recent advances in the understanding of mastocytosis: the...

Recent advances in the understanding of mastocytosis: the roleof KIT mutations*

Alberto Orfao,1 Andres C. Garcia-Montero,1 Laura Sanchez2 and Luis Escribano2 for the Spanish Network on Mastocytosis

(REMA)

1Centro de Investigacion del Cancer/IBMCC (CSIC/USAL), Departamento de Medicina and Servicio General de Citometrıa, University

of Salamanca, Salamanca, Spain, and 2Unidad de Mastocitosis, Hospital Ramon y Cajal, Madrid, Spain

Abstract

Mastocytosis is a heterogeneous disorder characterised by the

expansion and accumulation of mast cells in different organs

and tissues. Mast cell physiology is closely dependent on

activation of the stem cell factor/Kit signalling pathways and

accumulating evidences confirm the physiopathological key

role of activating KIT mutations (typically D816V) in mast-

ocytosis and their relationship with the clinical manifestations

of the disease. This paper reviews the most recent advances in

the understanding of the molecular mechanisms associated

with KIT mutations in mastocytosis, including recent data

about the use of new therapies targeting the Kit molecule and

its associated downstream signalling pathways.

Keywords: Mastocytosis, SCF/Kit signalling pathway, KIT

mutations, imatinib, tyrosine kinase inhibitors.

Mastocytosis is a relatively heterogeneous disorder charac-

terised by the expansion and accumulation of mature-

appearing mast cells (MC) in different organs and tissues

such as the skin, gastrointestinal tract, liver, spleen, bone

marrow (BM) and other lymphoid tissues. The disease was

first described as a rare form of urticaria (Nettleship & Tay,

1869), prior to the description of MC by Paul Ehrlich in 1879

(Ehrlich, 1879). The link between MC and urticaria pigmen-

tosa (UP) was quickly made (Unna, 1887). Since then, other

forms of mastocytosis have been described, such as mast cell

leukaemia (MCL), mastocytoma and systemic mastocytosis

(SM), among others (Valent, 2004). Knowledge of the

heterogeneous behaviour of the disease was further expanded

by the demonstration of both childhood and adult forms of

mastocytosis. Together, these observations called attention to

the need for a classification of mastocytosis and led to a first

proposal by Lennert and Parwaresch (1979). The Kiel

classification (Lennert & Parwaresch, 1979) was followed by

other attempts that tried to group mastocytosis into well-

defined clinico-biological entities and, in 1991, a first

consensus classification of mastocytosis was proposed (Met-

calfe, 1991). Since then, important biological markers of the

disease have been identified. Most relevant were the associa-

tions described between mastocytosis and increased serum

tryptase levels (Schwartz et al, 1987), the presence of the

D816V-activating KIT mutation (Furitsu et al, 1993; Longley

et al, 1995; Nagata et al, 1995) and an aberrant CD25+ and

CD2+ immunophenotype of BM MC (Escribano et al, 1998a).

The identification of these new biological markers has

facilitated a better understanding of the molecular mechanisms

of mastocytosis, contributed to improve the diagnosis and

classification of the disease and promote the search for new

molecular-targeted therapies. In line with this, in 2001 the

World Health Organisation (WHO) proposed new criteria for

the diagnosis and classification of mastocytosis (Valent et al,

2001).The WHO classification proposes a combination of

several major and minor criteria for the diagnosis of SM.

Accordingly, either one major (presence of dense infiltrates of

>15 MC in the BM or in other extracutaneous organs detected

by immunohistochemical analysis of tryptase expressing cells)

and one minor (abnormal MC morphology, KIT mutation at

codon 816, an aberrant CD25+ and/or CD2+ MC immuno-

phenotype and/or serum total tryptase of >20 ng/ml) or at

least three minor criteria, are required for the diagnosis of SM.

In turn, in the new WHO classification, mastocytosis was

grouped into seven different subtypes: cutaneous mastocytosis

(CM), indolent SM (ISM), aggressive SM (ASM), SM associ-

ated with a clonal haematopoietic non-MC disorder (SM-

AHNMD), MCL, MC sarcoma (MCS) and extracutaneous

mastocytoma (ECM); within some of these subgroups, provi-

sional variants were further described, e.g. isolated BM

mastocytosis (BMM) and smouldering SM (SSM) are new

provisional subvariants of ISM (Valent et al, 2001) (Table I).

More recently, an International Working Conference was held

in Vienna where a group of experts on mastocytosis proposed

new standards for clinical evaluations and diagnostic assays

(Valent et al, 2007a). In addition, this International Working

Correspondence: Alberto Orfao, MD, PhD, Centro de Investigacion del

Cancer (IBMCC), University of Salamanca/CSIC, Campus Miguel de

Unamuno, 37007 Salamanca, Spain. E-mail: [email protected]

*All authors have contributed equally to this manuscript.

review

ª 2007 The Authorsdoi:10.1111/j.1365-2141.2007.06619.x Journal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 138, 12–30

Tab

leI.

Cla

ssifi

cati

on

of

mas

tocy

tosi

s:su

mm

aris

edd

escr

ipti

on

of

the

mo

stre

leva

nt

clin

ical

and

bio

logi

cal

feat

ure

so

fth

ed

iffe

ren

tty

pes

of

mas

tocy

tosi

s(V

alen

tet

al,

2001

,20

07a)

.

Typ

e

Skin

lesi

on

s

Ext

racu

tan

eou

s

MC

lesi

on

sB

MM

Cb

urd

en

Ab

no

rmal

BM

MC

mo

rph

olo

gy

(MC

sub

typ

e)

BM

MC

clu

ster

s

(>15

MC

)

D81

6V

KIT

mu

tati

on

CD

25+

/CD

2+/)

MC

imm

un

op

hen

oty

pe

Seru

m

tryp

tase

(>20

ng/

ml)

Org

ano

meg

alie

s

Imp

aire

d

org

an

fun

ctio

nE

osi

no

ph

ilia

CM

+)

Lo

w)

))

/+)

/+)

/+)

))

MC

L)

+H

igh

(>20

%)

+(H

igh

grad

e)+

)/+

++

)/+

+)

/+

ISM

++

Lo

w+

(Aty

pic

alM

CI)

++

++

))

)/+

BM

M)

+L

ow

+(A

typ

ical

MC

I)+

++

+)

))

SSM

)/+

+L

ow

/in

term

edia

te+

(Aty

pic

alM

CI)

++

++

+)

)/+

ASM

)/+

+In

term

edia

te/h

igh

+(A

typ

ical

MC

I/II

)+

++

++

+)

SM–

AH

NM

D*

++

Lo

w/h

igh

+(A

typ

ical

MC

I/II

)+

++

)/+

)/+

)/+

)/+

WD

SM+

+L

ow

/hig

h)

+)�

))

))

)M

MU

S/M

MA

S)

+L

ow

+(A

typ

ical

MC

I))

)/+

�)

/+)

/+)

))

MC

S)

+L

ow

+(H

igh

grad

e))

)N

D)

/+)

)/+

)E

CM

)+

Lo

w+

(Lo

wgr

ade)

))

ND

)/+

))

/+)

BM

,b

on

em

arro

w;

MC

,m

ast

cell

s;C

M,

cuta

neo

us

mas

tocy

tosi

s;M

CL

,m

ast

cell

leu

kem

ia;

ISM

,in

do

len

tsy

stem

icm

asto

cyto

sis;

BM

M,

iso

late

dB

Mm

asto

cyto

sis;

SSM

,sm

ou

lder

ing

SM;

ASM

,ag

gres

sive

syst

emic

mas

tocy

tosi

s;SM

-AH

NM

D,

syst

emic

mas

tocy

tosi

sas

soci

ated

wit

ha

clo

nal

no

n-M

Cli

nea

geh

emat

olo

gica

ld

isea

se;

WD

SM,

wel

l-d

iffe

ren

tiat

edsy

stem

icm

asto

cyto

sis;

MM

US,

mo

no

clo

nal

MC

po

pu

lati

on

wit

hu

nd

efin

edsi

gnifi

can

ce;

MM

AS,

mo

no

clo

nal

MC

-act

ivat

ion

syn

dro

me;

MC

S,M

Csa

rco

ma;

EC

M,

extr

acu

tan

eou

sm

asto

cyto

ma.

Aty

pic

alM

CI:

Spin

dle

shap

edM

C,

MC

wit

ho

val

nu

cleu

sw

ith

or

wit

ho

ut

anex

cen

tric

po

siti

on

,o

rM

Cw

ith

hyp

ogr

anu

late

dcy

top

lasm

and

foca

lac

cum

ula

tio

no

fgr

anu

les

bu

tw

ith

ou

tsi

gns

of

deg

ran

ula

tio

n.

Aty

pic

alM

CII

:M

Cw

ith

bi-

or

po

lylo

bu

late

dn

ucl

eian

dh

ypo

gran

ula

ted

cyto

pla

smw

ith

ou

tsi

gns

of

deg

ran

ula

tio

n.

Cyt

op

ath

olo

gica

lsc

ore

:H

igh

Gra

de:

>20

%o

fM

Car

e‘m

etac

hro

mat

ic

bla

sts’

plu

sat

ypic

alM

CII

.Lo

wG

rad

e:<

10%

of

MC

are

‘met

ach

rom

atic

bla

sts’

plu

sat

ypic

alM

CII

.Dat

afr

om

Val

ent

etal

(200

1,20

07a)

.Sco

re:+

,det

ecte

din

mo

stca

ses;

)/+

,det

ecte

din

asu

bse

to

fca

ses;

),

no

td

etec

ted

or

det

ecte

dra

rely

.

*Su

bty

pes

of

SM-A

HN

MD

sho

uld

be

clas

sifi

edac

cord

ing

toth

ety

pe

of

AH

NM

Dan

do

fSM

foll

ow

ing

the

WH

O/F

AB

crit

eria

.�A

nat

ypic

alK

ITm

uta

tio

no

ther

than

D81

6Vis

freq

uen

tly

det

ecte

d.

Review

ª 2007 The AuthorsJournal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 138, 12–30 13

Conference discussed the differential diagnosis of new poorly

defined subgroups of patients with increased and/or altered

MC. Among others, the differential diagnosis between well-

differentiated SM (WDSM) and reactive MC hyperplasia, as

well as between cases with a monoclonal MC population with

undefined significance/monoclonal MC activation syndrome

(MMAS) and reactive MC hyperplasia, were considered

(Table I) (Valent et al, 2007a).

On the other hand, the identification of the presence of the

D816V KIT mutation, together with an extensive characteri-

sation of the immunophenotype of clonal MC from patients

with mastocytosis, represented a major step forward in the

understanding of the molecular mechanisms of the disease and

have accelerated the search for new therapies based on the use

of Kit-specific tyrosine kinase (TK) inhibitors and other

molecule-targeted agents, for those cases requiring cytoreduc-

tive therapy (Valent et al, 2005).

The D816V KIT mutation was first described by Furitsu et al

(1993) in the HMC-1 human MC line (Butterfield et al, 1988).

Later, it was shown that the D816V KIT mutation was also

present in SM patients (Nagata et al, 1995), where it was

repeatedly observed (Longley et al, 1995). However, the exact

frequency of the D816V KIT mutation has remained a matter

of debate, particularly for ISM patients, where it has been

reported to range from 31% (Pardanani et al, 2003a) to

virtually all patients (Fritsche-Polanz et al, 2001). Recently, our

group confirmed the presence of the D816V KIT mutation in

the great majority of adult SM patients (102/113 cases; 93%) in

fluorescence-activated cell sorted (FACS)-purified populations

of immunophenotypically aberrant MC; interestingly, in

around one quarter (3/11 cases; 27%) of those few cases

lacking the D816V KIT mutation, other mutations in the

tyrosine-kinase domain 2 (TK2) of KIT, were detected (Garcia-

Montero et al, 2006). Although these results require further

confirmation by other groups in large series of patients, they

support previous findings suggesting that the D816V KIT

mutation could represent a hallmark of the disease in adult

patients.

The present paper reviews the most recent advances in the

understanding of SM, focussing on the role of KIT mutations

to dissect the pathogenetic mechanisms of the disease.

Accordingly, the stem cell factor (SCF)/Kit signalling pathways

and the impact of KIT mutations on their behaviour, are

reviewed; then, the physiopathological role of the mutations of

KIT in SM and their relationship with the clinical manifesta-

tions of the disease are discussed; the final section reviews the

recent data regarding the use of new therapies targeting the Kit

molecule and other associated signalling pathways.

The stem cell factor/kit signalling pathway

The structure of the Kit molecule

Human KIT is a proto-oncogene that encodes for a trans-

membrane receptor (Kit) with intrinsic TK activity (Yarden

et al, 1987). Expression of the Kit protein has been reported in

both normal cells [e.g. haematopoietic progenitors (Simmons

et al, 1994), normal mature MC (Metcalfe, 2005), Cajal cells

(Huizinga et al, 1995), melanocytes (Halaban et al, 1993), and

germ cells (Strohmeyer et al, 1995)] and neoplastic cells from

gastrointestinal stromal tumours (GIST) (Andersson et al,

2002), seminomas (Strohmeyer et al, 1995), small cell lung

cancer (Sekido et al, 1991), colon cancer (Toyota et al, 1993),

neuroblastoma (Beck et al, 1995), breast cancer (Hines et al,

1995), acute myeloid leukaemia (AML) (Bene et al, 1998),

T-cell acute lymphoblastic leukaemia (ALL) (Bene et al, 1998),

multiple myeloma (Escribano et al, 1998b), myelodysplastic

syndromes (MDS) (Orfao et al, 2004), myeloproliferative

disorders (MPD) (Nakata et al, 1995), B-cell non-Hodgkin

lymphoma (Bravo et al, 2000) and B-cell precursor ALL (Bene

et al, 1998). In normal cells, Kit has been shown to play

a major role in haematopoiesis (in the differentiation of

erythroid, lymphoid, megakaryocytic and myeloid precursors)

(Nocka et al, 1989), gametogenesis (Kissel et al, 2000), MC

development and function (Metcalfe, 2005; Valent et al,

2005), melanogenesis (Nocka et al, 1989; Halaban et al,

1993) and gastrointestinal function (Miettinen & Lasota,

2005).

In humans, the KIT gene is located at chromosome 4q12,

in the pericentromeric region of the long-arm of chromosome

4 (Yarden et al, 1987), adjacent to the highly homologous

PDGFRA gene (Spritz et al, 1994). Genomic DNA of human

KIT spans approximately 89 kb and contains 21 exons which

are transcribed/translated into a type III TK receptor with

a molecular mass of 145 kD and 976 amino acids in length

(Giebel et al, 1992). The five immunoglobulin-like loops of

the extracellular domain of Kit are encoded by exons 1–9

(amino acid residues: 23–520), the transmembrane domain by

exon 10 (amino acids: 521–543), the juxtamembrane autoin-

hibitory domain by exon 11 (amino acids: 544–581) and the

TK domain is encoded by exons 13–21 (amino acids: 582–

937). The first three immunoglobulin (Ig)-like loops of the

extracellular domain form the binding site for SCF or Kit

ligand (Lev et al, 1993; Lemmon et al, 1997; Longley et al,

2001), while the fourth and fifth loops play a role in

stabilising the SCF-induced Kit dimer (Blechman et al, 1995;

Zhang et al, 2000); in addition, it has been proposed that the

fifth Ig-like Kit domain is also required for the proteolytic

cleavage from the cell surface (Broudy et al, 2001). The

autoinhibitory juxtamembrane domain contains alpha-helical

elements whose proper configuration is essential for the

downregulation of tyrosine phosporylation (Lev et al, 1993;

Hubbard, 2004; Mol et al, 2004). In turn, the kinase portion

of Kit is composed of two domains which are separated by

a kinase insert: (1) the TK1 domain is constituted by the

small N-terminal lobe that expands from amino acids 582–

684 and contains the ATP binding site, and; (2) the TK2

domain is formed by the large C-terminal lobe containing the

phosphotransferase site and the activation loop (amino acids:

810–839) (Fig 1).

Review

ª 2007 The Authors14 Journal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 138, 12–30

Kit signalling pathways

At present it is well known that activation of the SCF/Kit

signalling pathway is associated with multiple biological effects

depending on the activated cell. Among others, these effects

include cell proliferation, maturation/differentiation, suppres-

sion of apoptosis, degranulation and changes in the adhesion

properties and motility of the activated cells (Blume-Jensen

et al, 1991). To date, numerous interactions of Kit with

different adaptor proteins have been described. Such interac-

tions lead to Kit-mediated activation of several signal trans-

duction pathways in common to many other growth factor

receptors, such as those involving the phosphatydylinositol

triphosphate (PI3)-kinase, protein kinase C (PKC), Ras/

mitogen-activated protein kinase (MAPK), and Janus kinase

(JAK)/signal transducers and activators of transcription

(STAT) pathways, that are responsible for the ultimate effects

of binding of SCF to Kit.

Upon non-covalent binding of a dimer of SCF to the second

and third immunoglobulin loops of the extracellular domain,

Kit undergoes dimerisation (Blume-Jensen et al, 1991; Zhang

et al, 2000), followed by transphosphorylation of two tyrosine

residues in the autoinhibitory juxtamembrane segment (Y568

and Y570) (Hubbard, 2004; Mol et al, 2004) (Fig 1). These

molecular changes lead to a conformational modification of

the activation loop from a compact, inactive structure to an

extended and active conformation. Subsequent transphosph-

orylation of Y823 in the activation loop stabilises the enzyme

in its most active form (Mol et al, 2004) (Fig 1). In turn, the

activated intrinsic TK activity of Kit leads to auto-phosphory-

lation of other tyrosine residues that serve as docking sites for

signal transduction molecules containing Src homology 2

(SH2) domains and other phosphotyrosine-binding domains.

The presence of multiple phosphorylation sites in the Kit

sequence reflects the existence of several regulatory and

catalytic domains. Based on the localisation of the phospho

(p)-tyrosine binding sites, three preferential regulatory sites of

Kit have been identified with activating and/or inhibitory

effects on one or more downstream signalling transduction

pathways: (1) the juxtamembrane domain; (2) the tyrosine

kinase insert; and (3) the activation loop in the TK2 domain

(Fig 1). Despite all the information accumulated in recent

years about the different Kit-associated signalling pathways, it

should be noted that most reported results derive from the

Fig 1. Schematic representation of the structure of Kit and its binding to other proteins and adaptors through Kit phosphorylated tyrosine residues.

A summary of the downstream pathways activated by these interactions and their major biological effects, is also provided in the two columns in the

right. Plain lines (—–) represent the hypothetical docking site; arrows ( fi ) indicate activation, while crossed lines (’) indicate inhibitory effects.

The vertical arrows indicate increased (›) and decreased (fl) effects on different cell functions. References: a (Wollberg et al, 2003); b (Tauchi et al,

1994); c (Kozlowski et al, 1998); d (Linnekin et al, 1997); e (Timokhina et al, 1998); f (Price et al, 1997); g (Roskoski, 2005b); h (Lennartsson et al,

1999); i (Weiler et al, 1996); j (Thommes et al, 1999); k (Shivakrupa & Linnekin, 2005); l (Lev et al, 1992); m (Blume-Jensen et al, 1998); n (Tanaka

et al, 2005); o (Gommerman et al, 2000); p (Trieselmann et al, 2003); q (Lennartsson et al, 2003).

Review


study of cell line models and that some discrepancies exist

regarding the exact interactions associated with the activity of

the phosphorylated Kit molecule, which could be related to the

specific cell type analysed (Boissan et al, 2000). Figure 1

summarises the interactions that have been established

between different domains of Kit and other regulatory adaptor

proteins, and the downstream activated/inhibited cell signal-

ling pathways regulated by these proteins [for a more detailed

description of these interactions see (Ronnstrand, 2004) and

(Roskoski, 2005a)].

KIT activating mutations

Occurrence of different point mutations and in frame

deletions/insertions of KIT have been shown to cause altera-

tions of the downstream Kit signalling pathways that convert

the KIT proto-oncogene into an active, dysregulated (ligand-

independent) oncoprotein capable of inducing neoplastic

transformation of normal Kit expressing cells (Kitamura et al,

1995). Since the first description of the activating KIT

mutation in the HMC-1 human MC cell line (Furitsu et al,

1993), multiple KIT mutations have been reported in patients

with mastocytosis; many of these mutations are associated with

constitutional Kit phosphorylation and downstream activa-

tion, independent of SCF binding (Table II; Fig 2). In order to

better understand the impact of KIT mutations, a few years ago

Longley et al (2001) proposed that the activating KIT muta-

tions be classified into two major groups based on their

topological localisation: the ‘regulatory type’ and the ‘enzy-

matic pocket type’ mutations. The former KIT mutations

typically affect regulation of the kinase activity of the Kit

molecule by disrupting the autoinhibitory a-helix (Ma et al,

1999), affecting the binding of signal transducing or regulatory

molecules to Kit and/or inducing ligand-independent dimeri-

sation and activation; most frequently these ‘regulatory type’

mutations occur at the juxtamembrane domain of Kit. In turn,

the ‘enzymatic pocket type’ mutations directly affect the

enzymatic site at the TK2 activation loop and induce activation

of Kit in the absence of dimerisation of the receptor.

KIT mutations cluster in relatively small regions – most

frequently at exon 11 and 17 – leading to aminoacid changes at

the juxtamembrane and TK2 domain of Kit, respectively. Less

frequently, KIT mutations are detected at exons 2, 8, and 9 or

at exons 13 and 14, coding for the extracellular and TK1 Kit

domains (Table II; Fig 2). Although single point KIT muta-

tions or in frame deletions/insertions are found in most

patients (Taniguchi et al, 1999; Beghini et al, 2004; Garcia-

Montero et al, 2006), more than one mutation has been also

reported in a few cases (Furitsu et al, 1993; Buttner et al, 1998;

Andersson et al, 2002; Willmore-Payne et al, 2006). It is

currently well established that, apart from mastocytosis, KIT

mutations can also be frequently observed in other neoplastic

disorders. Interestingly, a careful analysis of the KIT mutations

shows a clear association between the type of KIT mutation

and specific disease groups (Table II; Fig 2). Accordingly, in

the great majority (>90%) of adult cases with SM, mutations

in the activation loop of KIT (most frequently D816V) are

detected in MC in association with an aberrant CD25+

phenotype (Garcia-Montero et al, 2006), except among those

few patients with WDSM whose BM MC are typically negative

for both the D816V KIT mutation and cell surface CD25/CD2

(Garcia-Montero et al, 2006). Interestingly, D816V-negative

SM patients frequently carry other KIT mutations in the

activation loop involving codons 815, 816, 817, 820 and 839

(Pignon et al, 1997; Longley et al, 1999; Sotlar et al, 2003;

Garcia-Montero et al, 2006). Despite this, a few KIT mutations

at exon 11 have been also reported in individual SM patients in

association with the typical D816V KIT mutation (Furitsu

et al, 1993; Buttner et al, 1998). In contrast, the D816V KIT

mutation has only been sporadically found among AML with

Inv(16) and AML with t(8,21) (Beghini et al, 2004) – where

coexistence of two independent diseases (AML and SM)

appears to be relatively frequent (Pullarkat et al, 2003;

Escribano et al, 2004) – and in rare cases of seminomas

(Sakuma et al, 2003; Kemmer et al, 2004) and germinomas

(Sakuma et al, 2004) but not in GIST, which are typically

D816V-negative and commonly show activating mutations at

the regulatory juxtamembrane region of Kit (exon 11) (Hirota

et al, 1998; Nakahara et al, 1998; Taniguchi et al, 1999;

Andersson et al, 2002) (Table II; Fig 2). Of note, mutation

of KIT in mastocytosis has also been associated with decreased

expression of Kit (CD117) on the cell surface, which could

probably be due to an increased cleavage and release of the

mutated Kit molecule into the extracellular compartment,

leading to increased soluble levels of CD117.

At present, the exact mechanisms leading to the association

between specific KIT mutations and unique groups of diseases

remain unknown. Overall, such association could be related

either to upregulation of different signal transduction path-

ways by distinct activating KIT mutations and/or to the

existence of cell-type specific Kit-associated downstream

signalling pathways and transcription factors. In line with the

first hypothesis, recent results show the occurrence of mast-

ocytosis-associated germ line KIT mutations (F522C, K509I

and D816V) in patients that developed severe forms of

mastocytosis (Akin et al, 2004; Garcia-Montero et al, 2006;

Zhang et al, 2006). Nevertheless, the observation that most

familial forms of mastocytosis show an indolent clinical course

(Longley et al, 1999; Tang et al, 2004) would indicate that the

type of mutation, rather than its pattern of expression

(germline versus somatic KIT mutations), could be responsible

for the clinical behaviour of the disease. In turn, mutation of

the PDGFRA gene has been found in the eosinophilic

compartment of some of those few cases diagnosed with

either SM associated with chronic eosinophilic leukaemia (SM-

CEL, a variant of SM-AHNMD) or GIST, who do not have KIT

mutations (Heinrich et al, 2003; Pardanani et al, 2004),

pointing out the involvement of common downstream Kit/

PDGFRA activating pathways in the development of different

diseases and the potential influence of the cell type-specific

Review


Table II. KIT mutations that have been reported in patients with mastocytosis in comparison with other non-mast cell neoplasias also carrying KIT

mutations.

Disease Domain Exon Mutation

Consequence of

mutation

Frequency

(%) Comments References

Mastocytosis Extracellular 8 del D419 Unknown <5 Familial SM Hartmann et al (2005)

9 K509I Unknown <5 Familial SM Zhang et al (2006)

Transmembrane 10 F522C Activating <5 SM Akin et al (2004)

10 A533D Activating <5 Familial CM Tang et al (2004)

Juxtamembrane 11 V559I Activating <5 ASM Nakagomi and Hirota (2007)

11 V560G Activating <5 ISM, MCL Furitsu et al (1993); Buttner et al (1998)

Activation loop 17 R815K Unknown <5 Paediatric UP Sotlar et al (2003)

17 D816V Activating >90 Adult SM Garcia-Montero et al (2006)

17 D816Y Activating <5 SM Longley et al (1999)

17 D816H Unknown <5 SM-AML Pullarkat et al (2003)

17 D816F Activating <5 SM Longley et al (1999)

17 I817V Unknown <5 WDSM Garcia-Montero et al (2006)

17 insV815_I816 Unknown <5 SM Garcia-Montero et al (2006)

17 D820G Unknown <5 ASM Pignon et al (1997)

17 E839K Inactivating <5 UP Longley et al (1999)

GIST Extracellular 8 del D419 Unknown <5 FamilialGIST Hartmann et al (2005)

9 insA502_Y503 Unknown <5 Lasota et al (2000)

Juxtamembrane 11 del in region

K550_E561

Activating 25–50 Hirota et al (1998); Nakahara et al

(1998); Taniguchi et al (1999);

Andersson et al (2002)

11 V559D Activating 16 Hirota et al (1998)

11 V560D Activating 40 Andersson et al (2002)

11 D579del Activating Nakahara et al (1998)

TK1 13 K642E Activating <5 Isozaki et al (2000); Lasota et al (2000)

Kinase insert 14 del K704_N705 Unknown <10 Andersson et al (2002)

15 del S715 Unknown >50 Andersson et al (2002)

MPD Extracellular 2 D52N Unknown 10 Nakata et al (1995)

AML Extracellular 8 D419del Unknown 30 Inv(16) Gari et al (1999)

8 del+ins 416–419 Unknown 9 Inv(16) Beghini et al (2004)

Transmembrane 10 V530I Unknown 14 Inv(16) Gari et al (1999)

Activation loop 17 D816V Activating 20 t(8;21) and

inv(16)

Beghini et al (2004)

17 D816Y Activating 10 Beghini et al (2004)

17 D816H Activating <5 Beghini et al (2004)

17 N822K Activating Kasumi-1 cells.

t(8;21)

Beghini et al (2002)

Nasal and

nasal-type

NK/T-cell

lymphoma

Juxtamembrane 11 V559I Unknown <5 Hongyo et al (2000)

11 E561K Unknown 8 Hongyo et al (2000)

Activation loop 17 D816N Unknown <5 Hongyo et al (2000)

17 V825A Unknown 30 Hongyo et al (2000)

Seminomas Juxtamembrane 11 W557C Unknown 6 Intracranial

germinoma

Sakuma et al (2004)

11 W557R Activating <5 Sakuma et al (2003)

11 L576P Activating <5 Willmore-Payne et al (2006)

Activation loop 17 D816V Activating 3–10 Sakuma et al (2003, 2004); Kemmer

et al (2004); Willmore-Payne et al (2006)

D816H Activating £5 Sakuma et al (2003); Kemmer et al (2004)

D816Y Activating <5 Willmore-Payne et al (2006)

17 D816E Activating <5 Willmore-Payne et al (2006)

17 D820H Unknown <5 Willmore-Payne et al (2006)

17 D820V Unknown 6 Intracranial

germinoma

Sakuma et al (2004)

17 N822Y Unknown 6 Intracranial

germinoma

Sakuma et al (2004)

Review


activated pathways and transcription factors on determining

their nature. Of note, among SM-CEL cases, FIP1L1/PDGFRA

mutation is a molecular marker of CEL, but not for the MC

component (Valent et al, 2007b). In line with this later

hypothesis, Kissel et al (2000) showed in a knock-in mouse

model that receptor-mediated PI3-kinase signalling is critical

for spermatogenesis and oogenesis, but not for haematopoi-

esis, melanogenesis and primordial germ cell development.

In turn, the potential association of specific KIT mutations

with specific subtypes of mastocytosis remains to be elucida-

ted. Despite the fact that the D816V KIT mutation is present in

>90% of SM, with the exception of rare cases of WDSM and

MCL (Garcia-Montero et al, 2006), the exact frequency of this

mutation in patients with CM remains unknown. Accordingly,

while a significant proportion of CM cases with a childhood

onset do not show the D816V KIT mutation (Verzijl et al,

Table II. (Continued).

Disease Domain Exon Mutation

Consequence of

mutation

Frequency

(%) Comments References

17 N822K Activating <5 Kemmer et al (2004)

17 Y823C Activating <5 Kemmer et al (2004)

17 Y823D Activating <5 Kemmer et al (2004)

17 Y823N Activating <5 Willmore-Payne et al (2006)

Melanoma Juxtamembrane 11 L576P Activating <5 Willmore-Payne et al (2005)

The following KIT mutations have also been found in piebaldism (Murakami et al, 2004): C136R, A178T, M318G, Q347X, M541L, W557X, E583K,

F584L, F584C, G601R, V620A, A621T, H650P, G664R, C788R, R791G, R796G, G812V, W835R, T847P, E861A, P869S, Y870C.

CM, cutaneous mastocytosis; SM, systemic mastocytosis; AML, acute myeloblastic leukaemia; ISM, indolent systemic mastocytosis; UP, urticaria

pigmentosa; CML, chronic myeloid leukaemia; MF, myelofibrosis; MPD, myeloproliferative disorder; ASM, aggressive systemic mastocytosis; WDSM,

well-differentiated systemic mastocytosis; TK1, Kit tyrosine kinase domain 1; MCL, mast cell leukaemia; GIST, gastrointestinal stromal tumour; NK,

natural-killer.

Fig 2. Schematic representation of the structure of Kit, illustrating the known function of its domains and the localisation of the more frequently

observed mutations in the KIT sequence, in association to a specific disease or group of diseases represented by round-circled symbols (A, acute

myeloid leukaemia; G, gastrointestinal stromal tumor; L, nasal and nasal-type NK/T-cell lymphoma; M, mastocytosis; Me, melanoma; P, myelo-

proliferative disorder; S, seminoma/germinoma). Asterisks (*) indicate point mutation sites and underlined amino acids represent either in frame

deletion or insertion sites.

Review


2007) and KIT mutations at codons 509, 533, 815, 816 and 839

have been reported in adult CM patients (Longley et al, 1999;

Sotlar et al, 2003; Tang et al, 2004; Zhang et al, 2006), the

greatest frequency of KIT mutation is still found at codon 816

(Sotlar et al, 2003; Yanagihori et al, 2005).

Altogether these observations support the notion that

genetic examination of the KIT mutational status of purified

MC from BM or other extracutaneous organs (peripheral

blood, spleen, liver, lymph nodes and pleural fluid), in

addition to lesional skin, is of great help for the differential

diagnosis of cutaneous versus SM.

Impact of KIT mutation/activation in SM

In normal mature MC, activation of Kit signalling through

SCF leads to an increased cell proliferation and survival,

changes MC migration and adhesion, MC degranulation and

mediator release. In SM patients such effects are typically

enhanced by the occurrence of activating KIT mutations.

Increased mast cell proliferation and survival One of the most

frequent and evident clinical manifestations of mastocytosis is

the increased expansion of the MC compartment and the

accumulation of neoplastic MC in different organs and tissues.

This could be related to both an increase in MC proliferation

and MC survival due to constitutive activation of Kit. In line

with this, studies performed in murine BM-derived cultured

MC have shown that activation of the PI3-kinase, p21Ras and

MAPK pathways is essential for SCF-induced MC proliferation,

the former two pathways (but not MAPK) being dependent on

the presence of Kit p-Y719 (corresponding to human Y721)

(Serve et al, 1995). Furthermore, the presence of p-Y821

(corresponding to human Y823) (Serve et al, 1995), which

stabilises Kit in its most active form (Mol et al, 2004), is

essential for Kit-mediated mitogenesis and survival, but it is

independent of PI3-kinase, p21Ras and MAPK activation (Serve

et al, 1995). In turn, in vitro studies performed with murine MC

also show that cell cycle progression in SCF-induced MC is

mediated by expression of cyclin D3 and pRb phosphorylation

(Itakura et al, 2001). More recently, Tanaka et al (2005) have

demonstrated constitutive activation and translocation to the

nucleus of NFjB, a PI3-kinase downstream protein, in the SCF-

independent HMC-1V560G,D816V cell line model. These

observations would support the role of the PI3-kinase/NFjB

pathway in neoplastic MC transformation, which could occur

because of an altered cell cycle regulation due to an abnormally

higher expression of cyclin D3 and pRb phosphorylation.

Moreover, the same authors showed that SCF-independent

HMC-1V560G,D816V cell proliferation is also mediated by the

PKC pathway, while it was apparently independent of MAPK

activation (Tanaka et al, 2005).

It is known that in vitro-derived (Mekori et al, 2001) or

ex vivo-isolated (Akin et al, 2003) and cultured normal human

MC undergo rapid apoptosis, if SCF is omitted from the

culture medium. This is due to the fact that MC survival is also

maintained by activation of SCF/Kit-associated signalling

pathways. Accordingly, PI3-kinase plays a key role in promo-

ting cell survival through activation of the Akt serine/threonine

kinase, which in turn leads to phosphorylation and inhibition

of Bad, a pro-apoptotic protein that promotes MC death

(Blume-Jensen et al, 1998). Regulation of the anti-apoptotic

activity of Kit by the PI3-kinase/Akt pathway is controlled by

phosphorylation of Y721 at its kinase insert domain (Blume-

Jensen et al, 1998; Shivakrupa & Linnekin, 2005); thus,

conformational changes caused by mutations at the Kit

activation loop (e.g. D816V) that cause activation of the PI3-

kinase pathway, may also contribute to MC transformation

through an enhanced cell survival. In addition, the mammalian

target of rapamycin (mTOR), a downstream serine/threonine

kinase target of Akt in the PI-3K pathway, is also constitutively

activated in HMC-1 cells carrying the D816V KIT mutation

(Gabillot-Carre et al, 2006). Of note, D816V MC isolated from

SM patients, but not normal human MC, are sensitive to

rapamycin (Gabillot-Carre et al, 2006), highlighting the role of

the PI3K/Akt/mTOR pathway in the abnormal cell growth,

proliferation and survival of neoplastic MC. Furthermore,

Stat-5, a Jak-2 downstream regulator of MC proliferation and

survival (Shelburne et al 2003, Ikeda et al 2005), is also

constitutively activated in multiple cell lines and MC from

patients carrying different activating KIT mutations (Growney

et al, 2005; Pan et al, 2007).

Despite the overall increased numbers of MC found in the

skin, BM and other tissues in patients with SM, a great

variation in the overall MC burden exists among individual

patients, even in cases carrying the same KIT mutation. In line

with this, KIT mutations have been identified not only in MC

from SM patients but they are also frequently detected in other

BM haematopoietic cell compartments, particularly among

CD34+ haematopoietic progenitor and precursor cells (HPC),

eosinophils and, to a lower extent, within the CD34)

neutrophil and monocytic precursors (Garcia-Montero et al,

2006). Interestingly, the frequency of cases showing involve-

ment of KIT mutation in BM cell compartments other than

MC is significantly lower among patients included within those

types of mastocytosis associated with a good prognosis, in

comparison with cases of ASM, MCL and SM-AHNMD.

Altogether, these results could suggest that SM patients

showing multilineage involvement of BM haematopoietic cells

could represent more advanced stages of the disease. However,

the relatively stable course of the disease in most SM patients

and the observation that the same KIT mutation (e.g. D816V)

can associate with indolent (good-prognosis) and malignant

tumours (Garcia-Montero et al, 2006) highlight the potential

role of other genetic and/or epigenetic factors in determining

the progression/outcome of the disease; further studies are

required in this regard.

Altered mast cell migration and adhesion The pattern of MC

involvement in SM typically reflects the tissue distribution of

normal MC (Valent et al, 2005). However, significant

Review


differences can be observed between distinct forms of the

disease. Accordingly, while skin involvement represents

a hallmark of CM and ISM, ASM and MCL patients

frequently show involvement of BM, spleen, liver, lymph

nodes and/or peripheral blood in the absence of cutaneous

lesions (Valent et al, 2001). Similarly, some ISM patients also

show recurrent anaphylaxia episodes together with BM

infiltration by MC, in the absence of cutaneous lesions (Akin

& Metcalfe, 2003). Altogether, these findings point out the

occurrence of variable patterns of involvement of different

tissues in mastocytosis.

It is currently well-established that MC migration to

peripheral tissues is also mediated by SCF/Kit signalling. SCF

alone or in combination with interleukin-3, is a potent

attractant for MC (Meininger et al, 1992). In addition, locally

produced SCF may also exert an inhibitory effect on the

chemotactic migration of MC induced by IgE-specific antigens,

contributing to the accumulation of MC at the peripheral sites

in allergic and non-allergic conditions (Sawada et al, 2005).

SCF-independent activating D816V KIT mutation may induce

alterations in this finely regulated mechanism, enhancing

chemotaxis of CD117+ (Kit+) cells (Taylor et al, 2001) and

inducing an abnormal accumulation of MC in different tissues

(e.g. BM and/or peripheral blood of ASM and MCL patients).

Activation of Kit has been also shown to mediate MC

adhesion to the extracellular matrix via fibronectin, through

the activation of fibronectin receptors on MC (Dastych &

Metcalfe, 1994); this effect is mediated by activation of PI3-

kinase (Serve et al, 1995). The key role of PI3-kinase in cell

adhesion has been shown to be dependent on p-Y719

(equivalent to human Y721) in the Kit kinase insert domain

of normal MC differentiated from murine BM haematopoietic

precursor cells, while residual adhesion activity is also partially

due to an Src-dependent PI3-kinase activation mechanism

(Serve et al, 1995). Surprisingly, Kit-mediated cell adhesion

appears to be independent of the presence of mutations at

Y821 (equivalent to human Y823) (Serve et al, 1995).

Augmented mast cell degranulation Over a decade ago, Costa

et al (1996) showed that the injection of SCF induced MC

degranulation and increased levels of MC tryptase and

histamine in normal subjects. However, recent findings

indicate that human MC degranulation is driven through

binding of IgE/antigen immunocomplexes to the high-affinity

IgE receptor (FceRI) on the surface of MC (Tkaczyk et al,

2004). SCF acts in synergy with antigens (Tkaczyk et al, 2004)

to markedly enhance degranulation and production of

cytokines by MC. SCF activates phospholipase C-c and

induces calcium mobilisation, leading to MC degranulation

when added together with the antigen (Hundley et al, 2004),

which in turn induces an FceRI-mediated activation of PKC.

This synergy in MC degranulation is mediated by tyrosine

phosphorylation of non-T-cell activation linker (NTAL) which

acts as a pivotal link between the signalling cascades following

Kit activation and cross-linking of FceRI (Tkaczyk et al, 2004).

Moreover, the Stat-5 molecule, a critical factor in IgE-induced

MC activation (Barnstein et al, 2006), is constitutively

phosphorylated in cells carrying activating KIT mutations

(Pan et al, 2007). Accordingly, constitutive, ligand-

independent Kit mutations would favour an enhanced MC

response against antigen and/or physical stimuli present in the

MC environment, leading to the release of different MC

mediators and the associated clinical symptoms (e.g. pruritus,

severe anaphylactic episodes and abdominal pain). In fact,

increased serum tryptase levels is a minor diagnostic criteria

for SM (Valent et al, 2001) and elevated serum tryptase levels

are associated with different subtypes of SM (Table I) (Valent

et al, 2001; Garcia-Montero et al, 2006). In addition, in SM

patients with recurrent anaphylaxia, serum tryptase levels also

show a significant increase during the anaphylactic episodes.

Although a clear relationship has been found between some

allergens and the occurrence of mastocytosis-associated

anaphylactic episodes (e.g. wasp venom), in many patients,

the stimuli responsible for massive MC degranulation remains

to be identified.

Kit-targeted therapy in mastocytosis

In the last decade major advances have been achieved in the

field of molecular-targeted therapy, in which drugs are selected

on the basis of specific molecular abnormalities causing

individual diseases. Among the new drugs developed, the

STI571 TK inhibitor (Imatinib mesylate or Gleevec) has been

considered as ‘a paradigm of targeted therapies’ (Druker, 2004)

representing a novel molecular approach to the treatment of

BCR/ABL+, PDGFR- and KIT-mutated malignancies.

Imatinib was first identified as a potent inhibitor of the c-abl

protein kinase and it was shown to have similar activity against

v-abl and both the p210 and p190 forms of bcr/abl (Druker

et al, 1996; Carroll et al, 1997; Beran et al, 1998). Moreover,

imatinib was found to inhibit the kinase activity of PDGFR aand b chains (Druker et al, 1996; Carroll et al, 1997).

Imatinib mesylate and Kit

In vitro studies have proven that imatinib inhibits wild type Kit

(wtKit) (Zermati et al, 2003) and suppresses proliferation of

the HMC-1V560G cell line, while it is ineffective on inhibiting the

growth of HMC-1V560G,D816V cells (Akin et al, 2003). Apart

from wtKit, Kit molecules carrying mutations in the extracel-

lular, transmembrane and juxtamembrane domains, such as

V560G (Akin et al, 2003), F522C (Akin et al, 2004) and K509I

(Zhang et al, 2006), remain sensitive to imatinib. In contrast,

several experiments have provided compelling evidence regard-

ing the resistance against the growth-inhibitory effects of

imatinib on cells carrying the D816V KIT mutation (Ma et al,

2002; Akin et al, 2003). In fact, imatinib did not show

preferential ex vivo cytotoxicity against neoplastic BM MC

obtained from patients with mastocytosis who carried the

D816V KIT mutation (Ma et al, 2002; Akin et al, 2003); in

Review


addition, structural changes in the Kit kinase domain induced

by the D816V KIT mutation have been identified as responsible

for preventing binding of imatinib to Kit (Mol et al, 2004).

Clinical studies using imatinib mesylate in mastocytosis

To date, 31 adult SM cases treated with imatinib mesylate have

been reported in the literature (Table III) and mutational

studies of KIT were performed in 27 of these patients. Of these,

13 corresponded to good-prognosis categories (10 ISM and 3

SSM), 12 were ASM (two were mastocytosis with a pediatric-

onset and either transmembrane or juxtamembrane KIT

mutations associated with an aggressive clinical course), one

case corresponded to a SM-AHNMD, another to a WDSM,

three cases were SM-CEL and the remaining case was SM with

FIP1L1/PDGFRA gene rearrangement [data on eosinophilia

was not provided by authors (Droogendijk et al, 2006)]. Such

a lack of homogeneous criteria in patients’ selection leads to

increased difficulty in adequately evaluating the response to

therapy. In any case, in line with the results of in vitro analyses,

these studies showed significant clinical responses to imatinib

in cases lacking the D816V mutation as well as in SM-AHNMC

carrying another imatinib-target, such as SM-CEL with

FIP1L1/PDGFRA gene rearrangements (Pardanani et al,

2003b; Elliott et al, 2004). However, overall complete response

(CR) was obtained in only 4/31 cases (13% of the cases

revised), corresponding to one-third of all cases lacking the

D816V KIT mutation (Pardanani et al, 2003b,c) and SM-CEL

patients carrying the FIP1L1/PDGFRA fusion gene (Elliott

et al, 2004). An additional CR was reported in a case of SM

associated with chronic myeloid leukaemia (Agis et al, 2005);

nevertheless, this patient had been previously treated with

hydroxycarbamide and, based on the effectiveness of hydrox-

ycarbamide in SM associated with MPD or MDS (Sheikh et al,

2006), the role of imatinib in inducing CR in this case could

not be accurately established (Agis et al, 2005). Finally,

sustained response to imatinib has also been obtained in

a rare case of WDSM carrying the F522C transmembrane KIT

mutation, associated with an aggressive course of the disease

(Akin et al, 2004) and in a case of familial mastocytosis

carrying the K509I juxtamembrane KIT mutation (Zhang et al,

2006). In the WDSM patient, a dramatic improvement in

clinical symptoms, bone pain, and quality of life, together with

a decrease in both BM MC infiltration (from 50% to <10%)

and serum tryptase levels (from 173 to 20 ng/ml), was noted;

at present, she remains alive under imatinib therapy, showing

good clinical condition in the absence of an increase in BM

MC and serum tryptase levels of 5–17 lg/l [J. Robyn,

Laboratory of Allergic Diseases (NIH/NIAID), Bethesda, MD,

USA, personal communication, December 2006]. Similar

results were obtained with imatinib therapy in the second

case. Interestingly, a clear predominance of BM MC showing

a ‘round-shape’ morphology – in the absence of CD25

expression in one of them (Akin et al, 2004) – was observed

in these two patients, suggesting that a careful examination of

both the morphology and immunophenotype of BM MC may

provide valuable criteria for the identification of this subtype

of mastocytosis, where mutational analysis of KIT could be of

great utility for predicting response to imatinib (Akin et al,

2004). In line with this, we have recently reported that most

WDSM patients do not carry the D816V KIT mutation

(Garcia-Montero et al, 2006).

Recently, Droogendijk et al (2006) reported on the occur-

rence of different degrees of response to imatinib administered

in combination with glucocorticoids in a group of D816V-

positive mastocytosis patients; the combined use of these two

drugs makes it difficult to evaluate response to imatinib, as

glucocorticoids alone may also decrease the neoplastic MC

burden, as well as the MC-mediator related symptoms –

headache, pruritus, flushing and mainly, abdominal discom-

fort. Thus, caution should be taken when considering the

effectiveness of imatinib in such cases.

These results, together with the good life expectancy and

quality of life of patients (see below), indicate that a risk-benefit

based therapy should be used in mastocytosis, even when

targeted-therapies are considered. As a consequence, in vitro

studies on the effectiveness of the drug, as well as on its short-

and long-term in vivo toxicity, are required. As mentioned

above, the D816V KIT mutation is found in the vast majority of

adult patients with sporadic SM (Garcia-Montero et al, 2006)

and thus, imatinib therapy will not be appropriate for most of

these patients. Furthermore, the life expectancy of 161 patients

suffering from pure CM, ISM, BMM and WDSM, after

a median follow-up of 152 months (range 6–476 months, with

45 cases having a follow up of >20 years), was similar to that

observed among individuals who have not mastocytosis

[Spanish Network on Mastocytosis (REMA), unpublished

data). This is particularly relevant because of the adverse

effects described after imatinib therapy, including cardiotox-

icity (Kerkela et al, 2006; Park et al, 2006), as well as the

development of different clonal abnormalities in CML patients

treated with imatinib (Bumm et al, 2003; Herens et al, 2003;

Medina et al, 2003; Meeus et al, 2003; O’Dwyer et al, 2003;

Alimena et al, 2004; Gozzetti et al, 2004; Guilbert-Douet et al,

2004) including trysomy 8 (O’Dwyer et al, 2003; Bernardeschi

et al, 2004; Terre et al, 2004; Tunca & Guran, 2005) and

exceptional cases of MDS and acute leukaemia (Alimena et al,

2004; Kovitz et al, 2006). In this sense, the experience gained in

the long-term follow-up of non-haematological malignant

diseases treated with imatinib, in which haematopoietic stem

cells are not involved, such as GIST, could provide some light

on the real risk of developing secondary haematological

malignancies in patients treated with imatinib mesylate.

Taking all these findings and considerations together, the

current REMA recommendations regarding the use of imatinib

therapy in mastocytosis only include (1) those exceptional cases

of ASM and MCL who are negative for the D816V KIT mutation;

(2) SM patients carrying juxtamembrane KIT mutations (e.g.

K509I and F522C) associated with an aggressive course of the

disease; and (3) aggressive cases of SM-AHNMD associated with

Review


Table III. Clinical characteristics and outcome of patients with mastocytosis treated with imatinib.

Case

number

Age

(years) Category Mutation

Previous

treatment/s

Concomitant

therapy Best response/Follow-up Reference

1 46 ISM None None None MR (CR) 10 months Pardanani et al (2003b,c)

2 31 ASM None HC, IFN None MR (CR) 19 months Pardanani et al (2003b,c)

3 72 ASM None HC None MR (CR) 1 month Pardanani et al (2003b,c)

4 61 ASM None CHOP None NR 3 months Pardanani et al (2003b,c)

5 45 ASM None IFN, Pred

2CdA

None MR (IR)

MR 10Æ5 months

Pardanani et al (2003b,c)

6 70 ASM None IFN None MR (IR) 8 months Pardanani et al (2003b,c)

7 30 ISM-CEL FIP1L1-PDGFRA HC, IFN None MR (CR) 19 months Elliott et al (2004)

8 50 SM FIP1L1-PDGFRA None in the

previous 6 months

Prednisone* MR (CR) 3 months Droogendijk et al (2006)

9 32 ISM-CEL

HIV+

FIP1L1-PDGFRA None None Response of SM not

evaluable

Merante et al (2006)

10 51 ISM-CEL FIP1L1-PDGFRA HC, CE, IFN None MR 24 months Florian et al (2006)

11 26 Familial ASM K509I IFN None PR (GPR) 24 months Zhang et al (2006)

12 25 WDSM F522C IFN None MR (IR) 39 months Akin et al (2004); J. Robyn,

personal communication

(12/10/2006)

13 43 SM -AHNMD

(CML)

D816V

bcr/abl

HU None MR (CR) 6 months Agis et al (2005)

14 78 ASM D816V IFN None NR 9 months Pardanani et al (2003b,c)

15 85 ASM D816V HU None NR 5 months Pardanani et al (2003b,c)

16 33 ASM D816V None in the

previous 6 months

None NR 4 months Musto et al (2004)

17 49 SSM D816V None in the

previous 6 months

Prednisone* MR (IR) 3 months Droogendijk et al (2006)

18 45 ISM D816V None in the

previous 6 months

Prednisone* NR 3 months Droogendijk et al (2006)


previous 6 months

Prednisone* MR (PCR) 3 months Droogendijk et al (2006)


previous 6 months



previous 6 months



previous 6 months



previous 6 months


24 73 ASM D816V IFN Prednisone* NR 3 months Droogendijk et al (2006)


previous 6 months



previous 6 months



previous 6 months


28 42 ASM ND IFN None NR 72 months Hennessy et al (2004)

29 80 ASM ND IFN, prednisone None NR 72 months, dead Hennessy et al (2004)

30 59 ISM ND None in the

previous 6 months

Prednisone* PR 3 months Droogendijk et al (2006)

31 60 ISM ND None in the

previous 6 months


H, hepatomegaly; S, splenomegaly; HC, hydroxycarbamide; IFN, interferon-a; ND, not done; CHOP, cyclophosphamide, doxorubicin, vincristine

and prednisone; CR, complete remission; MR, major response; NR, no response; PR, partial response; PCR, pure partial response; IR, incomplete

remission; BM, bone marrow; GPR, good partial response; ISM, indolent systemic mastocytosis; CEL, chronic eosinophilic leukaemia; ASM,

aggressive systemic mastocytosis; WDSM, well-differentiated systemic mastocytosis; CE, corticosteroids; 2CdA, cladribine.

*Prednisone treatment at 30 mg/d the first 2 weeks. Response criteria follow the guidelines proposed by Valent et al (2003).

Review


FIP1L1/PDGFRA gene rearrangements (SM-CEL). Of note, in

these latter cases, imatinib therapy should be prescribed to bring

eosinophil counts and thus the CEL-component under control,

whereas the SM component of SM-CEL typically behaves as an

indolent disease that does not require imatinib or any other

targeted or cytoreductive therapy.

Other tyrosine kinase inhibitors and mastocytosis

On the basis of experimental data, other TK inhibitors, such as

PKC412 (Gotlib et al, 2005; Gleixner et al, 2006) and dasatinib

(Schittenhelm et al, 2006; Shah et al, 2006), have also been

used to treat patients with mastocytosis (Table IV). Accord-

ingly, PKC412 was initially used on a compassionate basis in

a case of MCL, resulting in a good partial response associated

with marked improvement in the patient performance status,

resolution of organ dysfunction and a dramatic decrease in

both BM MC infiltration and circulating MC; however, after

3 months of therapy, progression to AML was observed

(Gotlib et al, 2005). A phase II study designed to assess

PKC412 efficacy and safety in ASM/MCL patients is currently

in progress (Gotlib et al, 2006); preliminary results of this

study showed partial (but not complete) responses in six of

nine ASM cases. In order to improve the efficacy of PKC412,

synergistic interactions with AMN107 therapy have been

evaluated in vitro showing induction of apoptosis and down-

regulation of CD2 and CD63 in both HMC-1V560G,D816V cells

and in primary neoplastic MC (Gleixner et al, 2006).

In a pilot Phase II trial for SM with dasatinib – primarily an

abl/src inhibitor with other TK activities (Verstovsek et al,

2006) – in which response was assessed after a minimum of

3 months (three cycles) of therapy, a total of 24 cases were

evaluated for response and toxicity; these included six patients

with ASM, four with SM-AHNMD – [two with chronic

myelomonocytic leukaemia, one with myelofibrosis (MF), one

with an hypereosinophilic syndrome (SM-HES)] and 14 with

ISM with uncontrolled symptoms despite optimal supportive

care measures. Only two patients (8%) showing a low MC

burden achieved CR – (one case each of SM-MF and SM-

HES), with a relatively limited overall response rate of 37%.

Other TK inhibitors that have shown cytotoxic activity in vitro

in both cell line and mouse models include semaxinib (SU5416)

and EXEL-0862; both compounds have been shown to be

effective in blocking the activity of Kit carrying the D814V

mouse mutation, equivalent to the human D816V KIT mutation

(Table IV). Accordingly, Kosmider et al (2006) have shown that

semaxinib is capable of inducing growth arrest and apoptosis in

murine cells carrying the D814V KIT mutation, by inhibiting Kit

autophosphorylation and activation of Akt, Erk1/Erk2 and Stat-

3 downstream signalling pathways. In turn, AP2346, a new

potent ATP-based inhibitor that targets the activation-loop of

Kit mutants, would selectively inhibit proliferation of human

D816V-positive cell lines without disrupting normal haemato-

poietic progenitor-cell growth (Corbin et al, 2005).

Other alternative molecular targeted-therapies inmastocytosis

Proteins of the Kit signalling pathway, other than the Kit

receptor itself, have been also evaluated as potential targets for

the treatment of patients with SM and D816V KIT mutation.

Accordingly, current in vitro observations suggest a therapeutic

Table IV. Recently developed molecular-targeting drugs capable of inhibiting wild-type and/or mutated Kit and their downstream signal

transduction pathways.

Drug Target Tumour/cell line KIT mutations References

Imatinib Kit GIST-T1 del (exon 11) Nakatani et al (2005)

Imatinib Kit BM MC F522C Akin et al (2004)

Imatinib Kit HMC-1 V560G Ma et al (2002)

17-AAG Hsp90 Kasumi-1, HMC-1

BM MC

N822K, D816V,

V560G

Fumo et al (2004); Yu et al (2006)

Geldanamycin Hsp90 GIST-T1 del (exon 11) Nakatani et al (2005)

IMD-0354 NF-jB HMC-1 D816V, V560G Tanaka et al (2005)

PKC412 TK HMC-1 and neoplastic MC (MCL) D816V Gotlib et al (2005); Gleixner et al (2006)

Dasatinib Src, Kit MC and leukemic cell lines D816 Schittenhelm et al (2006)

AP23464 Kit (Akt, Stat-3) HMC-1 D816V Corbin et al (2005)

AMN107 Kit, Abl, PDGFR HMC-1 V560G, D816V Gleixner et al (2006)

SU5416

(Semaxinib)

TK Erythroleukemic cells (from

Spi-1/PU.1 transgenic mouse)

D814V mouse* Kosmider et al (2006)

EXEL-0862 Kit, Stat-3, Stat-5 HMC-1

BM MC

V560G, D816V Pan et al (2007)

Rapamycin mTOR HMC-1

BM MC

D816V Gabillot-Carre et al (2006)

BM, bone marrow; TK, tyrosine kinase; Hsp90, heat shock protein 90; MC, mast cell; GIST-T1, gastrointestinal stromal tumour T1 cell line; HMC-1,

human mast cell-derived cell line; MCL, mast cell leukaemia.

*Mouse KIT mutation corresponding to human D816V.

Review


potential for some compounds that interfere in the Kit

signalling pathways. Among these compounds, the novel

IMD-0354 NF-jB inhibitor, together with the ansamycin

antibiotic derivatives 17-AAG and geldanamycin, which target

heat shock protein 90 (Hsp90), appear to be of particular

interest. Accordingly, IMD-0354 has been shown to induce

a complete suppression of proliferation of HMC-1D816V,V560G

cells, but not of cord blood-derived normal human MC, in

which NF-jB is not activated (Tanaka et al, 2005). In turn, the

use of some Hsp90 inhibitors (e.g. 17-AAG) which may also

target tyrosine phosphorylation of Kit (e.g. geldanamycin) has

been associated with an apoptotic effect and a decline in Kit

protein levels (Yu et al, 2006), as well as with an inhibition of

the interaction between Hsp90 and Kit in GIST-T1 cells

(Nakatani et al, 2005), respectively. Furthermore, encouraging

results have been obtained in the ex vivo treatment of

neoplastic BM MC from patients with SM with 17-allylami-

no-17-demethoxygeldanamycin (Fumo et al, 2004), the levels

and activity of both Kit and other downstream signalling

molecules (e.g. Akt and Stat-3) being downregulated in HMC-

1 cells after 17-AAG treatment (Fumo et al, 2004). These

results are in line with recent pharmacological evidences

supporting the notion that Hsp90 may contribute to the

stabilisation of Kit (Nakatani et al, 2005; Yu et al, 2006)

through an inhibitory effect on its proteosomal degradation.

In addition, several humanised monoclonal antibody

(hMAb) derivatives are being tested as potential targeted drugs

against neoplastic MC. The anti-CD44 hMAb A3D8 decreased

proliferation of both MC-derived cell lines and primary

neoplastic MC obtained from patients with MCL and SSM

(Boehm et al, 2005). Alemtuzumab, an anti-CD52 hMAb

antigen, has shown selective reduction in eosinophil counts

associated with clinical benefit in HES patients (Sefcick et al,

2004) and has been also tested in MC disorders (Santos et al,

2006). Mylotarg, an anti-CD33 hMAb conjugated to an

antitumoral antibiotic (calicheamicin) has been also effective

in vitro against MC (Krauth et al, 2007). In turn, LMB-2, an

anti-CD25 hMAb Fv fragment fused to truncated Pseudomonas

aeruginosa exotoxin A, produced reduction in CD25+ clonal

cell numbers from several haematological malignancies (Kre-

itman & Pastan, 2006), including MC in BM cultures obtained

from patients with mastocytosis (Escribano et al, 2006).

Despite their in vitro proven efficacy, some of these targeted

immunotoxins counteract growth of both normal and neo-

plastic MC (Krauth et al, 2007), while others produce signifi-

cant side effects including, infusion syndrome (Krauth et al,

2007), lymphopenia (Klastersky, 2006), hepatotoxicity and

vascular leak syndrome (Escribano et al, 2006). Therefore, the

exact benefit of most of these hMAb derivative drugs for the

treatment of severe MC disorders remains to be determined.

Concluding remarks

Overall, it can be concluded that mutation-associated constitu-

tive activation of Kit in mastocytosis may contribute to a better

understanding of the clinical manifestations of the disease.

Despite this, a significantly high variability in MC burden and

clinical symptoms and signs of the disease can not be explained

solely on the basis of the KIT mutation. Other factors, such as the

specific type of mutation, the presence of antigen/IgE immu-

nocomplexes in the MC microenvironment, the natural history

of the disease in individual patients, MC burden and associated/

secondary genetic alterations, could contribute to such hetero-

geneity and their real relevance deserves further investigation.

Independent of these factors, the recent development of new

drugs targeting Kit and other proteins involved in its down-

stream activation pathways, have opened new perspectives in the

treatment of SM patients requiring cytoreductive therapy.

Grants and financial support

This work was supported by grants from the Instituto de Salud

Carlos III (ISCIII), Fondo de Investigaciones Sanitarias (FIS)

of the Ministerio de Sanidad y Consumo, Spain (grants

PI050726, PI061377, PI05769, PI06529, REMA G03/007 and

RETICS RD06/0020/0035-FEDER) and from the Fundacion

MMA. ACG-M and LS are recipients of grants from FIS

(CP03/00035 and CMO3/0043, respectively).

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