Immunoglobulin

and

free

light

chain

abnormalities

in

Gaucher

disease

type

I

data

from

an

adult

cohort

of

patients

and

review

of

the

literature

Gaucher

disease

type

I

the

most

common

lysosomal

storage

disorder

is

associated

with

immunoglobulin

abnormalities

We

studied

the

prevalence

risk

factors

pathogenesis

and

effect

of

enzyme

relation

therapy

-LRB-

ERT

-RRB-

on

gammopathies

in

an

adult

Gaucher

disease

type

I

cohort

-LRB-

N

-RRB-

and

related

the

results

to

a

review

of

the

currently

available

literature

Polyclonal

gammopathies

and

monoclonal

gammopathy

of

undetermined

significance

-LRB-

MGUS

-RRB-

in

our

adult

GD

I

cohort

were

found

in

and

of

patients

These

results

are

similar

to

the

data

from

the

literature

and

correspond

to

the

increased

risk

of

multiple

myeloma

-LRB-

MM

-RRB-

that

has

been

described

The

prevalence

of

MGUS

in

our

cohort

increased

with

age

but

was

not

associated

with

disease

severity

or

exposure

time

The

serum

levels

of

free

light

chains

of

immunoglobulins

were

measured

and

were

not

found

predictive

for

the

development

of

MGUS

or

MM

Levels

of

pro-

as

well

as

anti-inflammatory

cytokines

growth

factors

and

chemokines

especially

those

involved

in

inflammation

and

B-cell

function

are

disturbed

in

GD

I

with

the

most

impressive

and

consisting

elevations

for

and

pulmonary

and

activation-regulated

chemokine

A

beneficial

effect

of

ERT

on

the

occurrence

and

progression

of

gammopathies

was

suggested

from

longitudinal

data

Introduction

Gaucher

disease

type

I

-LRB-

GD

I

OMIM

-RRB-

is

the

most

common

lysosomal

storage

disorder

The

disease

is

characterized

by

a

deficiency

of

the

lysosomal

enzyme

glucocerebrosidase

-LRB-

glucosylceramidase

-RRB-

which

leads

to

the

accumulation

of

glucocerebroside

in

macrophages

The

lipid

laden

macrophages

are

called

Gaucher

cells

and

are

mainly

found

in

liver

spleen

and

bone

marrow

resulting

in

the

most

important

clinical

features

hepatosplenomegaly

skeletal

disease

and

cytopenia

-LSB-

-RSB-

Since

GD

I

can

be

effectively

treated

with

recombinant

glucocerebrosidase

-LRB-

enzyme

replacement

therapy

ERT

imiglucerase

Genzyme

Cambridge

MA

USA

-RRB-

More

recently

substrate

reduction

therapy

-LRB-

miglustat

Actelion

Pharmaceuticals

Basel

Switzerland

-RRB-

has

been

registered

for

more

attenuated

disease

An

associated

feature

of

GD

I

is

the

high

frequency

of

polyclonal

and

monoclonal

gammopathies

-LSB-

-RSB-

In

the

general

population

polyclonal

hypergammaglobulinemia

is

a

common

clinical

finding

especially

in

the

setting

of

chronic

inflammation

-LSB-

-RSB-

resulting

from

an

overproduction

of

immunoglobulins

by

plasma

cells

or

B-lymphocytes

An

abnormal

increase

of

monoclonal

immunoglobulins

or

fragments

thereof

-LRB-

M

protein

-RRB-

can

be

found

in

pre-malignant

conditions

such

as

MGUS

-LRB-

monoclonal

gammopathy

of

undetermined

significance

-RRB-

and

malignancies

of

the

B-cell

lineage

such

as

multiple

myeloma

-LRB-

MM

-RRB-

primary

amyloidosis

Waldenström

macroglobulinemia

chronic

lymphocytic

leukemia

and

B-cell

lymphoma

MGUS

is

found

in

approximately

of

adults

over

the

age

of

increasing

to

at

the

age

of

-LSB-

-RSB-

Of

these

individuals

per

year

shows

progression

to

MM

or

a

related

malignancy

Accordingly

in

GD

I

the

risk

of

MM

is

strongly

increased

as

has

been

reported

in

recent

studies

-LSB-

-RSB-

The

risk

increases

with

advancing

age

-LSB-

-RSB-

but

whether

disease

severity

is

a

risk

factor

is

currently

unknown

The

mechanism

explaining

the

relationship

between

GD

and

immunoglobulin

abnormalities

is

far

from

understood

Studies

have

mainly

focused

on

cytokines

as

the

mediators

between

Gaucher

cells

surrounding

macrophages

and

B

cells

Plasma

levels

of

several

pro-inflammatory

cytokines

such

as

tumor

necrosis

factor

-LRB-

TNF

-RRB-

α

interleukin

-LRB-

IL

-RRB-

and

as

well

as

the

anti-inflammatory

were

found

to

be

increased

in

GD

to

variable

extents

-LSB-

-RSB-

and

as

well

as

hepatocyte

growth

factor

-LRB-

HGF

-RRB-

are

also

involved

in

the

pathogenesis

of

MM

-LSB-

-RSB-

Other

interesting

factors

known

to

be

highly

elevated

in

GD

patients

are

pulmonary

and

activation-regulated

chemokine

-LRB-

PARC

-RRB-

-LSB-

-RSB-

which

is

involved

in

initiating

the

adaptive

immune

response

Two

studies

have

reported

on

the

effect

of

ERT

with

some

decrease

in

polyclonal

gammopathies

-LSB-

-RSB-

and

a

decline

in

monoclonal

M-protein

level

in

a

single

case

-LSB-

-RSB-

A

relatively

new

method

to

identify

and

monitor

monoclonal

gammopathies

is

by

quantifying

immunoglobulin

free

light

chains

-LRB-

FLC

-RRB-

circulating

κ

and

λ

chains

that

are

not

bound

to

the

immunoglobulin

heavy

chains

using

a

highly

sensitive

automated

immunoassay

-LSB-

-RSB-

An

abnormal

FLC

ratio

is

an

important

independent

risk

factor

for

progression

of

MGUS

to

MM

or

a

related

disorder

-LSB-

-RSB-

In

addition

measuring

FLC

is

a

sensitive

additional

tool

to

identify

monoclonal

gammopathies

-LSB-

-RSB-

In

GD

I

in

which

the

incidence

of

MGUS

and

MM

is

highly

increased

-LSB-

-RSB-

the

FLC

assay

may

provide

a

useful

diagnostic

tool

for

early

detection

of

MGUS

and

MM

In

this

study

we

present

new

data

on

the

prevalence

of

polyclonal

and

monoclonal

gammopathies

from

a

large

adult

GD

I

cohort

and

review

the

currently

available

literature

Second

we

studied

whether

age

disease

severity

and

exposure

time

are

risk

factors

for

the

development

of

monoclonal

gammopathies

and

if

this

risk

is

subsequently

decreased

by

the

administration

of

ERT

In

addition

for

the

first

time

we

investigated

the

role

of

FLC

as

a

predictor

of

the

development

of

monoclonal

gammopathies

in

GD

I.

Finally

the

pathogenesis

of

immunoglobulin

abnormalities

in

GD

patients

was

studied

by

means

of

pro-

and

anti-inflammatory

cytokine

chemokine

and

growth

factor

levels

and

is

discussed

in

relation

to

previous

literature

Materials

and

methods

Patients

and

study

design

The

files

were

reviewed

and

archived

serum

samples

were

collected

of

all

adult

GD

type

I

patients

that

were

known

from

the

outpatient

clinic

for

inherited

metabolic

disorders

at

the

Academic

Medical

Centre

-LRB-

AMC

-RRB-

Amsterdam

The

Netherlands

-LRB-

N

-RRB-

The

diagnosis

GD

was

based

on

measurement

of

deficient

glucocerebrosidase

activity

in

leukocytes

and

genotyping

-LSB-

-RSB-

Of

the

patients

received

ERT

according

to

an

individualized

dosing

protocol

-LSB-

-RSB-

and

two

patients

received

substrate

reduction

therapy

Data

on

age

sex

splenectomy

genotype

severity

score

index

-LRB-

SSI

-LSB-

-RSB-

-RRB-

creatinine

urea

chitotriosidase

and

the

presence

and

type

of

immunoglobulin

class

of

monoclonal

proteins

were

collected

In

patients

with

a

monoclonal

gammopathy

exposure

time

was

defined

as

the

number

of

years

between

the

diagnosis

GD

I

and

the

diagnosis

monoclonal

gammopathy

or

start

of

ERT

whichever

came

first

In

patients

without

a

monoclonal

gammopathy

exposure

time

was

calculated

as

the

number

of

years

between

the

diagnosis

GD

I

and

start

of

therapy

or

in

patients

not

receiving

therapy

the

first

visit

to

our

clinic

MGUS

was

defined

as

the

presence

of

a

monoclonal

immunoglobulin

component

in

the

serum

-LRB-

as

determined

by

protein

electrophoresis

in

agar/agarose

gels

and

immunofixation

-RRB-

at

a

concentration

of

less

than

g/l

-LRB-

IgG

IgA

or

IgM

-RRB-

and

normal

serum

calcium

and

creatinine

-LSB-

-RSB-

Although

histology

is

required

to

distinguish

osteolytic

lesions

caused

by

MM

from

those

caused

by

GD

we

decided

to

classify

long-existing

lesions

as

being

GD

related

Bone

marrow

examinations

were

only

performed

when

a

previously

unknown

M

protein

was

discovered

or

in

case

of

a

sharp

rise

in

the

levels

of

immunoglobulins

or

FLC

In

case

of

MM

stage

was

determined

according

to

the

Durie

Salmon

-LSB-

-RSB-

and

International

Staging

System

-LRB-

ISS

-RRB-

criteria

-LSB-

-RSB-

We

investigated

whether

levels

of

FLC

could

be

predictive

for

the

development

of

MGUS

and

whether

these

levels

were

related

to

disease

severity

Therefore

we

measured

FLC

levels

in

serum

before

start

of

enzyme

replacement

therapy

in

ten

patients

with

mild

GD

-LRB-

SSI

-RRB-

and

ten

patients

with

severe

GD

-LRB-

SSI

-RRB-

all

without

a

monoclonal

gammopathy

In

addition

we

studied

whether

changes

in

cytokine

levels

and

in

FLC

and

immunoglobulins

at

baseline

and

during

ERT

were

related

to

the

presence

or

absence

of

a

monoclonal

gammopathy

Therefore

the

group

of

patients

with

a

monoclonal

gammopathy

was

matched

for

age

sex

spleen

status

SSI

and

the

use

of

ERT

to

a

control

group

of

GD

patients

without

a

monoclonal

gammopathy

Serial

measurements

of

levels

of

and

PARC

at

baseline

and

after

and

a

median

of

months

of

ERT

-LRB-

range

whatever

was

the

last

determination

-RRB-

were

performed

These

were

related

to

changes

in

immunoglobulins

and

free

light

chains

Hepatocyte

growth

factor

-LRB-

HGF

-RRB-

was

measured

only

at

baseline

One

patient

did

not

receive

therapy

the

first

hospital

visit

was

used

as

Assays

The

standard

enzyme

activity

assay

for

chitotriosidase

with

-LRB-

Sigma

St.

Louis

MO

USA

-RRB-

as

a

substrate

was

performed

at

pH

-LSB-

-RSB-

Serum

κ

and

λ

FLC

were

measured

using

FREELITE

reagents

-LRB-

The

Binding

Site

Ltd

Birmingham

England

-RRB-

on

a

BNII

nephelometer

-LRB-

Dade-Behring

Deerfield

IL

USA

-RRB-

-LSB-

-RSB-

Monoclonal

FLC

were

identified

as

values

for

κ

or

λ

that

exceeded

the

reference

ranges

-LRB-

κ

mg/l

λ

mg/l

-RRB-

and

produced

an

abnormal

κ/λ

ratio

-LRB-

or

-RRB-

Elevated

concentrations

of

FLC

without

an

abnormal

κ/λ

ratio

indicate

a

polyclonal

or

oligoclonal

increase

of

FLC

Levels

of

IgG

IgA

and

IgM

were

measured

by

immunoturbidimetric

assay

-LRB-

Roche

Tina-quant

-RRB-

HGF

and

PARC

were

measured

by

specific

enzyme-linked

immunosorbent

assays

-LRB-

ELISAs

-RRB-

according

to

the

instructions

of

the

manufacturer

-LRB-

and

Sanquin

Amsterdam

The

Netherlands

HGF

R&D

Systems

Minneapolis

MN

USA

PARC

Biosource

International

Camarillo

CA

USA

-RRB-

Statistics

Values

in

patients

are

given

as

medians

and

ranges

Differences

between

patient

groups

were

analyzed

by

the

Mann

Whitney

U

test

or

by

χ-square

test

Linear

mixed

models

were

used

to

assess

longitudinal

changes

Correlations

were

tested

by

the

rank-correlation

test

-LRB-

Spearman

coefficient

ρ

-RRB-

P

was

considered

to

represent

a

statistical

difference

Search

strategy

To

compare

the

prevalence

of

poly

and

monoclonal

gammopathies

in

our

cohort

with

earlier

case

series

and

to

study

the

relative

risk

of

MM

we

performed

a

MEDLINE

search

combining

the

MESH

terms

Gaucher

disease

with

either

hypergammaglobulinemia

paraproteinemias

and/or

multiple

myeloma

Literature

on

cytokines

chemokines

and

growth

factors

in

relation

to

the

development

of

immunoglobulin

abnormalities

in

GD

were

searched

combining

the

MESH

terms

Gaucher

disease

with

cytokines

and/or

chemokines

Case

reports

were

excluded

and

only

studies

that

included

at

least

ten

patients

were

selected

Results

Patients

The

patients

had

a

median

age

of

years

-LRB-

range

-RRB-

Thirty-two

of

patients

-LRB-

-RRB-

were

men

and

-LRB-

-RRB-

were

splenectomized

At

baseline

median

SSI

was

-LRB-

range

-RRB-

The

majority

of

patients

had

a

genotype

containing

-LRB-

-RRB-

with

being

the

most

common

combination

-LRB-

-RRB-

All

patients

had

creatinine

and

urea

values

within

the

normal

range

At

the

first

assessment

-LRB-

-RRB-

patients

had

normal

immunoglobulin

levels

-LRB-

-RRB-

patients

had

a

polyclonal

gammopathy

-LRB-

-RRB-

patients

had

MGUS

and

one

patient

had

MM

-LRB-

Table

-RRB-

During

follow-up

-LRB-

range

years

-RRB-

of

the

patients

with

normal

immunoglobulin

levels

or

a

polyclonal

gammopathy

developed

a

monoclonal

gammopathy

Table

of

Gaucher

disease

type

I

patients

with

a

monoclonal

gammopathyPatient

no.SexSxAge

in

or

at

deathTherapyMG

typeIg

progression

to

amyloidosis

and

MMFree

progression

to

amyloidosisIgGλ4MY60ERTMGUSIgGκ5MY67ERTMGUSIgGλ6MY59ERTMGUSIgAλ

IgGκ7FN67ERTMGUSIgAκ8MN56ERTMGUSIgGλ9MY65ERTMGUSIgGκ

IgGλ

Male

F

female

Sx

splenectomy

ERT

enzyme

replacement

therapy

MG

monoclonal

gammopathy

MM

multiple

myeloma

MGUS

monoclonal

gammopathy

of

undetermined

significance

Ig

immunoglobulin

Of

the

patients

with

MGUS

two

developed

MM

and/or

amyloidosis

after

approximately

years

of

ERT

-LRB-

and

-RRB-

Patient

was

diagnosed

with

MM

and

amyloidosis

months

after

start

of

ERT

Pulse

therapy

with

dexamethasone

was

started

Three

months

after

the

diagnosis

the

patient

died

from

cardiac

failure

At

months

after

start

of

ERT

patient

was

diagnosed

with

amyloidosis

She

received

melphalan

and

prednisone

but

died

months

later

from

cardiac

failure

Patient

was

diagnosed

with

MM

stage

IA

before

start

of

ERT

During

follow-up

IgG

levels

gradually

increased

After

years

of

ERT

treatment

with

reduced

dose

melphalan

and

prednisone

was

started

which

resulted

in

unacceptable

cytopenia

Subsequently

thalidomide

mg

daily

was

started

with

beneficial

effect

and

stable

disease

parameters

for

the

last

years

Of

the

ten

remaining

patients

with

non-progressive

MGUS

six

had

a

monoclonal

three

had

a

biclonal

and

one

a

triclonal

gammopathy

The

most

common

immunoglobulin

type

was

IgGκ

-LRB-

Table

results

previously

presented

in

part

in

-LSB-

-RSB-

-RRB-

Patients

with

a

monoclonal

gammopathy

were

significantly

older

than

patients

without

a

monoclonal

gammopathy

-LRB-

Table

-RRB-

Exposure

time

to

GD

defined

as

time

from

diagnosis

until

first

assessment

at

our

clinic

and

severity

of

disease

measures

assessed

by

SSI

and

chitotriosidase

at

baseline

were

comparable

Table

characteristics

of

Gaucher

type

I

patients

with

a

monoclonal

gammopathy

vs

patients

without

a

monoclonal

gammopathy

Without

MGWith

MGPNo

of

Age

in

or

at

-LRB-

-RRB-

-LRB-

-RRB-

No

of

male

-LRB-

-RRB-

-LRB-

-RRB-

NSYears

of

-LRB-

-RRB-

-LRB-

-RRB-

NSNo

of

-LRB-

-RRB-

-LRB-

-RRB-

NSSSI

at

-LRB-

-RRB-

-LRB-

-RRB-

NSChitotriosidase

-LRB-

nmol/ml

h

-RRB-

at

-LRB-

-RRB-

-LRB-

-RRB-

NSData

reflect

absolute

numbers

-LRB-

and

percentage

-RRB-

or

median

-LRB-

and

range

-RRB-

MG

Monoclonal

gammopathy

NS

not

significant

SSI

severity

score

index

A

search

for

studies

on

monoclonal

and

polyclonal

gammopathies

in

GD

I

resulted

in

five

series

-LSB-

-RSB-

Polyclonal

gammopathies

were

reported

in

and

monoclonal

gammopathies

in

of

GD

I

patients

-LRB-

Table

-RRB-

Table

on

the

prevalence

of

monoclonal

and

polyclonal

gammopathies

in

type

I

Gaucher

diseaseReferenceNo

of

Gaucher

patientsAgePolyclonal

gammopathiesMonoclonal

gammopathiesde

Fost

et

al.

-LSB-

-RSB-

-LRB-

-RRB-

-LRB-

-RRB-

Pratt

et

al.

-LSB-

-RSB-

-LRB-

-RRB-

-LRB-

-RRB-

Shoenfeld

et

al.

-LSB-

-RSB-

-LRB-

-RRB-

-LRB-

-RRB-

Marti

et

al.

-LSB-

-RSB-

-LRB-

-RRB-

-LRB-

-RRB-

Allen

et

al.

-LSB-

-RSB-

-LRB-

-RRB-

-LRB-

-RRB-

Brautbar

et

al.

-LSB-

-RSB-

-LRB-

-RRB-

Data

reflect

absolute

numbers

-LRB-

and

percentage

-RRB-

or

in

the

study

of

Marti

et

al.

mean

standard

deviation

Five

studies

have

described

the

prevalence

and/or

relative

risk

of

MM

in

GD

patients

-LSB-

-RSB-

Table

summarizes

the

data

from

these

studies

The

frequency

of

MM

in

these

cohorts

was

with

significantly

elevated

relative

risks

of

Table

on

the

prevalence

and

relative

risk

of

multiple

myeloma

in

type

I

Gaucher

diseaseReferenceNo

of

patientsEthnic

backgroundAgeControl

groupNo

of

patients

with

MMRR

of

MMde

Fost

et

al.

-LSB-

-RSB-

Cancer

-LRB-

-RRB-

-LRB-

CI

-RRB-

Lee

-LSB-

-RSB-

-LRB-

-RRB-

NDShiran

et

al.

-LSB-

-RSB-

individuals

from

the

same

-LRB-

-RRB-

NDZimran

et

al.

-LSB-

-RSB-

Cancer

-LRB-

-RRB-

NDRosenbloom

et

al.

-LSB-

-RSB-

Cancer

-LRB-

-RRB-

-LRB-

CI

-RRB-

Data

on

age

reflect

mean

standard

deviation

data

on

number

of

patients

with

multiple

myeloma

reflect

absolute

numbers

-LRB-

and

percentage

-RRB-

MM

Multiple

myeloma

RR

relative

risk

CI

confidence

interval

ND

not

done

Immunoglobulin

and

FLC

levels

In

GD

patients

from

the

Dutch

cohort

without

a

monoclonal

gammopathy

-LRB-

ten

with

mild

disease

SSI

and

ten

with

severe

disease

SSI

-RRB-

FLC

were

measured

before

start

of

therapy

-LRB-

Fig.

-RRB-

One

patient

was

found

to

have

a

slightly

abnormal

ratio

and

six

patients

had

an

increase

in

one

or

both

FLC

with

a

normal

ratio

There

were

no

significant

differences

in

κ

or

λ

FLC-levels

between

patients

with

severe

and

patients

with

mild

GD

I

During

follow-up

-LRB-

range

years

-RRB-

of

the

patients

developed

a

monoclonal

gammopathy

Fig.

levels

in

Gaucher

disease

type

I

patients

without

a

monoclonal

gammopathy

with

mild

disease

-LRB-

SSI

-RRB-

and

severe

disease

-LRB-

SSI

-RRB-

-LRB-

a

-RRB-

FLC

levels

in

Gaucher

disease

patients

with

a

monoclonal

gammopathy

and

matched

Gaucher

disease

controls

-LRB-

b

-RRB-

MG

Monoclonal

gammopathy

The

normal

range

for

κ

was

mg/l

and

for

λ

was

mg/l

The

normal

ratio

for

κ/λ

was

FLC

levels

were

measured

in

all

GD

I

patients

with

a

monoclonal

gammopathy

-LRB-

Table

-RRB-

and

matched

GD

I

controls

-LRB-

-RRB-

No

serum

was

available

of

patient

resulting

in

two

groups

of

patients

Baseline

immunoglobulin

levels

were

not

available

in

one

patient

with

a

monoclonal

gammopathy

-LRB-

-RRB-

and

five

patients

from

the

control

group

-LRB-

and

-RRB-

At

baseline

the

patients

who

already

had

or

would

develop

MM

and/or

amyloidosis

-LRB-

-RRB-

had

strongly

abnormal

FLC

κ/λ

ratios

-LRB-

Fig.

-RRB-

Of

the

nine

patients

with

MGUS

one

-LRB-

-RRB-

had

an

abnormal

FLC

κ/λ

ratio

and

four

showed

elevated

levels

of

FLC

κ

or

λ

but

with

a

normal

FLC

ratio

The

remaining

four

patients

with

MGUS

had

both

FLC

levels

as

well

as

a

FLC

κ/λ

ratio

within

the

normal

range

Six

of

the

patients

from

the

control

group

had

FLC

levels

and

a

FLC

κ/λ

ratio

within

the

normal

range

and

six

had

elevated

levels

of

one

or

both

chains

of

whom

only

one

-LRB-

-RRB-

patient

showed

an

abnormal

FLC

κ/λ

ratio

During

follow-up

-LRB-

range

years

-RRB-

of

the

patients

from

the

control

group

developed

a

monoclonal

gammopathy

Cytokines

chemokines

and

growth

factors

At

baseline

levels

were

within

the

normal

range

in

all

but

four

patients

-LRB-

Fig.

-RRB-

There

was

no

significant

difference

in

levels

between

patients

with

or

without

a

monoclonal

gammopathy

-LRB-

median

-LRB-

range

-RRB-

pg/ml

-LRB-

-RRB-

and

pg/ml

-LRB-

-RRB-

respectively

-RRB-

The

majority

of

patients

-LRB-

-RRB-

showed

elevated

levels

especially

in

the

group

of

patients

with

a

monoclonal

gammopathy

although

not

significantly

different

from

the

patients

without

a

monoclonal

gammopathy

-LRB-

pg/ml

-LRB-

-RRB-

and

pg/ml

-LRB-

-RRB-

respectively

P

-RRB-

PARC

levels

were

elevated

in

all

GD

patients

without

a

difference

between

patients

with

or

without

a

monoclonal

gammopathy

-LRB-

pg/ml

-LRB-

-RRB-

and

pg/ml

-LRB-

-RRB-

respectively

-RRB-

HGF

was

within

the

normal

range

in

most

-LRB-

-RRB-

patients

without

a

difference

between

patients

with

or

without

a

monoclonal

gammopathy

-LRB-

pg/ml

-LRB-

-RRB-

and

pg/ml

-LRB-

-RRB-

respectively

-RRB-

Fig.

levels

of

PARC

and

HGF

in

Gaucher

disease

patients

with

a

monoclonal

gammopathy

and

matched

Gaucher

disease

controls

Patients

with

multiple

myeloma

and/or

amyloidosis

are

marked

by

an

arrow

Dotted

lines

reflect

the

upper

limit

of

the

normal

range

IL

Interleukin

HGF

hepatocyte

growth

factor

PARC

pulmonary

and

activation-regulated

chemokine

MG

monoclonal

gammopathy

A

literature

search

resulted

in

seven

studies

on

cytokines

chemokines

and

growth

factors

in

GD

I

patients

-LRB-

Table

-RRB-

In

one

of

these

studies

mRNA

levels

were

measured

-LSB-

-RSB-

while

in

the

remaining

studies

plasma

levels

were

studied

If

possible

the

number

of

patients

with

cytokine

levels

exceeding

the

reference

range

was

determined

In

studies

in

which

normal

ranges

were

not

provided

-LSB-

-RSB-

we

defined

the

normal

range

as

mean

based

on

the

values

of

the

control

group

This

was

not

possible

in

the

study

of

Barak

et

al.

-LSB-

-RSB-

in

which

no

detailed

data

were

provided

Table

on

plasma

levels

of

cytokines

chemokines

and

growth

factors

in

Gaucher

type

I

patientsReferenceNo

of

GD

I

patientsAgeCytokinesResults

no

of

patients

-LRB-

-RRB-

with

elevated

levelsP

value

for

difference

with

mean

values

of

the

control

groupde

Fost

et

al.

-LSB-

-RSB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Michelakakis

-LSB-

-RSB-

-LRB-

-RRB-

NDAllen

et

al.

-LSB-

-RSB-

-LRB-

-RRB-

-LRB-

-RRB-

P

-LRB-

-RRB-

P

Lichtenstein

et

al.

-LSB-

-RSB-

-LRB-

-RRB-

P

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

NSHollak

et

al.

-LSB-

-RSB-

-LRB-

-RRB-

P

-LRB-

-RRB-

P

-LRB-

-RRB-

P

-LRB-

-RRB-

-LRB-

-RRB-

NSBarak

et

al.

-LSB-

-RSB-

levels

elevatedP

levels

elevatedP

levels

elevatedP

levels

elevatedP

-LRB-

-RRB-

NSBoot

et

al.

-LSB-

-RSB-

-LRB-

-RRB-

P

Altarescu

et

al.

-LSB-

-RSB-

-LRB-

-RRB-

NSNG

Not

given

ND

not

done

NS

not

significant

Longitudinal

changes

In

general

at

no

time

point

was

there

a

statistically

significant

difference

in

plasma

levels

of

any

of

the

factors

-LRB-

i.e.

IgG

IgA

IgM

FLC

κ

and

λ

and

PARC

-RRB-

between

patients

with

and

patients

without

a

monoclonal

gammopathy

-LRB-

Fig.

-RRB-

In

addition

using

mixed

model

analysis

in

neither

of

the

two

patient

groups

a

significant

decrease

or

increase

could

be

established

in

plasma

levels

of

any

of

the

measured

factors

The

only

exception

was

PARC

which

decreased

in

all

patients

except

for

one

patient

who

did

not

use

therapy

-LRB-

-RRB-

Changes

in

PARC

levels

could

not

be

related

to

changes

in

levels

of

immunoglobulins

or

free

light

chains

There

were

no

strong

correlations

-LRB-

i.e.

rho

-RRB-

between

any

of

the

factors

Fig.

changes

in

plasma

levels

of

immunoglobulin

heavy

and

free

light

chains

and

PARC

Dotted

lines

reflect

the

normal

range

Numbers

reflect

Gaucher

disease

patients

with

a

monoclonal

gammopathy

as

described

in

Table

-LRB-

-RRB-

and

matched

Gaucher

disease

controls

-LRB-

-RRB-

without

a

monoclonal

gammopathy

IL

Interleukin

PARC

pulmonary

and

activation-regulated

chemokine

More

specifically

immunoglobulin

levels

remained

stable

or

decreased

in

all

patients

except

for

patient

who

showed

an

increase

in

IgM

This

patient

was

different

from

other

MGUS

patients

because

she

did

not

receive

therapy

Free

light

chain

levels

were

more

variable

but

clearly

FLC

levels

of

patients

who

were

to

develop

or

already

suffered

from

MM

and/or

amyloidosis

remained

high

during

ERT

-LRB-

-RRB-

Although

in

most

patients

no

trends

could

be

distinguished

a

consistent

decrease

in

free

light

chains

and

immunogobulins

was

seen

for

example

in

patient

who

had

an

IgAκ

M

protein

This

patient

responded

very

well

to

ERT

but

other

patients

with

excellent

clinical

responses

did

not

always

show

a

similar

decrease

in

M-protein

levels

No

relationship

was

found

between

clinical

response

and

decrease

in

either

levels

of

immunoglobulins

or

FLCs

The

course

of

cytokines

did

not

show

a

relationship

with

changes

in

either

FLC

or

immunoglobulin

levels

although

in

the

untreated

patient

with

increasing

levels

of

IgM

-LRB-

-RRB-

also

an

increase

in

and

PARC

was

noted

On

the

contrary

a

spontaneous

decrease

in

was

found

in

patient

without

therapy

for

his

MM

In

patient

showed

a

clear

decrease

after

start

of

ERT

but

before

the

diagnosis

amyloidosis

and

MM

was

made

Discussion

In

addition

to

the

classical

symptoms

of

GD

I

such

as

hepatosplenomegaly

skeletal

disease

and

cytopenia

there

is

increasing

evidence

for

existence

of

co-morbidities

including

increased

risk

of

cancer

and

abnormal

immunoglobulin

profiles

In

fact

polyclonal

gammopathies

and

MGUS

occurred

in

and

respectively

of

our

adult

Gaucher

cohort

which

is

in

line

with

findings

from

the

literature

-LRB-

and

of

patients

-LSB-

-RSB-

-RRB-

In

addition

we

established

that

the

relative

number

of

biclonal

-LRB-

-RRB-

and

triclonal

-LRB-

-RRB-

gammopathies

within

the

MGUS

patients

was

especially

high

considering

that

in

general

biclonal

gammopathies

occur

in

of

subjects

with

MGUS

-LSB-

-RSB-

while

triclonal

gammopathies

occur

only

very

sporadically

-LSB-

-RSB-

The

types

of

immunoglobulin

heavy

and

light

chain

immunoglobulins

were

comparable

to

the

findings

for

MGUS

in

a

non-GD

population

with

a

predominance

of

IgGκ

-LSB-

-RSB-

As

in

the

general

population

the

prevalence

of

MGUS

in

our

cohort

increased

with

age

but

there

was

no

relationship

with

disease

severity

measures

as

assessed

by

exposure

time

baseline

SSI

and

chitotriosidase

levels

A

relationship

with

age

was

first

suggested

by

Pratt

et

al.

-LSB-

-RSB-

who

investigated

patients

aged

to

years

and

established

an

MGUS

in

four

patients

all

being

over

years

of

age

Indirectly

he

also

showed

an

association

with

severity

of

disease

since

all

patients

with

splenomegaly

had

either

a

monoclonal

or

polyclonal

gammopathy

Considering

the

relatively

high

risk

of

monoclonal

gammopathies

in

GD

the

question

was

raised

whether

sensitive

measuring

of

FLC

would

be

useful

for

the

early

detection

of

monoclonal

gammopathies

in

GD

I

patients

In

a

non-GD

population

MGUS

patients

showed

an

abnormal

FLC

ratio

in

of

cases

which

was

an

important

risk

factor

for

progression

to

MM

or

a

related

disorder

-LSB-

-RSB-

We

found

that

the

three

GD

patients

who

had

or

would

soon

develop

a

B-cell

malignancy

could

easily

be

identified

by

their

strongly

abnormal

FLC

κ/λ

ratios

It

was

however

not

possible

to

identify

GD

patients

with

MGUS

on

the

basis

of

abnormal

FLCs

since

of

GD

patients

with

MGUS

had

both

FLC

levels

as

well

as

a

FLC

κ/λ

ratio

within

the

normal

range

A

slightly

abnormal

ratio

was

not

predictive

for

progression

to

MM

or

MGUS

in

the

single

patient

without

a

monoclonal

gammopathy

even

after

years

of

follow-up

of

the

patients

had

a

decrease

in

renal

function

implicating

that

any

FLC

increase

was

indeed

due

to

increased

production

by

plasma

cells

MGUS

patients

are

at

risk

for

developing

MM

at

a

rate

of

of

patients

per

year

Accordingly

the

prevalence

of

MM

in

GD

I

cohorts

of

clearly

exceeds

the

general

prevalence

of

-LRB-

deduced

from

The

Surveillance

Epidemiology

and

End

Results

-LRB-

SEER

-RRB-

Program

of

the

National

Cancer

Institute

-LRB-

NCI

-RRB-

-RRB-

The

assumed

diversity

in

relative

risk

of

developing

MM

-LRB-

and

-RRB-

can

simply

be

explained

by

age

differences

in

the

cohorts

the

highest

number

of

MM

patients

was

clearly

established

in

the

highest

age

groups

In

a

recent

consensus

report

on

hematological

manifestations

of

Gaucher

disease

the

higher

incidence

of

multiple

myeloma

in

Gaucher

disease

is

acknowledged

and

it

is

recommended

that

Gaucher

patients

should

have

their

immunoglobulin

profile

determined

at

diagnosis

and

monitored

every

years

-LRB-

patients

years

-RRB-

or

every

year

-LRB-

patients

years

-RRB-

-LSB-

-RSB-

Hypotheses

on

the

pathogenesis

of

immunoglobulin

disorders

in

GD

have

mainly

focused

on

the

accumulated

glucocerebroside

as

the

causative

agent

for

chronic

stimulation

of

macrophages

surrounding

Gaucher

cells

Cytokines

of

macrophagic

origin

could

in

turn

stimulate

B

cells

leading

to

a

polyclonal

gammopathy

which

may

eventually

transform

directly

or

via

MGUS

into

MM

Interestingly

plasma

cells

can

be

found

in

close

contact

with

Gaucher

cells

suggestive

of

local

interaction

-LRB-

Fig.

-RRB-

Fig.

marrow

aspirate

showing

plasma

cells

surrounding

a

Gaucher

cell

in

a

patient

with

Gaucher

disease

type

I

and

multiple

myeloma

In

our

study

we

measured

and

HGF

three

factors

important

for

differentiation

and/or

proliferation

of

plasma

cells

In

addition

and

are

also

involved

in

inflammation

and

in

abnormal

bone

remodeling

in

MM

aspects

that

are

also

present

in

GD

We

found

elevated

levels

in

most

patients

especially

those

with

a

monoclonal

gammopathy

and

predominantly

normal

levels

of

and

HGF

Previous

studies

have

also

described

increased

levels

-LSB-

-RSB-

However

findings

on

in

GD

I

have

been

variable

-LSB-

-RSB-

Inconsistent

results

have

also

been

reported

for

the

pro-inflammatory

cytokines

TNF-α

and

and

the

chemokine

-LSB-

-RSB-

Although

not

included

in

this

study

increases

have

been

described

for

the

growth

factor

M-CSF

-LRB-

monocyte/macrophage-colony

stimulating

factor

-RRB-

the

monocyte/macrophage

activation

marker

-LRB-

soluble

-RRB-

-LSB-

-RSB-

-LRB-

soluble

receptor

-RRB-

and

-LRB-

receptor

antagonist

-RRB-

-LSB-

-RSB-

The

most

impressive

increase

both

in

our

study

as

well

in

as

in

the

study

by

Boot

et

al.

-LSB-

-RSB-

has

been

found

for

PARC

a

chemokine

that

is

assumed

to

be

involved

in

B-cell

differentiation

by

recruiting

T

cells

and

negative

mantle

zone

B

lymphocytes

to

antigen-presenting

cells

In

addition

to

the

in

vivo

studies

it

has

been

described

that

murine

macrophages

that

were

stimulated

in

vitro

by

glucocerebroside

release

the

pro-inflammatory

lymphocyte-activating

factor

-LRB-

LAF

-RRB-

in

a

dose-responsive

manner

This

effect

was

not

seen

on

incubation

with

galactocerebroside

sphingomyelin

and

ceramidetrihexoside

-LSB-

-RSB-

Possibly

only

minor

glycolipid

accumulation

is

sufficient

for

the

development

of

B-cell

derangement

In

fact

a

glucocerebrosidase

deficient

mouse

-LRB-

homozygote

-RRB-

showed

evidence

of

B-cell

proliferation

as

well

as

elevated

serum

IgG

levels

even

though

storage

cells

were

not

found

-LSB-

-RSB-

The

heterogeneity

in

plasma

cytokine

levels

that

have

been

found

could

be

explained

by

differences

in

patient

populations

and

cytokine

assays

In

addition

it

is

not

known

whether

plasma

levels

adequately

reflect

tissue

levels

Nevertheless

clearly

both

pro-

as

well

as

anti-inflammatory

cytokine

levels

are

disturbed

in

GD

I

It

is

unlikely

that

Gaucher

cells

directly

induce

inflammation

since

Gaucher

cells

have

a

phenotype

resembling

anti-inflammatory

alternatively

activated

macrophages

More

likely

pro-inflammatory

cytokines

are

produced

by

surrounding

macrophages

with

Gaucher

cells

compensating

for

the

inflammatory

compounds

-LSB-

-RSB-

Now

that

it

is

generally

accepted

that

ERT

effectively

reduces

hepatosplenomegaly

cytopenia

and

skeletal

disease

the

focus

should

be

shifted

to

new

challenges

including

the

prevention

and

treatment

of

Gaucher

associated

co-morbidities

Novel

data

from

our

study

suggest

a

beneficial

effect

of

ERT

on

the

occurrence

and

severity

of

gammopathies

in

GD

patients

First

of

the

patients

developed

MGUS

during

ERT

We

did

see

progression

of

MGUS

to

amyloidosis

and

MM

in

two

patients

However

since

both

patients

developed

these

diseases

relatively

shortly

after

start

of

ERT

one

could

envision

that

irreversible

changes

leading

to

malignant

transformation

had

already

taken

place

and

could

not

be

corrected

by

ERT

Second

we

found

that

immunoglobulin

levels

either

decreased

or

remained

stable

in

both

patients

with

and

without

a

monoclonal

gammopathy

The

only

patient

with

a

strong

increase

in

immunoglobulins

-LRB-

IgM

-RRB-

was

a

patient

not

using

ERT

A

clear

influence

of

ERT

on

levels

of

FLC

and

was

not

found

in

our

study

A

previous

report

also

found

a

decrease

in

immunoglobulin

levels

during

ERT

although

only

in

patients

with

a

polyclonal

gammopathy

and

not

in

patients

with

a

monoclonal

gammopathy

-LSB-

-RSB-

Decreases

in

the

levels

of

-LSB-

-RSB-

M-CSF

and

PARC

-LSB-

-RSB-

have

been

described

Nevertheless

to

draw

firm

conclusions

on

a

possible

positive

effect

of

ERT

on

gammopathies

in

GD

I

our

presumptions

should

be

confirmed

in

larger

studies

This

would

require

an

international

multicenter

effort

that

should

aim

to

establish

whether

indeed

these

long-term

complications

can

be

prevented

by

ERT

and

whether

this

would

justify

the

early

start

of

treatment

In

summary

there

is

a

high

prevalence

in

GD

I

of

both

polyclonal

as

well

as

monoclonal

gammopathies

including

MM

The

risk

of

these

diseases

increases

with

age

Mechanisms

causing

gammopathies

remain

to

be

elucidated

but

include

the

disturbance

of

cytokine

levels

involved

in

inflammation

and

B-cell

function

ERT

is

likely

to

have

a

beneficial

effect

in

preventing

the

occurrence

and

the

progression

of

gammopathies

O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O B O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

gaucher disease
mgus
multiple myeloma
free light chains
cytokines