Telomeres and telomerase

in mature B-cell disorders

Elisa Genuardi PhD, Torino, Italy, EU

What is my

destiny?

Somatic cell destiny

replicative senescence

MORTAL

mitosis

somatic cells

MORTAL

NECROSIS

APOPTOSIS

TELOMERE

effective sensor of genetic fitness from a dangerous open end

Telomeric DNA and telomere-specific binding

proteins have an essential role in stabilizing

chromosome ends by forming a cap structure that

protects chromosome ends from degradation and

terminal fusions.

Telomeres and Cell Senescence

Adapted from Shay JW and Wright WE, Journal of pathology 2007

Telomere shortening: late life effects

PROLIFERATIVE STRESS OXYDATIVE STRESS

TELOMERE SHORTENING

AND REPLICATIVE SENESCENCE

FUNCTIONAL EXHAUSTION

Genetic instability

Epigenetic interactions

Telomerase reactivation

Compensative proliferation

Telomere position effect

TRANSFORMATION

Tumor-suppressing function of replicative senescence

replicative senescence

GENETIC INSTABILITY

crisis

MORTAL

mitosis

somatic cells

telomere length Hayflick’s limit

tumor cells

Rb1

p53

IMMORTAL

Telomere stabilization

TELOMERASE ACTIVATION

A succesful phenotype for a cancer cell

Short

Telomeres High H-TERT

Prevention of crisis

(immortalization)‏

Extra-telomeric effects *

(oncogene-like properties)‏ Genetic

Instability

Epigenetic

effects

(TPE)‏

Senescence

associated

(pro-

inflammatory)‏

behaviors

* This might explain why choosing non telomerase-based telomere protection strategies such as ALT is a highly minoritarian choice

by cancer cells

All these aspects might contribute to maximize the

malignant properties of cancer cells

Telomerase and escape to cell senescence

SOME TISSUES MUST ESCAPE CELL SENESCENCE TO PERFORM THEIR FUNCTIONS

Germinal cells

Some embryonic tissues

lymphoid cells

Other adult tissues (to a lesser extent)

These “ESCAPE” Mechanisms can (or must) be exploited by tumor cells

Low level of TA

Skvortzov D.A. Acta Naturae 2009

Telomere and telomerase in normal lymphoid compartment

Telomere in B-cell tumours: ALL, MCL, MM

The example of CLL

Telomere and telomerase in lymphoid tumors

Telomere and Telomerase in normal lymphoid compartment

(Aubert G. Physiol Rev. 2008‏)

telomerase

Telomere and telomerase in normal lymphoid compartment

Lymphocytes high telomerase activation even during advanced phases of differentiation

T-cells

• telomerase unable to fully protect telomeres from proliferation induced

erosion (aging of immune system)‏ (Weng NP PNAS 1995 and J Exp Med 1996)‏

• expecially evident in case of activation against common antigens (C.tetanii, C.albicans)‏ (Burns JB J Immunol 2000)‏

• TL in memory T-cells < TL in T-naive (Bodnar AG Exp Cell Res 96, Weng NP Immunol Rev 97)‏

Telomerase and B-cell development

B-cell precursor TA +

naive B-cell TA +/-

memory B-cell TA +/-

extra GC activation TA ++

GC maturation TA +++++

high proliferation

no TL erosion

Telomere and telomerase in normal lymphoid compartment

Lymphocytes high telomerase activation even during advanced phases of differentiation

B-cells

• physiologic age-related shortening lower compared to T-cells (Son NH J Immunol 2000)‏

• absence of telomere erosion during GC differentiation process (only somatic district where extensive cell prolife- ration occurs without telomeric attrition)‏ (Weng NP PNAS 1997 and Immunity 1998, Hu BT J Immunol 1997)‏

• TL in memory B-cells‏≤‏TL‏in‏B-naive (Son NH Mech Ageing Dev 2003)‏

...However...

TELOMERIC EROSION OCCURS IN LYMPHOCYTES

extra-physiological stressors

• extensive proliferation

• inflammation

• viral infections (i.e. CMV)‏

• sex hormones

• ROS

• genetic polymporphisms of

telomere related proteins

• mechanisms occurring in

hematopoietic stem cells ageing

• mechanisms associated with

lymphocytes specific functions

physiological mechanisms

Two pathways to cancer

Cancer

Critical telomere shortening

Loose regulation of the senescence checkpoint

Telomere and telomerase in normal lymphoid compartment

Telomere in B-cell tumours

Telomere in mature B-cell disorders: MCL and MM

The example of CLL

TELOMERE in B-CELL TUMOURS

high proliferative and differentiative potential of GC lymphocytes

high TA and TL conservation in GC

telomeric

checkpoint

released

increased risk of neoplastic transformation in GC

B-cell tumours arise frequently in phases with physiological telomerase activation

TELOMERE in B-CELL TUMOURS Telomere in B-cell tumours

(Hiyama K J Immunol 1995, Hodes RJ Immunology 2002)‏

TL heterogeneity in lymphoproliderative diseases is not related to

patient age

TRF (

bp)

0

2000

4000

6000

8000

10000

12000A B

FL

age (years)

20 30 40 50 60 70 80 90

TRF (

bp)

2000

4000

6000

8000

10000

12000

MZL

age (years)

20 30 40 50 60 70 80 90

TRF (

bp)

2000

4000

6000

8000

10000

12000

r ² = 0.0267

r ² = 0.0103

age (years)

20 30 40 50 60 70 80 90

TRF (

bp)

2000

4000

6000

8000

10000

12000

r ² = 0.2205

MCL

age (years)20 30 40 50 60 70 80 90

TRF (

bp)

2000

4000

6000

8000

10000

12000

BL

MM

age (years)

20 30 40 50 60 70 80 90

TRF (

bp)

2000

4000

6000

8000

10000

12000

MM

age (years)

20 30 40 50 60 70 80 90

TRF (

bp)

2000

4000

6000

8000

10000

12000

DLCL

r ² =0.3949

r ² = 0.0005

r ² = 0.0121

2D Graph 9

age (years)

20 30 40 50 60 70 80 90

TRF (

bp)

2000

4000

6000

8000

10000

12000

all

r ² = 0.0783

CLL

age (years)

20 30 40 50 60 70 80 90

TRF (

bp)

2000

4000

6000

8000

10000

12000

r ² = 0.0891

90th percentile

75th percentile

25th percentile

10th percentile

mean

median

St.Dev.

(Lansdorp P Blood 2008;

Ladetto M Blood 2004)‏

TL in mature B-cell lymphoproliferative disorders

GC unexp NHL

n° of cell divisions

telomere

shortening

GC-derived NHL GC-lymphomagenesis

pre-GC-lymphomagenesis

Telomere in B-cell tumours

GC unexperienced GC experienced

Healthy B naive 8300 bp 9000bp

Tumor B-cells 3900 bp 7500bp

TL shortening -4400 bp -1500 bp

TL is not a mere histopathogenetic marker

MULTIPLE FACTORS MIGHT INFLUENCE THE TELOMERE STATUS OF LYMPHOID TUMOURS

prolipherative history (number of replication cycles)‏

histopathogenesis (GC vs non GC experience)‏

oxidative stress

oncogenes (c-myc)‏

tumour suppressor genes (ATM)‏

disease progression (MGUS vs MM)‏

Telomere and telomerase in normal lymphoid compartment

Telomere in B-cell tumours

Telomere in mature B-cell disorders: MCL and MM

The example of CLL

MCL patients show significantly shorter TL indicating the occurrence of telomere dysfuction

TL and MCL

(Cottliar A. Eur J Haeatol 2009)

(Jebaraj BM, Blood 2013)‏

In MCL patients TL was not associated with any of clinical or biologic factor rilevant for pathogenesis or prognostication and disease

outcome

(Jebaraj BM, Blood 2013)

TL and MM

MM plasma cells (MMPCs) showed significantly shorter Telomere length

and high Telomerase activity

(Wu KD, Blood 2003;

Cottliar A. Eur J of Haematol 2003)

Telomere and telomerase in normal lymphoid compartment

Telomere in B-cell tumours

The example of CLL

...Telomere shortening is a crucial event in tumorigenesis but it

isn’t‏a‏disease‏predictor....

…IN‏CONTRAST‏TO….

The example of CLL

PROGNOSTIC BIOMARKERS

Binet status

Genetic abnormalities

IGVH mutational status

p53, CD38, ZAP-70, CD49d

Stratification according to these parameters does not fully explain the

heterogeneity of CLL.

HAS TELOMERE LENGTH A POTENTIAL ROLE IN THE BIOLOGY AND

CLINICAL OUTCOME OF CLL?

Prognostic role of TL in CLL

(Bechter OE Cancer Res 1998)‏

SHORT TELOMERES ARE ASSOCIATED WITH A WORSE OUTCOME

58 B-CLL pts

median TL 6.0 kb

TL < 6.0 kb associates to inferior OS and Binet

B-C

high TA (TRAP) associates to poor

outcome

TL and TRAP are inversely correlated

Correlation between TL and IGHV-MS

LONG TELOMERES ARE ASSOCIATED TO A VH MUTATED STATUS...

(Hultdin M Br J Cancer 03)‏ (Damle RN Blood 2004)‏

Correlation between TL and IGHV-MS

…AND‏ARE‏PREDICTIVE‏OF‏A‏BETTER‏OVERALL‏SURVIVAL

(Hultdin M Br J Cancer 03)‏ (Grabowski P Blood 05)‏

Correlation between TL and IGHV-MS TL is not a mere surrogate marker for IGHV-MS

TL IS A STRONGER PREDICTOR COMPARED TO VH-MUTATIONAL STATUS

0 24 48 72 96 120 144 168 192 216 240 264 288

0

20

40

60

80

100

p < 0.0001

months

Perc

en

t su

rviv

al

0 24 48 72 96 120 144 168 192 216 240

0

20

40

60

80

100

p < 0.0001

months

Perc

en

t su

rviv

al

0 12 24 36 48 60 72 84 96 108 120 132 144

0

20

40

60

80

100

p < 0.0001

months

Perc

en

t su

rviv

al

0 24 48 72 96 120 144 168 192 216 240 264 288

0

20

40

60

80

100

p < 0.0001

months

Perc

en

t su

rviv

al

0 24 48 72 96 120 144 168 192 216 240

0

20

40

60

80

100

p < 0.005

months

Perc

en

t su

rviv

al

0 12 24 36 48 60 72 84 96 108 120 132 144

0

20

40

60

80

100

p < 0.0005

months

Perc

en

t su

rviv

al

IGHVMS

TL

OS TTFT PFS

(Ricca I Leukemia 2007)‏

Correlation between TL and IGHV-MS distribution of TL among VH mutated and unmutated pts

1000150020002500300035004000450050005500600065007000750080008500900095001000010500

U

M

TRF (bp)

(Ricca I Leukemia 2007)‏

27/201 (42%) pts UM with long TL

discordance between TL and IGHV-MS is not uncommon

Correlation between TL and IGHV-MS survival analysis in UM pts according to TL

In discordant cases TL IS A STRONGER PREDICTOR than IGHV-MS

OS PFS

(Ricca I Leukemia 2007)‏

Shortly telomeres are associated with NOTCH 1 and SF3B1 gene mutations

(Mansouri L, Am J Hematol 2013)‏

validation on a series of 401 pts

blinded analysis of results

191 pts Turin University

LEARNING SERIES

210 pts Novara Amedeo Avogadro

University

BLINDED

VALIDATION

SERIES

(Rossi D Leukemia 2009)‏

Validation on a great series of 401 pts

Definition of the optimal cut-off value

ROC analysis

5000 bp: best TL cut-off value

(Rossi D Leukemia 2009)‏

Area 95% CI p

.797 .068-.914 .<000

Confirm the prognostic value of TL in terms of clinical outcome

TFS OS

TL is an independent predictor of TFS and OS

(Rossi D Leukemia 2009)‏

Prediction of additional outcome events

(Richter Syndrome transformation)‏

TL is an independent predictor of risk of Richter transformation

(Rossi D Leukemia 2009)‏

...however data on TL dynamics over time are scant and anedoctal...

study of telomere dynamics during the natural history of CLL

(Sellmann L. Int J Hematol 2011

Mansouri L, Am J Hematol 2013)‏

Telomere length can predict OS at diagnosis and when measured at the time of follow up

median time between

TL determinations:

42 months

1° TL determination: 88 pts at diagnosis

2° TL determination:

63 pts in WW phase

2° TL determination:

25 pts at relapse

median time between

TL determinations:

56 months

TFS analysis descriptive evaluation of telomere dynamics

over time

(Bernocco E. ASH 2011)‏

PROGRESSIVE TELOMERE SHORTENING IS PART OF THE NATURAL HISTORY

OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) AND IMPACTS CLINICAL

OUTCOME

TL shorten over time compared to baseline

(Bernocco E. ASH 2011)‏

telomere loss is greater in cases with higher baseline TL (only modest additional erosion in pts with short TL at diagnosis)

MUTATED UNMUTATED

-2000

-1000

0

1000 IgH status p<0.05

YL

(bp

‏(

whole population

no correlation with available

clinical and biological

parameters except IGHV-MS

MUTATED UNMUTATED

-2000

-1000

0

1000 IgH status p=0.02

YL

(bp

‏(

WW patients

rapid telomere erosion associates

with IGHV-mutated status and

unfavourable cytogenetics

(Bernocco E. ASH 2011)‏

rapid TL erosion is linked to an

inferior TFS

(Bernocco E. ASH 2011)‏

patients with long and stable

telomeres have an excellent TFS

Telomere disruption represents a critical step associated to CLL

progression

What is the link between dysfunctional telomeres and CLL progression?

1) high telomerase activity is a signature of poor survival in B-CLL patients (Terrin L Leukemia 2007)‏

2) telomerase expression is linked to the presence of short telomeres (Bechter OE Cancer Res 1998, Damle RN

Blood 04)

3) global changes of the telomere related protein with a deregulation of telomeric protein

machinery (Poncet D Blood 2008)

Titia DeLange Science 2009

Est1A

Pinto AR Front Biosci 2011 David J Clin Cancer Res 2007

Rpa1

SHELTERIN TELOMERASE

COMPLEX TELOMERE RECRUITORS of

DNA-REPAIRING COMPLEX

POT1 mutations cause telomere disfunctions in CLL

POT1 somatic mutation emerged

as the second most frequently

mutated gene

POT1 is exclusively mutated in

IgHV unmutated patients

Telomerase complex dysfunctions

genomic instability and

progression to crisis phase

(Ramsay AJ Nat Genet 2013)‏

crisis-like phenomena associated with

dysfunctional telomeres play a role in

CLL progression

(Lin TT Blood 2011)‏

(Lin TT Blood 2011)‏

Single-molecule Telomere Length Analysis (STELA) revealed:

presence of critically short telomeres was associated with severe genomic instability

(complete telomeric loss events, fusion events and genomic rearrangements in telomeric

regions)

(Lin TT Blood 2011)‏

DNA sequencing in pts with dysfunctional telomeres telomere fusion shows a characteristic mutational profile

reduced number of TTAGGG repeats subtelomeric deletions

microhomology

...moreover the presence of dysfunctional telomeres was more common BUT NOT EXCLUSIVELY found in

patients with poor prognostic features...

TELOMERIC DYSFUNCTIONS MIGHT BE INVOLVED AT EARLY STAGE OF CLL PROGRESSION

Telomere distrupt, CLL progresses

(Ladetto M, Blood 2010)‏

Thanks‏to…

LYMPHOMA unit

Simone Ferrero

Barbara Mantoan

Daniela Barbero

Luigia Monitillo

Daniela Drandi

Roberta Benedetto

Giulia Verardo

Federica Cavallo

Paola Ghione

and the clinical team

Haematology division

UNIVERSITÀ DI TORINO - Prof. Mario Boccadoro

Dott.ssa Paola Omedè

FISH-Cytofluorimetric

Staff

Dott. Roberto Passera

Dott.Marco Ladetto

Dott.ssa Elisa Bernocco