Genomics of hairy cell leukemia and its

translation into new diagnostic and

therapeutic opportunities

Brunangelo Falini M.D.

Institute of Hematology, University of Perugia

Perugia, Italy

Conflict of interests: nothing to disclose.

Homing of HCL cells to selected body sites

Spleen: pre-IFN era BM: “fried-egg pattern”

Red pulp involvementLiver: sinusoidal pattern

PB smear: hairy morphology

Distinct mature B-cell leukemia entity:

- Adults/older people, predominantly male (M/F ratio= 4:1)

- Indolent course. Presenting with progressive pancytopenia and

splenomegaly, usually no lymphadenopathy

- Highly responsive to purine analogs (2-CDA, Pentostatin)

Leukemic cells show:

- Distinctive features (hairy morphology)

- Unique dissemination pattern (PB , BM, spleen, liver)

- Unique immunophenotype (CD20+, CD25+, CD11c+, CD103+)

- Mutated IgV genes (~85% cases)

- Probable derivation from memory B cell

- Unknown underlying genetic lesion (difficult to study: pancytopenia,

dry tap, no splenectomy in the purine analogues era)

Hairy cell leukemia (WHO 2008)

Whole exome sequencing of one HCL patient

Purified leukemic

hairy cells

Purified normal cells

(granulocytes)

In-solution exome

capture

In-solution exome

capture

Illumina

sequencing

Illumina

sequencing

Discovery of BRAF mutation in HCL

BRAF

Serine/threonine kinase of the RAS-RAF-MEK-ERK pathway.

BRAF is activated by RAS in a GTP dependent manner.

BRAF is a major activator of MEK 1/2 that in turns activates

ERK, leads to nuclear ERK translocation and promotes

proliferation and survival

BRAF is the kinase-encoding oncogene most frequently

mutated in human cancers: ~50% melanomas

~40% papillary thryoid carcinomas

≤10% colon, prostate, lung, ovary

Rare in hematological tumors

(57% LCH, 4% multiple myeloma)

> 90% mutations are T1796A transversion (exon 15) leading to

BRAF-V600E. This phosphomimetic substitution (valine with

glutamate) leads to constitutive activation of MAPK pathway

RTK

RAS

MEK

ERK

BRAF

SurvivalProliferation

P

P

Ligand

V600E

BRAF-V600E mutation is the disease-defining genetic event in HCL:

- First activating point mutation of a kinase-encoding gene with high

recurrence in B-cell lymphomas

- Somatic and present in the entire tumor cell clone

- Present in 100% of HCL cases, encompassing the whole clinical

spectrum of the disease. Disease-defining genetic event of HCL

- Stable at relapse up to many years and following several lines of

therapy

- Absent in HCL-variant and splenic MZ lymphoma

BRAF-V600E causes continuous phosphorylation of MEK and ERK

with consequent constitutive activation of MAPK pathway

BRAF mutations in Hairy Cell Leukemia (Tiacci et al, NEJM 2001)

Diagnostic implications of genomic studies

(GEP and WES) in HCL

News specific markers

Annexin A1 IImmunohistochemistry

BRAF-V600E Molecular assays

Improved

diagnosis

HCL-specific genes

CB CC N MHCL FL BL B-CLLDLBCL MCL

Normal B cells B cell lymphomas

- ANXA1

- FLT3

- CD63

- SCNB1- SDC3

- FGF2

- CCND1

- IL3Ra

- TIMP1

- MYF6

- PLXNC1

- FGFR1

- PTPRM

- SYT1

- TIMP4

- arrestin BPreviously known

IHC performed

- EPB41L2

- CXCR5

- GAS7

(Falini B., Lancet 363:1869, 2004)

0% 1

2

3

4

5

6

7

8

9

10

11

12

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

3.1

%

1.6

%

0.8

%

0.2

%

0.1

%

0.0

5

% 1

2

3

4

5

6

7

8

9

10

11

12

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

Mutant

BRAF

amplicon

Wild-type

BRAF

amplicon

Mutant

BRAF

amplicon

Wild-type

BRAF

amplicon

Mutant BRAF alleles* HCL pre-therapy

(1%-10% cells)

HCL-variant SMZL

Allele-specific PCR for the qualitative detection

of BRAF-V600E in whole-blood samples

* Serial DNA dilution of two cell lines, one homozygous for BRAF-V600E and one wild-type

HCL post-

therapy

(0.1% cells)

(Tiacci, Schiavoni et al. Blood 2012;119:192)

MATURE B-CELL NEOPLASMS

Chronic lymphocytic leukemia /Small

lymphocytic lymphoma

B-cell prolymphocytic leukaemia

Splenic marginal zone lymphoma

Hairy cell leukemia

Splenic lymphoma/leukaemia, unclassifiable

(HCL variant, splenic diffuse red pulp B-cell

lymphoma)

Lymphoplasmacytic lymphoma

Waldenström macroglobulinemia

Heavy chain diseases (alpha, gamma, mu)

Plasma cell myeloma

Solitary plasmacytoma of bone

Extraosseous plasmacytoma

Extranodal marginal zone B-cell lymphoma

of mucosa-associated lymphoid tissue

(MALT lymphoma)

Nodal marginal zone B-cell lymphoma

Pediatric type nodal MZL

Follicular lymphoma

Pediatric type follicular lymphoma

Primary cutaneous follicle centre lymphoma

Mantle cell lymphoma

RAS

RTK

V600E

MEK

ERK

RAF

Survival

Proliferation

Transformation

VEMURAFENIB

DABRAFENIB

Effect of BRAF-V600E inhibitors on constitutively activated MAPK

pathway in HCL

Increased

phosphorylation

Dr. Lessia Santi - Abstract S1300: Sunday, 8.00-8.15 AM, Room Red 1+2 (NW-1)

Session: NHL & HL – Biology: Novel genetics and signaling)

Analysis of:

GEP, morphology,

apoptosis of HCL

vs HCL-like cells

N=23 HCL

N=10 HCL-like

The BRAF inhibitor Vemurafenib causes dose-dependent

MEK-ERK dephosphorylation

**

* Same results with Dabrafenib and Trametinib.

** Blot exposure in HCL-like cells: dozens of minutes (versus dozen of seconds in HCL cells).

The BRAF inhibitor Vemurafenib does not induce MEK-ERK

dephosphorylation in HCL-like neoplasms *

Vehicle Vemurafenib

48 h 72 h 48 h 72 h

*in melanoma and colon carcinoma (Pratilas et al, PNAS 2009)

Vehicle versus Vemurafenib

NES 1.83

p-value 0.002

Genes induced (n=48) by the BRAFV600E-MEK-ERKpathway in melanoma and colon carcinoma

HCL (n=6)

*

48 genesIN HCL, VEMURAFENIB SILENCES

THE BRAF-MEK-ERK PATHWAY

TRANSCRIPTIONAL OUTPUT *

Gene set enrichment

analysis

BRAF inhibition changes the GEP of hairy cell leukemia

30 genes upregulated* by

Vemurafenib:

105 genes dowregulated* by Vemurafenib:

- MAFF, ETV5, DUSP6, EGR1, SPRY2

- CCND1, Actin beta, trombospondin 1 (THBS1)

(red: known trascriptional targets of the MAPK

pathway)

* ≥ 2 fold; corrected p-value <0.05

HCL-specific genes

CB CC N MHCL FL BL B-CLLDLBCL MCL

Normal B cells B cell lymphomas

- ANXA1

- FLT3

- CD63

- SCNB1- SDC3

- FGF2

- CCND1

- AIF1

- TIMP1

- MYF6

- PLXNC1

- FGFR1

- PTPRM

- SYT1

- Beta Actin

- Arrestin B

- CXCR5

- GAS7

- Trombospondin

- SPRY2

Loss of the HCL-specific gene expression

signature* after BRAF-V600E inhibition

*Basso et al, JEM 2004

BRAF inhibition changes the GEP of hairy cell leukemia

30 genes upregulated* by

Vemurafenib:

105 genes dowregulated * by Vemurafenib:

- MAFF, ETV5, DUSP6, EGR1, SPRY2

- CCND1, Actin beta, trombospondin 1 (THBS1)

- CD25 (IL2RA), TRAP (ACP5)

* ≥ 2 fold; corrected p-value <0.05

Ctrl. Drug-1μM

Ctrl.

Ctrl.

Drug-1μM Drug-1μM

Ctrl.

Drug-1μM

Double staining: phalloidin* / ANXA5*

HCL cells HCL-like cells

Loss of hairy morphology in primary HCL cells

exposed to Vemurafenib for 48 hours

Phalloidin (green): marker for F-Actin; ANXA5 (red): marker for apoptosis

MEK and ERK

dephosphorylation

(minutes/hours)

Loss of the HCL

expression

signature and

morphology

(2-3 days)

Double staining phalloidin / ANXA5

Santini

PLX 4032 Axon

PLX 4032 Roche

1000

1000

DM

SO

90’ 6h

ERK1/2

p-ERK1/2

1000

1000

DM

SO

1000

1000

DM

SO

30’

1000

1000

DM

SO

90’ 6h

1000

1000

DM

SO

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1000

DM

SO

30’

1000

1000

DM

SO

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1000

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SO

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1000

DM

SO

p-MEK1/2

30’ 90’ 6h

1000

1000

DM

SO

1000

1000

DM

SO

1000

1000

DM

SOMEK1/2

30’ 90’ 6h

pERK

Santini

PLX 4032 Axon

PLX 4032 Roche

1000

1000

DM

SO

90’ 6h

ERK1/2

p-ERK1/2

1000

1000

DM

SO

1000

1000

DM

SO

30’

1000

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90’ 6h

1000

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30’

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p-MEK1/2

30’ 90’ 6h

1000

1000

DM

SO

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1000

DM

SO

1000

1000

DM

SO

MEK1/2

30’ 90’ 6h

pMEK

0.5 hr 1.5 hr 6 hr

Drug-1μm Drug-1μm Drug-1μm

Ctrl. Drug-1μM

Apoptosis

(3-4 days)

0 20 40 60 80 100 120

1

2

Series1

0 20 40 60 80 100 120

1

2

Series1

100806040200

% live cells (ANXA5-/PI-)

Ctrl.

Drug 1μm

120

Effects of BRAF inhibition in in vitro-cultured primary HCL cells

HCL-PG01 CLINICAL TRIAL

Sponsor: Institute of Hematology, Perugia (PI: B. Falini)

Vemurafenib (Zelboraf – Roche): oral, ATP-competitive, reversible inhibitor of

the active conformation of the BRAF kinase

Results presented by Enrico Tiacci at the

Presidential Symposium, EHA meeting

(Milan, 12-15 June, 2014)

Main inclusion criteria:

1) HCL patients refractory to purine analogs

2) HCL patients relapsed after purine analogs therapy (≤ 2ys CR/PR

after the 1st cycle or ≤ 4ys CR/PR after 2nd or more cycles)

3) HCL patients with severe side effects during/after previous purine

analog therapy

Recruitment of the 28 patients was started on May 2011 and was

completed on April 2012.

HCL-PG01 CLINICAL TRIAL

(N=28 patients)

Vemurafenib

960 mg twice/day

(8 weeks)

CR

Stop therapy X 2 weeks

Stop drug

Vemurafenib

960 mg twice/day

(4 weeks)

no CRCR

no CR

Vemurafenib

960 mg twice/day

(4 weeks)

Stop drug

Stop drug

CR= According to standard criteria

(normalization PB count and

absent HCs by morphology)

Vemurafenib in HCL: conclusions

- The drug is highly active in refractory/relapsed HCL

(> 90% overall response rate). Patients are in follow-

up for duration of response.

- The drug is generally well tolerated. Side effects are

similar to those observed in melanoma trials

- The drug is not myelotoxic

- Vemurafenib-resistant HCL cells are detectable in

all cases.

BRAF inhibitors in HCL: open questions

- Which are the mechanisms underlying resistance

to Vemurafenib in HCL ?

- Therapy: which are the next steps ?

Dual targeting of MAPK: BRAF + MEK inhibitors.

Non myelotoxic therapy: BRAF inhibitors + Rituximab.

PERUGIA (COORDINATING CENTER):

B. Falini (PI)

E. Tiacci

L. De Carolis

S. Ascani

F. Falzetti

M.P. Martelli

F. Falcinelli

S. Ballanti

P. Sportoletti

OTHER CENTERS AND COLLABORATORS:

P.L. Zinzani, S.A. Pileri (Bologna)

A. Pulsoni, R. Foà (Rome)

M. Mazzucco, F. Zaia (Udine)

M. Cimminiello, M. Pizzuti (Potenza)

D. Rossi, G.L. Gaidano (Novara)

G. Motta, F. Di Raimondo (Catania)

M. Varettoni, L. Arcaini (Pavia)

A. Anastasia, G. Rossi (Brescia)

A. Ambrosetti, G. Pizzolo (Verona)

A. M. Carella (Genova)

A. Rambaldi (Bergamo)

P. Leoni (Ancona)

V. Fraticelli (Campobasso)

HCL-PG01 CLINICAL TRIAL - ACKNOWLEDGMENTS