor treatment response

The p53 protein is crucial for regulation of the cell cycle arrest or apoptosis

TP53 dysfunction – del(17p) and/or TP53 mutation

The most important independent prognostic marker

High risk of progression within 1-2 years

Median overall survival 3-5 years

Important predictive marker (resistance to FCR, alkylating agents, purine analogues…)

Valid also in „novel mutation era“

New therapeutic options (inhibition of BcR signaling, Bcl2 inhibition)

TP53 dysfunction in CLL

Frequency of TP53 aberrations: Prognostic and predictive marker

Fre

qu

en

cy (

%)

Frequency of TP53 aberrations increases with advanced disease stage and therapy

Chromosomal aberrations are prognostically relevant in CLL

Döhner H, et al. The New England Journal of Medicine, 2000

12q trisomy

N = 325

Time (months)

Ove

rall

Surv

ival

(%

)

del 17p MS 32 months

del 11q

Normal

0

80

24 48 72 96 120 144 168

60

40

20

0

100

Isolated del13q

HIERARCHICAL MODEL OF CHROMOSOMAL ABNORMALITIES KARYOTYPE No. OF PATIENTS (%) 17p deletion 23 (7%) 11q deletion 56 (17%) 12q trisomy 47 (14%) Normal karyotype 57 (18%) 13q deletion as sole abnormality 117 (36%) Various abnormalities 25 (8%)

Types of TP53 defects in CLL – comparison of different studies

del(17p) sole

TP53 mutation

sole

Del(17p) &

TP53 mutation

Frequency increases during disease development

Related to previous therapy

Dicker et al. Leukemia 2009

Zenz et al. Blood 2008

Rossi et al. CCR, 2009 Malcikova et al. Blood, 2009

LRF CLL4 (Clb vs F vs FC)

Gonzalez et al. JCO, 2011

GCLLSG CLL4 trial (F vs FC)

Zenz et al. JCO, 2010

Independent prognostic impact of del(17p) and TP53 mutations

Analysis of both del(17p) by FISH and TP53 mutation recommmended!

CLL8 trial (FC vs FCR)

Stilgenbauer et al. Blood 2014

Stengel et al, Leukemia 2017

Development and testing of TP53 mutations in CLL

Overall survival according the TP53 mutation status in relapse

The risk of TP53 mutation acquisition at 5 years from diagnosis:

Untreated: 1%, Treated: 17%

P<0.0001

Malcikova et al., Leukemia 2015, Landau et al., Nature 2015, Rossi et al., Blood 2013, Ouillette et al., CCR 2013, Zenz et al., Blood 2008

When to test TP53 abnormalities?

At diagnosis (optional) Before 1st treatment Before subsequent treatment lines

Currently recommended methods:

Sanger sequencing with or without prescreening Next generation sequencing

Updated Recommendations – Leukemia 2018

Which material? Which exons? Methods – Sanger sequencing and Next Generation sequencing Interpretation and reporting

Recommendations for TP53 mutation analysis in CLL

http://www.ericll.org/eric-recommendations/

Manual for TP53 mutational analysis

Persistence

TREATMENT

Slow

expansion

RELAPSE 1 RELAPSE 2-n

NO TREATMENT

Small subclones detectable by NGS

TP53 wt

TP53 mut

Current recommendation – to report mutations present at >10%

Ultra-deep next generation sequencing allows detection of very small subclones – mutations present in <1% of cancer cells

QUESTIONS:

Should be patients with small subclones treated similarly to patients with dominant TP53-mutated subclone?

What detection sensitivity do we really need?

Minor TP53 mutated subclones expand mainly after therapy and are subjected to clonal evolution

Rossi et al., Blood 2014; Malcikova et al., Leukemia 2015; Landau et al., Nature 2015, Nadeu et al., Blood 2016, Amin et al., Clin Cancer Res 2016, Lazarian et al., Int J Canc 2016

Presence of minor subclonal TP53 mutation represents a warning – could expand during the disease course

Therapy provides a selection advantage for more aggressive clones

Rossi et al., Blood 2014

Minor subclonal TP53 mutations in CLL are clinically important

Rossi et al., Blood 2014

Small TP53 mutated subclones have the same unfavorable prognostic impact as clonal TP53 defects in CLL

TP53 mutant clones have negative impact on overall survival

Nadeu et al., Blood 2016

Clonal and subclonal mutations of TP53 and NOTCH1, clonal mutations of SF3B1, and ATM mutations in CLL have an impact on clinical outcome.

Clonal evolution in longitudinal samples occurs before and after treatment and may have an unfavorable impact on overall survival.

Quesada et al., BMC Medicine 2017

Many novel prognostically relevant genes in CLL …

Prognostic models combine cytogenetics and mutation analysis

N=26

(4.0%)

NOTCH1 M

SF3B1 M

BIRC3 M

del13q14

+12

TP53 M

del11q22-q23 MYD88 M

BIRC3 del del17p13

*

* * *

Rossi et al., Blood 2013

The integration of mutations and cytogenetic lesions improves the accuracy of survival prediction in CLL.

ERIC TP53 NETWORK

Thessaloniki

Brno

Uppsala/Stockholm

Ulm

Belfast

Paris

Novara

Salamanca

Copenhagen

Amsterdam

Bellinzona

11 Training Centres

3 Certifying Centres

ERIC TP53 Network www.ericll.org/pages/networks/TP53Network

Educational activities (workshops) and laboratory

certification of TP53 mutation analysis (already certified 125 centers from 25 countries)

ERIC Publications on TP53 analysis

Comprehensive profiling and analysis of TP53 mutation in CLL

TP53 mutation profile in chronic lymphocytic leukemia: evidence for a disease specific profile from a comprehensive analysis of

268 mutations. T Zenz, D Vollmer, M Trbusek, J Smardova, A Benner, T Soussi, H Helfrich, M Heuberger, P Hoth, M Fuge, T Denzel, S

Häbe, J Malcikova, P Kuglik, S Truong, N Patten, L Wu, D Oscier, R Ibbotson, A Gardiner, I Tracy, K Lin, A Pettitt, S Pospisilova, J Mayer, M

Hallek, H Döhner, S Stilgenbauer, European Research Initiative on CLL (ERIC): Leukemia, 2010.

ERIC recommendations on TP53 mutation analysis in Chronic Lymphocytic Leukemia.

Pospisilova S, Gonzalez D, Malcikova J, Trbusek M, Rossi D, Kater AP, Cymbalista F, Eichhorst B, Hallek M, Döhner H, Hillmen P, van Oers

M, Gribben J, Ghia P, Montserrat E, Stilgenbauer S, Zenz T.: Leukemia, 2012.

Assessment of TP53 functionality in chronic lymphocytic leukaemia by different assays; an ERIC-wide approach. Te Raa GD,

Malčiková J, Mraz M, Trbusek M, Le Garff-Tavernier M, Merle-Béral H, Greil R, Merkel O, Pospisilova S, Lin K, Pettitt AR, Stankovic T, van

Oers MH, Eldering E, Stilgenbauer S, Zenz T, Kater AP; European Research Initiative on CLL (ERIC): Brit. J. Haematology, 2014.

Innovation in the prognostication of chronic lymphocytic leukemia: how far beyond TP53 gene analysis can we go?

Pospisilova S, Sutton LA, Malcikova J, Tausch E, Rossi D, Montserrat E, Moreno C, Stamatopoulos K, Gaidano G, Rosenquist R, Ghia P;

European Research Initiative on CLL (ERIC): Haematologica, 2016.

ERIC Recommendations for TP53 Mutation Analysis in Chronic Lymphocytic Leukemia – Update on Methodological Approaches

and Results Interpretation. Malcikova J, Tausch E, Rossi D, Sutton LA, Soussi T, Zenz T, Kater AP, Niemann CU, Gonzalez D, Davi F,

Gonzalez Diaz M, Moreno C, Gaidano G, Stamatopoulos K, Rosenquist R, Stilgenbauer S, Ghia P, Pospisilova S.:, Leukemia (2018)

Stresa (Italy), Lake Magiorre

November 7-8, 2017

2nd ERIC Workshop on TP53 analysis in CLL

Clinicians

37%

Diagnosticians

51% Both

12%

Survey ERIC + Gilead: participants

• Questionnaires were collected

at the occasion of 2nd ERIC

workshop on TP53 analysis

in CLL (Stresa, Italy, November

2017)

• 140 participants composed of

more diagnosticians than

clinicians from 28 countries

N Mean

proportion of

patients (%)

95% CI (%)

For all patients with CLL: in what proportion of patients do you…

conduct sequencing for TP53 variants at diagnosis? 67 21 14, 29

conduct sequencing for TP53 variants before first-line treatment? 65 88 81, 95

conduct sequencing for TP53 variants in the relapsed/refractory

setting? 62 83 76, 91

For patients testing negative for del(17p): in what proportion of patients do you…

conduct sequencing for TP53 variants at diagnosis? 67 36 26, 47

conduct sequencing for TP53 variants before first-line treatment? 67 87 80, 95

conduct sequencing for TP53 variants in the relapsed/refractory

setting? 65 84 76, 92

Current practice

Clinical section

0

20

40

60

80

100

Stage of patient journey

% o

f re

spo

nd

en

ts (

N=

69

) Prior to initiation of first-line treatment

In parallel with testing for del(17p)

Prior to initiation of second or subsequent lines of treatment, only ifpatient was previously negative for del(17p) or a TP53 variantPrior to initiation of second or subsequent lines of treatment,regardless of previous TP53 statusWhen considering including a patient in a clinical trial

Following refractory disease

When considering allogeneic stem cell transplantation

In patients who test negative for del(17p)

Following relapse

When considering a specific treatment option

At diagnosis

In parallel with other genetic mutations

Other

In patients who test positive for del(17p)

Following identification of another genetic mutation

When in the patient journey is TP53 variant status typically assessed? Clinical section

0

10

20

30

40

50

60

70

80

90

100

% o

f re

sp

ondents

(N

=69)

Treatment option

Ibrutinib Idelalisib Venetoclax

In your clinical practice, which of the targeted agents do you have access to for use

within their licensed indications in CLL?

Clinical section

Yes, international guidelines

Yes, national guidelines

Yes, institutional/ho

spital guidelines

I am not sure

No, none of the above

51

%

13%

10

%

6%

20

%

In your clinical practice, do you follow guidelines on TP53 variant screening?

A variety of international,

national and institutional

guidelines are followed

Clinical section

Sanger sequencing only

NGS** only

Sanger sequencing and

NGS only*

Sanger sequencing,

NGS and other

I am not sure Other only

DNA

RNA

Both I am not sure

*8% of those respondents use NGS for research purposes only

**Next Generation Sequencing

28%

25%

38%

6%

89%

3% 3% 5%

Which TP53 sequencing method do you use? Type of nucleic acid used for TP53 analysis

19%

55% 57% 100% 87%

52%

87% of diagnosticians analyse exons 4–10 (N=83)

100% 100% 98%

100%

Which TP53 exons do you analyze?

Laboratory section

99%

ERIC note: Keep in mind that analyzing at least exons 4-10 is now recommended (Leukemia 2018). Analysis of all

coding exons (exons 2-11) is an optimum. Always include splice sites (+/-2bp in introns)!

0

5

10

15

20

25

30

35

40

45

% o

f re

sp

ondents

(N

=88) >80% of tests are turned around

within 4 weeks

≤2 weeks 2-4

weeks

4-8

weeks

I am not sure

Time

What is your turnaround time between receiving the sample and reporting the result?

Laboratory section

Jitka Malčíková

Šárka Pavlová

Karla Plevová

Marie Jarošová

Boris Tichý

Michael Doubek

THANK YOU

FOR YOUR ATTENTION !

Paolo Ghia

Kostas Stamatopoulos

Richard Rosenquist

Emili Montserrat

Carol Moreno

Arnon Kater

Lesley Ann Sutton

ERIC office in Barcelona

ACKNOWLEDGEMENT