Serum

proteome

analysis

for

profiling

protein

markers

associated

with

carcinogenesis

and

lymph

node

metastasis

in

nasopharyngeal

carcinoma

Nasopharyngeal

carcinoma

-LRB-

NPC

-RRB-

one

of

the

most

common

cancers

in

population

with

Chinese

or

Asian

progeny

poses

a

serious

health

problem

for

southern

China

It

is

unfortunate

that

most

NPC

victims

have

had

lymph

node

metastasis

-LRB-

LNM

-RRB-

when

first

diagnosed

We

believe

that

the

based

serum

proteome

analysis

can

be

useful

in

discovering

new

biomarkers

that

may

aid

in

the

diagnosis

and

therapy

of

NPC

patients

To

filter

the

tumor

specific

antigen

markers

of

NPC

sera

from

healthy

volunteers

non-LNM

NPC

patients

and

LNM

NPC

patients

were

selected

for

screening

study

using

combined

with

MS.

Pretreatment

strategy

including

sonication

albumin

and

immunoglobulin

G

-LRB-

IgG

-RRB-

depletion

was

adopted

for

screening

differentially

expressed

proteins

of

low

abundance

in

serum

By

image

analysis

and

MALDI-TOF-MS

identification

twenty-three

protein

spots

were

differentially

expressed

Three

of

them

were

further

validated

in

the

sera

using

enzyme-linked

immunosorbent

assay

-LRB-

ELISA

-RRB-

Our

research

demonstrates

that

and

SAA

confirmed

with

ELISA

at

sera

and

immunohistochemistry

are

potential

NPC

metastasis-specific

serum

biomarkers

which

may

be

of

great

underlying

significance

in

clinical

detection

and

management

of

NPC

Introduction

Nasopharyngeal

carcinoma

-LRB-

NPC

-RRB-

is

a

rare

malignancy

in

most

parts

of

the

world

though

it

is

highly

prevalent

in

Southern

Asia

where

the

incidence

is

about

a

higher

than

in

other

populations

It

is

one

of

the

most

confusing

commonly

misdiagnosed

and

poorly

understood

diseases

Previous

studies

show

that

the

cancer

is

an

Epstein-Barr

virus-associated

malignancy

with

a

remarkable

racial

and

geographical

distribution

The

etiology

of

NPC

is

thought

to

be

associated

with

a

complex

interaction

of

genetic

viral

environmental

and

dietary

factors

Thanks

to

the

advancements

in

genomics

proteomics

and

bioinformatics

in

recent

decades

the

etiology

carcinogenesis

and

progression

of

the

disease

is

better

understood

Research

into

these

components

may

unravel

the

pathways

in

NPC

development

and

potentially

decipher

the

molecular

characteristics

of

the

malignancy

-LSB-

-RSB-

NPC

is

an

insidious

tumor

and

is

usually

at

the

stage

of

metastasis

involving

lymph

nodes

or

other

organs

before

it

can

be

found

Thus

early

and

accurate

diagnosis

is

very

important

for

therapy

and

prognosis

of

NPC

patients

Unfortunately

no

effective

method

of

accurately

diagnosing

new-onset

NPC

is

available

now

We

presume

if

specific

serum

biomarkers

associated

with

NPC

metastasis

can

be

identified

on

the

basis

of

advancements

in

genomics

proteomics

and

bioinformatics

an

approach

to

the

early

detection

and

monitoring

of

NPC

may

be

found

comparative

proteome

analysis

is

a

new

technology

for

the

separation

and

identification

of

disease-specific

proteins

and

it

has

been

applied

successfully

to

screen

potential

biomarkers

for

NPC

in

cell

lines

and

tumor

tissues

-LSB-

-RSB-

In

our

previous

studies

we

employed

proteomic

techniques

to

study

protein

changes

of

a

poorly

differentiated

squamous

carcinoma

cell

line

of

human

NPC

cells

induced

by

-LRB-

TPA

-RRB-

It

is

likely

that

TPA

promotes

NPC

in

necessary

cooperation

with

EBV

or

that

it

may

function

as

an

antiproliferative

or

differentiative

revulsant

in

noninitiated

cells

-LSB-

-RSB-

However

if

a

marker

can

be

detected

only

in

surgical

specimens

its

clinical

significance

is

limited

especially

for

early

screening

or

diagnosis

Recently

Saeid

R.

et

al.

reported

their

pioneering

work

on

NPC

serum

analysis

by

without

any

pretreatment

revealing

the

enhanced

expression

of

relatively

abundant

proteins

as

ceruloplasmin

-LRB-

CPL

-RRB-

-LSB-

-RSB-

In

order

to

find

relatively

low-abundant

serum

proteins

which

may

be

more

valuable

in

predicting

NPC

progression

we

pretreated

serum

with

sonication

albumin

and

IgG

depletion

before

analysis

By

comparing

image

analyses

for

healthy

volunteers

non-LNM

NPC

and

LNM

NPC

patients

we

may

identify

the

differentially

expressed

proteins

so

that

specific

serum

biomarkers

associated

with

NPC

metastasis

can

be

found

Materials

and

methods

Subjects

Serum

samples

from

healthy

volunteers

and

NPC

patients

were

available

for

comparative

proteomic

analysis

Samples

from

NPC

patients

were

obtained

before

any

clinical

treatment

All

the

patients

had

a

nasopharyngeal

biopsy

and

NPC

was

confirmed

by

two

independent

pathologists

unaware

of

the

patient

's

condition

Careful

medical

examinations

and

CT

scan

determined

non-LNM

cases

and

LNM

cases

in

the

NPC

patients

Clinical

data

of

all

the

subjects

for

and

ELISA

are

shown

in

Table

In

the

screening

study

serum

samples

from

the

same

group

were

pooled

to

smooth

intrinsic

individual

differences

and

enhance

common

characteristic

traits

only

related

to

disease

status

-LSB-

-RSB-

aliquoted

and

stored

at

°C

before

analysis

In

the

validation

study

of

the

patients

received

a

mean

of

months

of

follow-up

-LRB-

range

months

-RRB-

after

radiotherapy

and/or

chemotherapy

Six

of

the

followed-up

patients

died

of

NPC

and

the

other

survived

the

follow-up

period

Table

samples

in

the

studySamples

for

for

ELISAHealthyNPCHealthyNPCLymph

node

-LRB-

Male/Female

-RRB-

-LRB-

SD

-RRB-

Depletion

of

high-abundance

proteins

Sigma-Aldrich

ProteoPrep

Immunoaffinity

Albumin

and

IgG

Depletion

Kit

was

used

for

depletion

of

proteins

albumin

and

IgGs

allowing

visualization

of

co-migrating

proteins

with

albumin

and

IgG

on

a

gel

and

also

higher

sample

loads

for

improved

visualization

of

lower

abundance

proteins

A

total

of

μl

of

serum

was

diluted

with

μl

of

equilibration

buffer

from

the

depletion

kit

-LRB-

Sigma

-RRB-

and

sonicated

using

four

ultrasonic

bursts

of

s

each

Between

sonications

the

samples

were

chilled

on

ice

Then

serum

albumin

and

IgG

was

depleted

according

to

the

manufacturer

's

instructions

After

collection

of

the

bound

proteins

the

majority

-LRB-

-RRB-

of

the

unbound

proteins

were

left

in

the

depleted

serum

sample

Modified

Bradford

assay

was

used

to

measure

the

protein

concentrations

of

all

serum

samples

-LSB-

-RSB-

analysis

The

Immobiline

Dry

strip

-LRB-

pH

length

cm

Bio-Rad

-RRB-

was

rehydrated

with

mg

protein

in

ml

rehydration

buffer

containing

M

urea

M

thiourea

CHAPS

mM

DTT

mM

tributylphosphine

IPG

buffer

and

bromophenol

blue

for

h

at

room

temperature

IEF

was

performed

using

Protean

IEF

cell

-LRB-

Bio-Rad

Hercules

CA

USA

-RRB-

with

a

total

of

kVh

Then

the

strip

was

subjected

to

two-step

equilibration

in

equilibration

buffer

containing

M

urea

glycerol

SDS

and

mM

Tris

HCl

-LRB-

pH

-RRB-

with

w/v

DTT

for

the

first

step

and

w/v

iodoacetamide

for

the

second

step

The

second-dimensional

SDS-PAGE

gel

-LRB-

T

C

and

-RRB-

was

carried

out

using

a

Protean

II

xi

cell

-LRB-

Bio-Rad

-RRB-

according

to

the

following

procedure

min

at

a

constant

current

of

mA

followed

by

mA

per

gel

until

the

bromophenol

blue

front

reached

the

bottom

of

the

gel

Subsequently

the

gels

were

stained

with

MS

accommodated

silver

staining

-LSB-

-RSB-

of

each

sample

was

run

in

triplicate

to

minimize

run-to-run

variation

The

silver-stained

gel

were

scanned

using

a

Power-Look

imaging

scanner

-LRB-

Umax

Dallas

TX

USA

-RRB-

and

analyzed

using

PDQuest

software

package

-LRB-

Bio-Rad

-RRB-

In-gel

enzymatic

digestion

Protein

spots

were

excised

from

gel

with

an

operating

knife

blade

destained

twice

with

mM

potassium

ferricyanide

and

mM

sodium

thiosulfate

-LRB-

v/v

-RRB-

and

then

equilibrated

in

mM

to

pH

After

dehydrating

with

ACN

and

drying

in

at

°C

for

min

the

gel

pieces

were

rehydrated

in

ml

trypsin

solution

-LRB-

μg/ml

in

mM

-RRB-

at

°C

for

min

and

incubated

at

°C

overnight

Peptides

were

extracted

twice

using

TFA

in

CAN

and

dried

with

MALDI-TOF-MS

identification

The

peptide

mixtures

were

solubilized

with

TFA

with

saturated

-LRB-

CHCA

-RRB-

solution

in

acetonitrile

as

the

matrix

and

analyzed

using

M@LDI

R

-LRB-

Micromass

Manchester

UK

-RRB-

Mass

spectra

were

externally

calibrated

with

lock

mass

Da

and

internally

calibrated

with

autodigested

peaks

of

trypsin

-LRB-

MH

Da

-RRB-

Protein

identification

and

database

searching

Protein

identification

using

peptide

mass

fingerprinting

-LRB-

PMF

-RRB-

was

performed

by

the

MASCOT

search

engine

-LRB-

MatrixSicence

Ltd.

London

UK

-RRB-

against

the

MSDB

protein

database

The

errors

in

peptide

masses

were

in

the

range

of

ppm

One

missed

tryptic

cleavage

site

per

peptide

was

allowed

during

the

search

Proteins

matching

more

than

four

peptides

and

with

a

MASCOT

score

higher

than

were

considered

significant

-LRB-

P

-RRB-

Carboamidomethylation

of

cysteine

was

selected

as

the

static

modification

and

oxidation

of

methionine

as

the

differential

modification

Protein

identification

results

were

filtered

with

peakErazor

software

Enzyme-linked

immunosorbent

assay

-LRB-

ELISA

-RRB-

confirmation

ELISAs

were

conducted

to

confirm

the

protein

identification

and

differential

expression

of

SAA

and

in

sera

from

the

three

groups

Measurements

were

done

by

commercially

available

ELISA

kits

-LRB-

StressXpress

ELISA

Kit

Stressgen

Biotechnologies

Victoria

High

sensitivity

ELISA

Kit

Bender

Medsystems

Company

Australian

Human

SAA

ELISA

Kit

YES

Biotech

Laboratories

Ltd

Canada

-RRB-

in

accordance

with

the

manufacturer

's

instructions

All

sera

were

stored

at

°C

before

they

were

measured

Both

standards

and

samples

were

run

in

duplicate

The

main

protocols

were

as

follows

monoclonal

coating

antibody

was

adsorbed

onto

microwells

target

protein

present

in

the

sample

or

standard

bound

to

antibodies

was

adsorbed

to

the

microwells

second

antibody

was

added

and

bound

to

target

protein

captured

by

the

first

antibody

following

incubation

unbound

enzyme

was

removed

during

a

wash

step

a

color

reaction

was

formed

and

absorbance

was

measured

at

nm

The

standard

curve

was

used

to

determine

the

concentration

of

target

protein

in

an

unknown

sample

Immunohistochemistry

-LRB-

IHC

-RRB-

assay

The

previous

studies

showed

that

in

serum

could

be

released

by

membrane-associated

of

cancer

cell

-LSB-

-RSB-

However

SAA

is

synthesized

and

secreted

into

serum

by

hepatocytes

in

the

liver

during

the

acute

phase

-LSB-

-RSB-

In

the

present

study

therefore

expressions

of

only

and

were

detected

in

tissue

by

IHC

assay

Three-micrometer

sections

of

paraffin-embedded

tissues

were

cut

for

a

prior

antigen

retrieval

step

in

boiling

mM

citrate

buffer

pH

for

min

The

fixed

tissues

were

subjected

to

immunostaining

using

an

ultrasensitive

S-P

technique

-LRB-

MaiXin

China

-RRB-

Briefly

the

slides

were

incubated

overnight

at

°C

with

goat

anti-ICAM

and

polyclonal

antibody

-LRB-

-RRB-

-LRB-

Wuhan

Boster

Biological

Technology

Co.

Ltd.

China

-RRB-

The

concentrations

of

the

primary

antibodies

used

have

been

optimized

previously

After

washing

with

PBS

three

times

they

were

treated

with

biotin-conjugated

second

antibody

before

adding

streptavidin-peroxidase

For

color

reaction

diaminobenzidine

-LRB-

DAB

-RRB-

was

used

All

series

included

positive

controls

For

negative

controls

the

antibody

was

replaced

by

PBS

To

evaluate

the

expression

the

cellular

localization

of

immunoreactivity

was

determined

and

scored

for

both

intensity

-LRB-

negative

weak

moderate

and

strong

-RRB-

and

proportion

-LRB-

and

-RRB-

of

stained

cells

To

allow

comparison

of

immunostaining

with

pathological

stage

and

metastasis

for

each

cancer

integer

values

were

assigned

to

the

scores

of

intensity

-LRB-

-RRB-

and

proportion

of

tumor

cells

stained

-LRB-

-RRB-

If

the

evaluations

did

not

agree

the

specimens

were

reevaluated

and

then

classified

according

to

the

assessments

given

most

frequently

by

the

observers

The

proportion

and

intensity

scores

were

then

added

to

obtain

a

total

score

which

was

labeled

as

one

of

the

four

following

categories

-LRB-

a

-RRB-

high

positive

-LRB-

-RRB-

total

score

of

-LRB-

b

-RRB-

moderate

positive

-LRB-

-RRB-

total

score

of

-LRB-

c

-RRB-

low

positive

-LRB-

-RRB-

total

score

of

-LRB-

d

-RRB-

negative

-LRB-

-RRB-

total

score

of

Statistical

analysis

All

the

analyses

performed

were

descriptive

The

serum

levels

of

protein

expressions

were

compared

using

one-way

ANOVA

test

Associations

between

immunohistochemical

scores

and

clinicopathological

variables

of

tissue

specimens

were

evaluated

by

Fisher

's

exact

test

or

χ2

test

PDQuest

software

was

used

to

establish

a

master

gel

representing

each

group

-LSB-

-RSB-

For

differential

expression

of

matched

gels

protein

spots

whose

intensities

were

either

increased

or

decreased

two-fold

or

greater

were

marked

and

then

confirmed

by

manual

inspection

of

all

relevant

gel

not

only

those

included

in

the

matchsets

to

ensure

consistency

Quantitative

analysis

was

performed

using

the

Student

's

t-test

among

three

groups

of

gels

All

statistical

analysis

was

performed

with

SPSS

software

and

P

value

were

considered

statistically

significant

Results

Quantitative

comparison

and

identification

of

protein

spots

on

gels

Among

the

three

groups

we

found

twenty-three

spots

significantly

altered

-LRB-

P

-RRB-

or

absent/emergent

of

which

were

identified

successfully

-LRB-

Fig.

-RRB-

The

corresponding

detailed

differential

expression

patterns

are

shown

in

Fig.

Quantitative

comparison

and

MS

identification

of

the

differentially

expressed

proteins

are

shown

in

Fig.

and

summarized

in

Table

respectively

Overall

transferrin

-LRB-

TRF

-RRB-

and

transthyretin

-LRB-

TTR

-RRB-

were

down-regulated

and

-LRB-

-RRB-

serum

amyloid

protein

precursor

-LRB-

SAA

-RRB-

soluble

intercellular

adhesion

-LRB-

-RRB-

and

lysine-specific

histone

-LRB-

-RRB-

were

up-regulated

in

two

NPC

groups

compared

with

the

healthy

group

Furthermore

increased

expression

levels

of

and

SAA

were

observed

in

LNM

NPC

as

compared

with

non-LNM

NPC

However

heat

shock

protein

-LRB-

-RRB-

and

cytochrome

-LRB-

-RRB-

were

expressed

significantly

and

constantly

only

in

LNM

NPC

patients

The

MALDI-TOF-MS

mass

spectra

are

shown

in

Fig.

Fig.

patterns

of

albumin

and

IgG

depleted

serum

from

healthy

-LRB-

a

-RRB-

non-LNM

NPC

-LRB-

b

-RRB-

and

LNM

NPC

groups

-LRB-

c

-RRB-

In

the

images

spots

indicate

up-regulation

and

down-regulation

in

NPC

group

Black

arrows

indicate

protein

spots

that

were

identified

successfully

by

MSFig

patterns

of

the

thirteen

differentially

expressed

proteins

among

healthy

non-LNM

and

LNM

NPC

groups

-LRB-

a

-RRB-

Corresponding

gel

positions

are

shown

in

Fig.

Magnified

maps

demonstrating

the

different

expressions

of

selected

proteins

are

listed

in

Table

The

densitometric

analysis

of

each

protein

was

calculated

from

nine

different

gels

using

PDQuest

software

package

-LRB-

b

-RRB-

Each

bar

represents

the

mean

S.D.

of

intensity

with

significant

differences

between

groups

found

by

two-tailed

student

's

test

Note

that

this

panel

has

a

different

vertical

-LRB-

y

-RRB-

axis

and

a

break

in

the

axis

to

accommodate

the

poor

performance

of

the

subject

spot

No.

The

break

region

is

from

to

P

compared

with

healthy

group

P

compared

with

non-LNM

NPC

groupTable

results

of

differentially

expressed

proteins

among

healthy

volunteers

non-LNM

NPC

and

LNM

NPC

patientsNo.aProtein

descriptionMSDB

IDProtein

levelbMASCOT

scorecSequence

coveraged

-LRB-

-RRB-

Theoretical

Arachidonate

Cytochrome

Serum

amyloid

protein

precursorYLHUS

Hemoglobin

beta

subunits

are

histone

demethylase

Heat

shock

kDa

protein

-LRB-

-RRB-

protein

aThe

numbers

indicate

the

spot

positions

in

gel

as

shown

in

Figs.

and

up-regulation

in

NPC

patients

down-regulation

in

NPC

patientscBy

MALDI-TOF

MS

analysisdCalculated

by

amino

acid

counteCalculated

from

the

database

entry

without

any

processingFig

of

protein

spots

No.

and

respectively

representing

-LRB-

panel

A

-RRB-

SAA

-LRB-

panel

B

-RRB-

and

-LRB-

panel

C

-RRB-

which

were

expressed

differentially

between

the

groups

of

non-LNM

and

LNM

NPC

-LRB-

MASCOT

scores

are

and

respectively

P

-RRB-

ELISA

confirmation

Figures

and

show

the

ELISA

results

of

SAA

and

performed

in

the

three

groups

There

were

statistically

significant

differences

between

health

and

NPC

groups

-LRB-

P

-RRB-

as

well

as

between

non-LNM

and

LNM

NPC

groups

-LRB-

P

-RRB-

-LRB-

Fig.

-RRB-

And

with

the

TNM

stage

advanced

the

serum

levels

of

and

were

detected

significantly

elevated

but

that

of

SAA

not

-LRB-

Fig.

-RRB-

Furthermore

bivariate

correlation

analysis

showed

a

significant

relationship

between

the

serum

levels

of

the

three

proteins

-LRB-

P

-RRB-

The

follow-up

showed

that

the

serum

levels

of

and

but

not

of

SAA

were

strongly

associated

with

NPC

mortality

-LRB-

Student

's

t-test

P

and

respectively

-RRB-

Our

ELISA

findings

were

consistent

with

results

of

and

silver-staining

Fig.

comparison

of

and

SAA

in

sera

among

healthy

non-LNM

NPC

and

LNM

NPC

groups

Each

bar

represents

the

mean

S.D.

of

the

concentration

with

significant

differences

between

groups

found

by

one-way

ANOVA

test

Note

that

this

panel

has

a

different

vertical

-LRB-

y

-RRB-

axis

and

a

break

in

the

axis

to

accommodate

the

poor

performance

of

the

subject

SAA

The

break

region

is

from

to

μg/l

P

compared

with

healthy

group

P

compared

with

non-LNM

NPC

groupFig

comparison

of

and

SAA

in

sera

among

NPC

patients

at

different

TNM

stage

Each

bar

represents

the

mean

S.D.

of

the

concentration

with

significant

differences

between

groups

found

by

one-way

ANOVA

test

Note

that

this

panel

has

a

different

vertical

-LRB-

y

-RRB-

axis

and

a

break

in

the

axis

to

accommodate

the

poor

performance

of

the

subject

SAA

The

break

region

is

from

to

μg/l

P

compared

with

NPC

patients

at

TNM

stage

I

P

compared

with

NPC

patients

at

TNM

stage

II

P

compared

with

NPC

patients

at

TNM

stage

III

IHC

assay

In

NPC

patients

and

were

observed

in

tumor

cells

but

not

in

stromal

endothelial

or

inflammatory

cells

The

immunoreactivity

was

located

in

both

membrane

and

cytoplasm

for

and

only

in

cytoplasm

for

-LRB-

Fig.

-RRB-

The

immunoreactivities

of

and

were

observed

in

-LRB-

-RRB-

and

-LRB-

-RRB-

of

the

cases

of

NPC

respectively

The

correlation

of

positive

rates

showing

and

immunoreactivity

with

clinicpathological

characteristics

of

the

cases

is

summarized

in

Table

Statistically

positive

expression

rates

of

and

were

significantly

correlated

with

lymph

node

metastasis

-LRB-

LNM

-RRB-

of

NPC

Furthermore

we

found

that

positive

expression

rates

of

-LRB-

-RRB-

and

-LRB-

-RRB-

were

high

in

the

cases

that

died

of

NPC

during

the

follow-up

period

-LRB-

P

-RRB-

The

results

manifested

that

and

expressions

were

strongly

associated

with

NPC

metastasis

and

mortality

indicating

poor

prognosis

Fig.

detection

of

and

in

NPC

-LRB-

Magnification

-RRB-

The

immunoreactivity

was

located

in

both

membrane

and

cytoplasmic

stainings

for

but

only

in

cytoplasmic

staining

for

The

black

arrows

indicate

the

target

proteins

stained

in

brown-yellow

in

the

NPC

nest

The

red

arrow

in

E

shows

negative

immunoreactivity

of

normal

nasopharyngeal

epithelia

a

negative

expression

of

b

and

c

positive

expressions

of

d

negative

expression

of

e

and

f

positive

expressions

of

a

b

d

and

e

non-LNM

NPC

C

and

F

LNM

NPCTable

of

NPC

patients

dependent

on

and

expressionNegativePositive

-LRB-

-RRB-

P

valueNegativePositive

-LRB-

-RRB-

P

valueGender

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

AgeMean

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Lymph

node

status

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

TNM

stage

I

-LRB-

-RRB-

-LRB-

-RRB-

III

-LRB-

-RRB-

-LRB-

-RRB-

Survival

rate

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Note

Values

in

parentheses

are

percentages

Discussion

Serum

is

a

complex

body

fluid

containing

a

large

diversity

of

proteins

More

than

different

proteins

are

present

in

the

human

serum

and

many

of

them

are

secreted

or

shed

by

cells

during

different

physiology

or

pathology

processes

-LSB-

-RSB-

Consequently

proteomics

has

raised

great

expectations

for

the

discovery

of

biomarkers

to

improve

diagnosis

or

classification

of

a

wide

range

of

diseases

including

cancers

-LSB-

-RSB-

Serum

is

expected

to

be

an

excellent

source

of

protein

biomarkers

because

it

circulates

through

or

comes

in

contact

with

all

tissues

During

this

contact

it

is

likely

to

pick

up

proteins

secreted

or

shed

by

tissues

which

has

recently

been

tested

and

confirmed

-LSB-

-RSB-

However

serum

has

been

termed

as

the

most

complex

human

proteome

-LSB-

-RSB-

with

considerable

differences

in

the

concentrations

of

individual

proteins

ranging

from

several

milligrams

to

less

than

one

pictogram

per

milliliter

-LSB-

-RSB-

Another

analytical

challenge

for

biomarker

discovery

arises

from

the

high

variability

in

the

concentration

and

state

of

modification

of

some

human

plasma

proteins

between

different

individuals

-LSB-

-RSB-

Despite

these

limitations

human

serum

holds

immense

diagnostic

potential

In

the

last

decade

several

large-scale

projects

have

been

initiated

aimed

at

characterizing

the

human

plasma/serum

proteome

In

the

mean

time

several

serum

proteomic

studies

on

NPC

have

been

reported

Cho

et

al.

performed

protein

chip

profiling

analysis

with

surface-enhanced

laser

desorption

ionization

time-of-flight

mass

spectrometry

-LRB-

SELDI-TOF-MS

-RRB-

technology

on

sera

from

NPC

patients

and

demonstrated

that

SAA

may

be

a

potentially

usefully

biomarker

for

NPC

-LSB-

-RSB-

However

the

technology

for

discovery

of

new

cancer

biomarkers

has

recently

been

questioned

for

its

flaws

such

as

its

qualitative

nature

high

identification

error

rate

poor

reproducibility

and

nonspecific

absorption

matrices

-LSB-

-RSB-

comparative

proteome

analysis

though

a

new

technology

for

the

separation

and

identification

of

disease-specific

proteins

has

been

applied

successfully

to

screen

potential

biomarkers

for

NPC

in

cell

lines

and

tumor

tissues

-LSB-

-RSB-

Recently

Saeid

et

al.

reported

their

pioneering

work

on

NPC

serum

analysis

by

revealing

the

enhanced

expression

of

such

relatively

abundant

proteins

as

ceruloplasmin

-LRB-

CPL

-RRB-

-LSB-

-RSB-

However

in

study

abundant

proteins

such

as

albumin

and

IgG

that

account

for

approximately

of

the

total

serum

proteins

-LSB-

-RSB-

mask

other

proteins

that

migrate

to

the

surrounding

areas

and

limit

the

loading

amount

of

serum

As

albumin

and

IgG

are

known

to

function

as

carriers

and

transporters

of

important

proteins

such

as

hormones

cytokines

and

lipoproteins

within

the

blood

-LSB-

-RSB-

the

depletion

of

these

two

highly

abundant

proteins

may

result

in

the

loss

of

potentially

important

proteins

bound

to

them

at

the

same

time

In

order

to

release

those

adsorbed

or

bound

proteins

we

sonicated

the

diluted

sera

before

the

depletion

and

desalting

steps

as

suggested

by

Quero

et

al.

-LSB-

-RSB-

The

improvements

we

made

in

sample

preparation

enabled

us

to

find

some

valuable

low

abundant

proteins

Of

the

successfully

identified

protein

spots

we

focused

on

up-regulated

proteins

in

NPC

for

further

validation

We

also

accumulated

certain

knowledge

about

three

proteins

by

literature

profiling

Finally

we

decided

to

further

investigate

and

SAA

which

seemed

more

associated

with

our

research

interest

using

both

ELISA

and

IHC

to

validate

their

differential

expressions

Intriguingly

most

of

these

identified

proteins

have

been

reported

to

be

associated

with

carcinogenesis

and

tumor

metastasis

Our

research

is

herein

chiefly

concerned

with

the

functional

implications

of

the

three

proteins

to

NPC

at

the

serum

level

Although

the

source

of

has

not

been

fully

elucidated

researches

show

it

can

be

released

by

cancer

cells

-LSB-

-RSB-

as

well

as

by

peripheral

blood

mononuclear

endothelial

and

fibroblastic

cells

-LSB-

-RSB-

Proteolytic

cleavage

of

membrane-bound

may

be

the

most

likely

mechanism

for

the

generation

of

-LSB-

-RSB-

In

patients

with

certain

malignancies

the

serum

titers

have

been

found

elevated

in

association

with

tumor

growth

and

distant

metastasis

of

malignant

melanoma

-LSB-

-RSB-

lung

-LSB-

-RSB-

breast

-LSB-

-RSB-

gastric

-LSB-

-RSB-

hepatocellular

-LSB-

-RSB-

and

colorectal

cancers

-LSB-

-RSB-

Poor

survival

of

cancer

patients

correlated

with

a

high

level

of

serum

has

also

been

demonstrated

-LSB-

-RSB-

In

our

study

the

positive

expression

of

tissue

and

levels

of

serum

were

significantly

correlated

to

the

presence

progression

metastasis

and

mortality

of

NPC

possesses

most

of

the

necessary

extracellular

structures

to

retain

the

functional

activities

of

-LSB-

-RSB-

It

has

been

reported

that

on

the

surface

of

cancer

cells

or

antigen

presenting

cells

-LRB-

i.e.

macrophages

-RRB-

is

a

costimulatory

factor

that

stabilizes

T-cell

receptor-mediated

binding

between

these

cells

and

T

lymphocytes

-LSB-

-RSB-

would

work

as

an

immunosuppressive

agent

by

blocking

on

T

lymphocytes

thus

rendering

it

less

available

for

binding

with

on

the

surface

of

cancer

cells

-LSB-

-RSB-

In

this

manner

the

shedding

of

may

speed

up

the

metastatic

process

by

escaping

host

immune

surveillance

This

probably

presents

an

additional

potential

mechanism

accounting

for

high

serum

levels

of

in

NPC

patients

who

have

metastasized

via

hematogenous

and

lymphatic

routes

As

serum

may

be

useful

for

monitoring

hematogenous

metastasis

measuring

the

serum

level

might

be

potentially

significant

in

clinic

SAA

is

an

acute-phase

protein

with

various

isoforms

in

a

molecular

mass

range

of

In

normal

individuals

SAA

is

produced

by

hepatocytes

in

the

liver

-LSB-

-RSB-

After

secreted

into

serum

it

rapidly

binds

to

high-density

lipoprotein

with

of

the

protein

particles

bound

-LSB-

-RSB-

A

review

of

the

literature

shows

that

only

a

low

level

of

SAA

can

be

found

in

the

sera

of

healthy

individuals

despite

the

ubiquitous

nature

of

SAA

-LSB-

-RSB-

This

is

in

sharp

contrast

to

the

patients

with

neoplastic

diseases

such

as

those

with

renal

-LSB-

-RSB-

and

colorectal

-LSB-

-RSB-

cancers

who

showed

dramatic

elevation

of

serum

SAA

The

prognostic

significance

of

SAA

for

other

cancers

has

also

been

found

by

conventional

radioimmunoassay

in

line

with

the

findings

of

this

study

-LSB-

-RSB-

Cho

et

al.

demonstrated

that

SAA

may

be

a

potentially

usefully

biomarker

for

NPC

-LSB-

-RSB-

The

report

fully

confirmed

relatively

huge

concentrations

of

SAA

in

serum

-LRB-

g/l

-RRB-

that

are

thousands

of

times

higher

than

classical

cancer

biomarkers

-LRB-

such

as

CEA

PSA

etc.

-RRB-

originating

from

tumor

cells

In

a

similar

way

we

further

confirmed

that

serum

SAA

was

much

elevated

in

NPC

patients

particularly

at

the

process

of

lymph

node

metastases

when

compared

with

non-LNM

patients

However

such

biomarkers

-LRB-

acute-phase

reactants

-RRB-

are

not

commonly

considered

as

cancer-specific

ones

and

expected

to

be

elevated

in

other

malignant

diseases

or

inflammatory

diseases

as

well

-LSB-

-RSB-

In

previous

reports

SAA

was

found

elevated

in

different

malignancies

such

as

cancers

of

kidney

colon

and

prostate

as

well

as

in

leukemias

and

lymphomas

-LSB-

-RSB-

Therefore

SAA

may

represent

a

cancer

epiphenomenon

unlikely

to

be

of

much

clinical

use

in

diagnosing

and

monitoring

cancer

-LSB-

-RSB-

but

it

will

be

interesting

to

explore

whether

the

rapid

production

of

SAA

in

the

liver

or

the

epithelia

of

different

organs

-LSB-

-RSB-

may

be

related

to

the

stimulation

by

cytokines

abundantly

present

in

the

NPC

cells

It

is

also

meaningful

to

investigate

how

SAA

is

produced

at

different

stages

of

clinical

manifestation

in

NPC

patients

is

the

main

protein

produced

during

cellular

response

to

varied

stresses

such

as

heat

shock

ischemia/reperfusion

and

oxidative

changes

-LSB-

-RSB-

Because

confers

cell

protection

against

different

stresses

it

has

been

hypothesized

that

it

plays

a

protective

role

in

tumor

growth

in

vivo

-LSB-

-RSB-

Indeed

is

overexpressed

in

human

tumors

of

varied

origins

-LSB-

-RSB-

such

as

colorectal

-LSB-

-RSB-

breast

-LSB-

-RSB-

prostate

-LSB-

-RSB-

liver

-LSB-

-RSB-

cancers

and

melanoma

-LSB-

-RSB-

LNM

and

poor

survival

of

cancer

patients

correlated

with

overexpression

has

also

been

documented

-LSB-

-RSB-

It

is

suggested

that

is

needed

for

in

vivo

tumor

progression

In

addition

may

be

released

from

tumor

cells

involving

involves

both

active

secretion

and

passive

release

from

necrotic

cells

-LSB-

-RSB-

Physiological

mechanisms

include

co-secretion

in

exosomes

of

with

TRF

and

the

active

secretion

by

the

nonclassical

pathway

employed

by

cytokines

-LSB-

-RSB-

Therefore

is

detectable

in

serum

so

that

it

could

potentially

be

used

as

a

biomarker

for

diagnosis

or

disease

classification

Abe

et

al.

reported

that

is

a

marker

of

prostate

cancer

and

may

be

used

in

conjunction

with

PSA

to

identify

patients

with

early-stage

prostate

cancer

-LSB-

-RSB-

However

to

the

best

of

our

knowledge

the

association

between

serum

level

of

and

NPC

status

has

not

been

reported

Since

our

findings

demonstrated

that

there

was

a

modest

association

between

serum

level

and

NPC

staging

the

serum

level

should

not

be

considered

as

an

independent

prognostic

factor

in

NPC

patients

although

it

might

be

a

prognostic

predictor

by

univariate

analysis

In

short

serum

may

have

an

adjunctive

clinical

value

in

monitoring

tumor

progression

and

evaluating

prognosis

in

NPC

patients

though

its

clinical

application

as

a

major

tumor

marker

is

limited

In

conclusion

our

serum

proteomic

analysis

using

a

comprehensive

pretreatment

strategy

provides

a

practical

and

exemplary

tool

of

screening

progression-associated

serum

proteins

in

NPC

research

After

comparing

image

analyses

for

healthy

volunteers

non-LNM

NPC

and

LNM

NPC

patients

we

successfully

identified

differentially

expressed

protein

spots

We

further

explored

the

differential

expressions

of

three

of

the

proteins

namely

and

SAA

by

ELISA

at

serum

and

IHC

at

tissue

though

more

work

should

be

completed

to

pinpoint

the

direct

correlation

between

serum

level

of

sICAM

and

and

their

expression

level

in

the

tissues

We

suggest

that

the

three

proteins

may

be

potential

serum

biomarkers

which

can

serve

as

effective

target

points

for

early

diagnosis

and

therapy

of

NPC

patients

though

further

clinical

research

should

be

done

before

the

potential

come

true

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

nasopharyngeal carcinoma
serum proteome
carcinogenesis
lymph node metastasis


The effect of stimulation technique on sympathetic skin responses in healthy subjects The aim of this study was to collect normative data for sympathetic skin responses -LRB- SSR -RRB- elicited by electrical stimulus of the ipsilateral and contralateral peripheral nerves , and by magnetic stimulus of cervical cord . SSRs were measured at the mid-palm of both hands following electrical stimulation of the left median nerve at the wrist and magnetic stimulation at the neck in 40 healthy adult volunteers -LRB- mean age 52.2 ± 12.2 years , 19 males -RRB- . The onset latency , peak latency , amplitude and area were estimated in `` P '' type responses -LRB- i.e. , waveforms with a larger positive , compared to negative , component -RRB- . SSR onset and peak latency were prolonged when the electrical stimulus was applied at the contralateral side -LRB- i.e. , the SSR recorded in the right palm P < 0.001 -RRB- . The onset latency was similar on both sides during cervical magnetic stimulation . However , peak latency was faster on the left side -LRB- P < 0.03 -RRB- . Comparison of electrical and magnetic stimulation revealed that both the onset and peak latency were shorter with magnetic stimulation -LRB- P < 0.001 -RRB- . The latency of a SSR varies depending on what type of stimulation is used and where the stimulus is applied . Electrically generated SSRs have a longer delay and the delay is prolonged at the contralateral side . These factors should be taken into account when interpreting SSR data . Introduction Sympathetic skin response -LRB- SSR -RRB- testing is commonly used to assess sympathetic function -LSB- 18 -RSB- . Abnormal SSR occur in both central -LSB- 7 , 8 -RSB- and peripheral autonomic dysfunction -LSB- 1 , 16 , 21 -RSB- . The SSR is a complex polysynaptic reflex , which courses through a common centrifugal path via sympathetic post-ganglionic unmyelinated fibers -LSB- 2 , 24 -RSB- . However , its exact pathway through the central nervous system is not fully understood . A variety of stimuli can generate a SSR , which illustrates the complexity of the afferent inputs to the reflex . Although measuring SSR is relatively simple , interpreting these measurements is not so straightforward . Different afferent stimuli may well lead to a wide range of normal SSR values . It is difficult to attribute abnormalities of SSR to sympathetic dysfunction alone , particularly in cases of polyneuropathy , due to the potential influences by somatic fibers sensory inputs -LSB- 24 -RSB- . The aim of this study was to collect normative SSR data . SSRs were measured in both mid-palms following electrical stimulation of the left medial nerve and magnetic stimulation of the neck in healthy controls . SSR onset and peak latency , amplitude and area were analyzed . Electrically generated SSR in the ipsilateral -LRB- left -RRB- and contralateral -LRB- right -RRB- palms were compared to the SSR evoked by magnetic stimulation . Methods Subjects Forty healthy adult volunteers -LSB- 9 males/21 females , mean age 52.21 ± 12.23 -LRB- range 28 -- 75 -RRB- years , height 172 ± 11.3 cm -RSB- were studied . All subjects were healthy , with no history of cardiovascular or neurological disease . All subjects denied previous alcohol or drug abuse . None of the subjects had contraindications for magnetic stimulation , such as metal prosthesis or pacemaker . All participants gave written informed consent prior to the start of the study . Procedure The study was performed using a 2-channel Counterpoint , Medtronic-Dantec -LRB- Medtronic-Dantec Electronics , Skovlunde , Denmark -RRB- electromyographic unit . SSR recordings were based on the technique described by Shahani et alal . -LSB- 20 -RSB- . Briefly , subjects lay supine in an air-conditioned , quiet room with temperature maintained at 24 °C and skin temperature at 33 ± 0.5 °C . The same investigator performed all experiments , and care was taken to avoid all external stimulation . Active standard disk ECG Ag/AG/Cl electrodes were attached to the mid-palm of both hands and referenced to the corresponding dorsal surfaces . Skin temperature was verified as being above 32 °C by measuring close to the recording electrodes . The recording sites were the same in all experiments . Electrical stimulation Single square-wave electrical pulses of 0.1 milliseconds duration were applied to the left median nerve at the wrist at irregular intervals -LRB- 30 -- 60 seconds -RRB- to produce a SSR . The sweep speed was 1,000 milliseconds/D , the amplifiers sensitivity was 100 μV/div and the amplifier filters were set at 0.5 Hz and 2 kHz for the low and high frequency filter , respectively . Five trials were recorded simultaneously from both palms and on each side the response with the largest amplitude and shortest latency were taken . A fixed stimulus intensity of 60 mA was used throughout . Magnetic stimulation A Magstim 200 stimulator , equipped with a circular coil of 9 cm inner diameter and a 2.0-T maximum magnetic field strength , was used to apply magnetic stimulation at the cervical area . The center of the circular coil was placed in the midline over the 7th cervical spinous process . The current was set to anti-clockwise flow in the coil -LRB- side A visible -RRB- and the stimulus intensity fixed at 60 % of maximum output . Five stimuli were delivered randomly every 20 -- 30 seconds . Analysis of waveforms To avoid habituation 5 minutes rest was given between electrical and magnetic stimulation . To maintain uniformity of the results only `` P '' type responses -LRB- waveforms with a larger positive , compared to negative , component -RRB- were analyzed . Figure 1 shows how the components of the waveform were analyzed . The onset latency was taken from the time of the stimulus artefact to the start of the positive deflection . The peak latency was taken from the time of the stimulus artefact to the peak of the positive deflection . The peak-to-peak amplitude and the area under the SSR curve were also measured . Figure 1A typical sympathetic skin response recording . Typical SSR recordings taken from the right palm -LRB- upper trace -RRB- and left palm -LRB- lower trace -RRB- -LRB- a -RRB- following electrical stimulus of left median nerve and -LRB- b -RRB- magnetic stimulation of cervical cord Statistical analysis Differences between stimulus procedures in the same subjects were determined by using paired t-tests . The tests were two tailed . Statistical significance was set at P < 0.05 . All analyses were performed using SPSS for Windows -LRB- 10.0 ; SPSS Inc. , Chicago , IL -RRB- . Results Clear SSR recordings with a main positive component -LRB- i.e. , P waveforms -RRB- were obtained in all participants . Latency Mean SSR onset and peak latency on the right side , which was contralateral to the electrical stimulus , was significantly prolonged compared to the left side , which was ipsilateral to the electrical stimuli -LRB- P < 0.001 , Tables 1 and 2 -RRB- . No differences in the onset latency were found between right and left sides following cervical magnetic stimulation . However , a weak interside difference in peak latency -LRB- P < 0.03 -RRB- was observed -LRB- faster on the left side -RRB- . Comparison between electrical and magnetic stimulus revealed that both onset and peak latency on either side were significantly shorter for magnetic stimulation -LRB- P < 0.001 , Tables 1 and 2 -RRB- . Table 1Normative sympathetic skin response data in healthy subjectsElectrical ipsilateral -LRB- L -RRB- Electrical contralateral -LRB- R -RRB- Magnetic -LRB- L -RRB- Magnetic -LRB- R -RRB- Latency-onset -LRB- seconds -RRB- 1.79 ± 0.221.90 ± 0.24 a1 .63 ± 0.25 c1 .66 ± 0.28dLatency-peak -LRB- seconds -RRB- 2.54 ± 0.332.68 ± 0.38 a2 .40 ± 0.27 c2 .43 ± 0.28 b , dAmplitude -LRB- mV -RRB- 2.67 ± 1.492.35 ± 1.32 a2 .74 ± 1.612.70 ± 1.69 Area -LRB- mV seconds -RRB- 2.85 ± 1.532.63 ± 1.413.24 ± 1.993.22 ± 1.96 dAll data are mean ± SD . Electrical stimulation was applied at the left medial nerve of the wrist . SSR were recorded in both the left -LRB- ipsilateral -RRB- and right -LRB- contralateral -RRB- palms . Statistically significant difference -LRB- P < 0.05 -RRB- a Electrical stimulus -- between sidesb Magnetic stimulus -- between sidesc Left side -- between electrical and magnetic stimulusd Right side -- between electrical and magnetic stimulusTable 2The difference in sympathetic skin response onset latencies evoked by electrical and magnetic stimulationLatency difference -LRB- milliseconds -RRB- Electrical stimulation . R -- L105 ± 85 -LRB- 0 -- 320 -RRB- * Magnetic stimulation . R -- L27 ± 86 -LRB- − 110 -- 330 -RRB- Left electrical -- magnetic158 ± 183 -LRB- − 50 -- 640 -RRB- * Right electrical -- magnetic237 ± 201 -LRB- 0 -- 780 -RRB- * Differences in SSR onset latency measurements during 3 types of stimulation -LRB- left and right electrical stimulation and magnetic stimulation -RRB- . All data are mean ± SD -LRB- and range -RRB- . L , left ; R , right . * Significance of P < 0.05 -LRB- comparison by one sample t-test -RRB- Amplitude and area During electrical stimulation amplitude was higher of the stimulated -LRB- left -RRB- side compared to the non-stimulated side . Amplitude was similar during magnetic and contralateral electrical stimulation . The area of the SSR measured at the right side was greater for magnetic stimulation compared to electrical stimulation . Discussion The main finding of this study was that the latency of a SSR depends on whether an electrical or magnetic evoking stimulus is used . We also found a mean latency difference between contralateral and ipsilateral evoked electrical responses of 105 milliseconds . SSR are generated through a complex somato-sympathetic reflex with spinal , bulbar and suprabulbar components -LSB- 13 -RSB- . Until now , they have not been precisely defined in humans -LSB- 26 -RSB- . SSR can be evoked by various types of stimuli including auditory , deep breathing , painful sensation of the limbs or face etc. , and are therefore mediated by different afferent pathways -LSB- 2 -RSB- . Moreover , SSR are evoked by impulses that originate in a different area to which the response is produced . For example , electrical pulses over the sternal skin can be used to elicit SSR in the palms -LSB- 19 -RSB- , which highlight the complexity of the internal neuronal network involving polysynaptic relays and connections between different vertical levels along the neuraxis and in both sides of the human body -LSB- 11 -RSB- . It is , therefore , not surprising that measurement of a SSR depends on the site and the type of stimulus used to initiate the reflex . The longer latency in the contralateral side of 105 milliseconds may be explained by the crossing over of the signal from one side of the body to the other . The cross over time of around 100 milliseconds suggests that the reflex circuits are supraspinal -LSB- 13 -RSB- . Data from animal models indicate that the crossing occurs at multiple levels , including the brainstem and spinal cord -LSB- 26 -RSB- . However , patients with cervical or high thoracic lesions resulting in complete tetraplegia or paraplegia have no SSR from the hands and feet -LSB- 6 -RSB- , suggesting that supraspinal centers are essential in the generation of SSR . A constant latency to alternatively electrical stimulation of median nerves was suggested in a previous study -LSB- 3 -RSB- . However , latency was measured with 20 consecutive electrical stimulations , and thus the effects of adaptation -LRB- or habituation -RRB- can not be excluded . Magnetic stimulation may be most reproducible method to evoke SSR -LSB- 14 -RSB- . In theory , strong stimulation generated by the magnetic field close to the spine avoids any possible interference by affected somatic sensory fibers , providing a measure of efferent sympathetic function alone . In the case of the lower motor neurons , it has been showed that cervical magnetic stimulation excites the motor roots at or very near the exit foramen or even further distally -LSB- 5 -RSB- . In respect of SSRs , magnetic stimulation could , in analogy to motor neurons , excite the sensory roots at their entry -LSB- 14 -RSB- or directly activate the sympathetic trunks within the spinal canal -LSB- 25 -RSB- . In our study , the latency of the SSR evoked by magnetic stimulation was shorter than the corresponding values of the electrically elicited responses . The shorter latency of the magnetically generated SSR could be caused by impulses bypassing the peripheral afferent part of the reflex . Given that the difference between electrical stimulation at the wrist and magnetic stimulation at the neck was 158 milliseconds and assuming that the average distance from wrist to C7 is 70 cm , the conduction velocity of afferent somatic fibers is calculated to be approximately 5 m/second . Previous studies suggested that the efferent path is served by sympathetic B and C fibers conducting at approximately 2 m/second -LSB- 12 , 17 -RSB- , whereas the afferent path is served by somatic myelinated sensory groups II and III conducting at rates of 50 -- 5 m/second -LSB- 12 -RSB- . In good agreement with our results , other investigators have reported a latency difference between electrically and magnetically elicited SSR of 80 milliseconds -LSB- 14 -RSB- and 130 milliseconds -LSB- 4 -RSB- , with the former being always longer . Based on our results , it can be assumed that the SSR onset latency to spinal cord magnetic stimulation was the shortest , whereas that to contralateral peripheral nerve electrical stimulation was the longest . Furthermore , the difference in latency between SSR to magnetic and ipsilateral electrical stimuli was close to that between SSR to ipsilateral and contralateral electrical stimuli . A satisfactory explanation for the observed differences would not be offered without the risk of being highly speculative . The morphology is another element of SSRs instability . Two main types of SSR waveform were previously recognized ; P type which has a larger positive component and N type with a larger negative component -LSB- 15 , 22 , 23 -RSB- . It has been shown that strength of stimulation was positively correlated with P type amplitude and inconsistently with N type and that strong stimuli probably produced P type responses -LSB- 22 -RSB- . To ensure similarity of measurements and facilitate comparison of responses to different kinds of stimuli , we arbitrarily choose to study only subjects with predominant P type responses . In none of our participants the change of stimulation from electrical -LRB- ipsilateral and contralateral -RRB- to magnetic resulted in switching of P to N type of SSR . This can be the consequence of using relatively high intensity in electrical and magnetic stimulus . The latency values in our subjects were slightly higher than those in previous reports . In those reports , the mean SSR latency from the palm to electrical stimulation of the median nerve ranged between 1.27 seconds and 1.51 seconds -LSB- 4 , 10 , 14 , 24 -RSB- . This discrepancy should be attributed to a methodological difference . The negative onset of the response is generated by the electrical activation of the sweat gland . The peak of the negative phase is probably variable and generated by the relationship between sweat production and the surround tissue -LSB- 24 -RSB- . Under the particular recording conditions applied , the majority of the examined individuals had potentials with an initial positive peak . Therefore , instead of measuring the onset latency to the first negative peak , which is most often used , we measured the onset latency from stimulus artifact to the onset of the main positive wave irrespective of the existence or not of a proceeding small negative component . As regards to the peak latency values , they showed similar to onset latency differences in this study . Thus , peak latency is not suggested as an additional parameter , which could be useful in clinical practice . Amplitude is the SSR parameter that has been reported to be highly variable . It can be influenced by several factors such as temperature , time of the day , emotion and for this reason several authors have not used it as measure of disease -LSB- 2 , 9 -RSB- . The relative stability of amplitude and area measurements among the SSRs evoked by different stimuli in our subjects is noticeable . These findings could be attributed to the stable conditions of recordings -LRB- single session for all SSR recordings in each subject and long interstimulus interval to avoid habituation -RRB- . There are certain potential limitations in the study design . First , the fact that SSRs were not assessed for reproducibility in individual subjects . However , the phenomenon of habituation -LSB- 3 -RSB- did not allow us to compare measurements -LRB- particularly for the amplitude -RRB- of consecutive responses in a single individual . Second , since it is known that a variety of stimuli are capable of producing SSR , startle could intermix with the electrical stimulus and , mostly , auditory could interfere with the magnetic stimulus , leading to shorter SSR latencies . A study comparing parameters of SSR elicited by pure auditory as opposed to other types of stimulation should clarify this point . However , in a previous study , where ear-plugs were used to exclude auditory stimuli , the SSR latency measurements to neck magnetic stimulation were even shorter than in our subjects -LSB- 14 -RSB- , suggesting that the intermixed auditory stimulation is not a major limitation . Finally , habituation , in a broad sense including the central processing and familiarity , can not be completely ruled out by a 5-minute interval between techniques , although intervals of 40 -- 60 seconds between trials were reported elsewhere -LSB- 10 , 14 , 22 -RSB- . Application of electrical followed by magnetic stimulation in some subjects and the inverse procedure in others might have been used , although this would have increased the complexity of the study design . In brief , SSRs ' have been introduced as an easy to perform and painless mean for the evaluation of post-ganglionic sympathetic function -LSB- 20 -RSB- . In the filed of autonomic nervous system where the diagnostic options are limited , and despite the expression of certain doubts , mainly related to the large diversion of individual values , SSR remains at present the most widely applied neurophysiological test for the sympathetic nervous system . According to the results of the present study , latency which is the most widely quoted parameter , varied depending on the stimulation type , being shorter at ipsilateral than contralateral electrical stimulus and even less short when magnetic stimulation at the neck was applied . Further studies on the challenging issue of SSR waveform and its possible clinical relevance are necessary to explore it in detail .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

sympathetic skin response
methods
autonomic nervous system

galvanic skin response
sudomotor function