-LSB-

-RSB-

Adenine

is

a

suitable

radioligand

for

the

labeling

of

G

protein-coupled

adenine

receptors

but

shows

high

affinity

to

bacterial

contaminations

in

buffer

solutions

-LSB-

-RSB-

Adenine

has

previously

been

used

to

label

the

newly

discovered

G

protein-coupled

murine

adenine

receptors

Recent

reports

have

questioned

the

suitability

of

-LSB-

-RSB-

adenine

for

adenine

receptor

binding

studies

because

of

curious

results

e.g.

high

specific

binding

even

in

the

absence

of

mammalian

protein

In

this

study

we

showed

that

specific

-LSB-

-RSB-

adenine

binding

to

various

mammalian

membrane

preparations

increased

linearly

with

protein

concentration

Furthermore

we

found

that

Tris-buffer

solutions

typically

used

for

radioligand

binding

studies

-LRB-

mM

pH

-RRB-

that

have

not

been

freshly

prepared

but

stored

at

°C

for

some

time

may

contain

bacterial

contaminations

that

exhibit

high

affinity

binding

for

-LSB-

-RSB-

adenine

Specific

binding

is

abolished

by

heating

the

contaminated

buffer

or

filtering

it

through

filters

Three

different

aerobic

gram-negative

bacteria

were

isolated

from

a

contaminated

buffer

solution

and

identified

as

Achromobacter

xylosoxidans

A.

denitrificans

and

Acinetobacter

lwoffii

A.

xylosoxidans

a

common

bacterium

that

can

cause

nosocomial

infections

showed

a

particularly

high

affinity

for

-LSB-

-RSB-

adenine

in

the

low

nanomolar

range

Structure

activity

relationships

revealed

that

hypoxanthine

also

bound

with

high

affinity

to

A.

xylosoxidans

whereas

other

nucleobases

-LRB-

uracil

xanthine

-RRB-

and

nucleosides

-LRB-

adenosine

uridine

-RRB-

did

not

The

nature

of

the

labeled

site

in

bacteria

is

not

known

but

preliminary

results

indicate

that

it

may

be

a

high-affinity

purine

transporter

We

conclude

that

-LSB-

-RSB-

adenine

is

a

well-suitable

radioligand

for

adenine

receptor

binding

studies

but

that

bacterial

contamination

of

the

employed

buffer

solutions

must

be

avoided

Introduction

Purinergic

receptors

play

an

important

role

in

transmembrane

signaling

-LSB-

-RSB-

Currently

two

distinct

families

are

officially

recognized

by

the

International

Union

of

Pharmacology

-LRB-

IUPHAR

-RRB-

i.e.

receptors

for

the

purine

nucleoside

adenosine

-LRB-

or

adenosine

receptors

-RRB-

and

receptors

for

purine

and/or

pyrimidine

nucleotides

-LRB-

receptors

-RRB-

-LSB-

-RSB-

Whereas

the

receptor

family

comprises

four

subtypes

and

all

of

which

are

G

protein-coupled

receptors

-LRB-

GPCRs

-RRB-

the

family

is

further

subdivided

into

two

subfamilies

-LRB-

GPCRs

-RRB-

and

-LRB-

ligand-gated

ion

channels

-RRB-

-LSB-

-RSB-

In

addition

to

the

nucleoside

-LRB-

adenosine

-RRB-

and

nucleotide

receptors

a

receptor

for

the

nucleobase

adenine

has

recently

been

discovered

by

a

reverse

pharmacological

approach

identifying

adenine

as

the

natural

ligand

for

a

rat

orphan

GPCR

-LSB-

-RSB-

A

mouse

orthologue

-LRB-

-RRB-

of

the

rat

adenine

receptor

was

subsequently

identified

by

sequence

comparison

-LSB-

-RSB-

Very

recently

a

new

adenine

receptor

has

been

cloned

from

mice

showing

identity

in

its

amino

acid

sequence

to

and

to

the

rat

adenine

receptor

indicating

that

the

new

receptor

is

a

distinct

adenine

receptor

subtype

-LRB-

Genbank

nucleotide

sequence

accession

numbers

new

mouse

adenine

receptor

rat

adenine

receptor

-RRB-

-LSB-

submitted

-RSB-

Both

adenine

receptors

that

have

been

pharmacologically

characterized

are

coupled

to

the

inhibition

of

adenylate

cyclase

by

Gi

protein

-LSB-

-RSB-

So

far

no

human

receptor

for

adenine

has

been

identified

although

initial

clues

for

the

possible

existence

of

human

adenine

receptors

have

been

found

-LSB-

-RSB-

Adenine

receptors

are

structurally

unrelated

to

and

receptors

and

therefore

constitute

a

new

family

of

purinergic

receptors

for

which

we

proposed

the

designation

-LRB-

P

zero

-RRB-

receptors

-LSB-

-RSB-

based

on

the

structural

relationships

of

the

physiological

agonists

adenine

-LRB-

-RRB-

representing

a

partial

structure

of

adenosine

-LRB-

-RRB-

and

adenosine

again

being

a

partial

structure

of

adenine

nucleotides

-LRB-

-RRB-

such

as

adenosine

triphosphate

-LRB-

ATP

-RRB-

or

adenosine

diphosphate

-LRB-

ADP

-RRB-

Radioligand

binding

studies

are

widely

used

to

characterize

GPCRs

on

the

protein

level

-LSB-

-RSB-

Adenine

the

natural

ligand

of

adenine

receptors

is

commercially

available

in

tritium-labeled

form

-LRB-

-LSB-

-RSB-

adenine

-RRB-

and

has

been

used

to

characterize

recombinant

rat

adenine

receptors

expressed

in

Chinese

hamster

ovary

-LRB-

CHO

-RRB-

cells

-LSB-

-RSB-

as

well

as

natively

expressed

rat

adenine

receptors

in

rat

brain

-LSB-

-RSB-

using

membrane

preparations

The

radioligand

has

been

shown

to

be

stable

under

the

incubation

conditions

-LSB-

-RSB-

Furthermore

mouse

adenine

receptors

natively

expressed

in

-LRB-

neuroblastoma

glioma

hybrid

-RRB-

cell

membranes

were

labeled

by

-LSB-

-RSB-

adenine

-LSB-

-RSB-

Recently

we

successfully

applied

-LSB-

-RSB-

adenine

binding

to

detect

the

mouse

adenine

receptor

protein

recombinantly

expressed

in

insect

cell

membranes

which

constitute

a

background

because

they

do

not

endogenously

express

any

high

affinity

binding

site

for

adenine

-LSB-

-RSB-

Two

recent

poster

presentations

reported

on

problems

with

-LSB-

-RSB-

adenine

binding

to

adenine

receptors

In

one

study

using

whole

rat

brain

membrane

preparations

a

high-affinity

binding

site

was

detected

-LRB-

apparent

Ki

nM

-RRB-

-LSB-

-RSB-

However

a

very

high

Bmax

value

was

found

-LRB-

pmol/mg

protein

-RRB-

and

the

binding

was

almost

completely

blocked

by

μM

of

hypoxanthine

and

abolished

in

the

absence

of

indicating

that

the

detected

binding

site

was

not

identical

with

the

G

protein-coupled

rat

adenine

receptor

-LSB-

-RSB-

In

another

study

high

affinity

binding

of

-LSB-

-RSB-

adenine

was

detected

even

in

the

absence

of

added

protein

and

the

authors

suggested

that

-LSB-

-RSB-

adenine

bound

in

a

highly

specific

manner

to

the

glass

fiber

filters

used

in

the

filtration

assays

-LSB-

-RSB-

values

were

determined

for

five

adenine

derivatives

and

three

compounds

-LSB-

adenine

-LRB-

nM

-LSB-

-RSB-

rat

nM

-LSB-

-RSB-

nM

-LSB-

-RSB-

-RRB-

-LRB-

μM

-LSB-

-RSB-

rat

μM

-LSB-

-RSB-

-RRB-

-LRB-

μM

-LSB-

-RSB-

rat

μM

-LSB-

-RSB-

-RSB-

showed

similar

values

as

those

previously

determined

for

the

rat

adenine

receptor

but

two

compounds

-LSB-

deoxyadenosine

-LRB-

μM

-LSB-

-RSB-

rat

μM

-LSB-

-RSB-

-RRB-

-LRB-

μM

-LSB-

-RSB-

rat

μM

-LSB-

-RSB-

-RSB-

had

much

lower

affinities

for

the

unknown

binding

site

labeled

in

the

absence

of

added

protein

-LSB-

-RSB-

than

for

the

rat

adenine

receptor

as

previously

determined

-LSB-

-RSB-

indicating

that

the

unknown

binding

sites

labeled

in

the

absence

of

added

rat

tissue

were

very

different

Based

on

their

results

the

IJzerman

group

had

suggested

to

avoid

the

use

of

-LSB-

-RSB-

adenine

as

a

radiolabeled

probe

for

the

adenine

receptor

due

to

its

putative

specific

high-affinity

binding

to

glass

fiber

filters

-LSB-

-RSB-

That

report

prompted

us

to

carefully

reanalyze

radioligand

binding

data

obtained

with

-LSB-

-RSB-

adenine

in

our

laboratory

with

the

goal

to

find

out

the

reason

for

the

problems

encountered

in

other

laboratories

As

a

matter

of

fact

we

had

occasionally

observed

unusually

high

counts

in

a

few

experiments

which

could

however

be

avoided

by

repeating

the

experiments

under

carefully

controlled

experimental

conditions

including

the

use

of

freshly

prepared

buffer

solutions

We

have

now

performed

a

systematic

study

clearly

showing

that

-LSB-

-RSB-

adenine

is

a

suitable

radioligand

for

the

labeling

of

adenine

receptors

in

various

cells

and

tissues

if

bacterial

contaminations

are

excluded

Furthermore

we

identified

three

common

gram-negative

aerobic

bacteria

that

grow

in

cold

Tris

buffer

and

express

high-affinity

binding

sites

for

-LSB-

-RSB-

adenine

Materials

and

methods

Chemicals

-LSB-

-RSB-

Adenine

-LRB-

Ci/mmol

-RRB-

was

obtained

from

Amersham

Biosciences

-LRB-

Munich

Germany

-RRB-

Tris

was

obtained

from

Acros

Organics

-LRB-

Leverkusen

Germany

-RRB-

and

dimethyl

sulfoxide

-LRB-

DMSO

-RRB-

was

from

Fluka

-LRB-

Switzerland

-RRB-

All

other

chemicals

and

reagents

were

obtained

from

Sigma

unless

otherwise

noted

Cell

culture

Human

embryonic

kidney

-LRB-

HEK

-RRB-

and

cells

were

grown

as

monolayers

at

°C

-LRB-

-RRB-

in

Dulbecco

's

modified

Eagle

medium

-LRB-

DMEM

-RRB-

containing

fetal

calf

serum

U/ml

penicillin

μg/ml

streptomycin

and

mM

L-glutamine

Membrane

preparations

Frozen

rat

brains

were

obtained

from

Pel

Freez

-LRB-

Rogers

AR

USA

-RRB-

and

thawed

at

°C

Cortex

and

striata

were

dissected

and

membrane

fractions

were

prepared

as

previously

described

-LSB-

-RSB-

Membrane

preparations

from

cells

and

cells

were

prepared

as

described

-LSB-

-RSB-

Membrane

preparations

from

bacteria

were

obtained

after

growing

them

on

agar

plates

and

subsequent

amplification

of

single

strains

in

Lennox

broth

-LRB-

LB

-RRB-

medium

over

night

Membranes

were

then

prepared

in

analogy

to

the

procedures

used

for

mammalian-cell

membranes

-LSB-

-RSB-

Protein

concentrations

were

determined

according

to

the

method

of

Lowry

-LSB-

-RSB-

-LSB-

-RSB-

Adenine

binding

assays

Adenine

binding

assays

were

carried

out

as

previously

described

-LSB-

-RSB-

however

in

the

absence

of

and

ethyleneglycoltetraacetic

acid

-LRB-

EGTA

-RRB-

unless

otherwise

noted

as

and

EGTA

were

found

to

have

no

effect

on

determined

Ki

values

-LRB-

data

not

shown

-RRB-

In

brief

membrane

preparations

-LRB-

μg

of

protein

unless

otherwise

indicated

-RRB-

were

incubated

with

nM

-LSB-

-RSB-

adenine

in

mM

Tris-HCl

pH

in

a

total

volume

of

μl

Inhibition

curves

were

determined

using

six

to

nine

different

concentrations

of

adenine

spanning

three

orders

of

magnitude

Three

separate

experiments

were

performed

each

in

triplicate

unless

otherwise

noted

Nonspecific

binding

was

determined

in

the

presence

of

μM

unlabeled

adenine

Incubations

were

carried

out

for

h

at

room

temperature

and

terminated

by

rapid

filtration

through

GF/B

glass

fiber

filters

-LRB-

Whatman

Dassel

Germany

-RRB-

Filters

were

washed

three

times

ml

each

with

freshly

prepared

ice-cold

mM

Tris-HCl

buffer

pH

and

immediately

transferred

to

mini

vials

Scintillation

cocktail

-LRB-

Ultima

Gold

Canberra

Packard

ml

-RRB-

was

added

and

after

an

incubation

of

h

filter-bound

radioactivity

was

measured

by

liquid

scintillation

counting

at

an

efficiency

of

In

some

experiments

the

addition

of

mammalian

protein

was

omitted

and

replaced

by

μl

of

different

buffer

samples

For

competition

experiments

with

bacteria

either

μl

of

or

dilution

of

an

overnight

culture

of

the

bacteria

in

Tris

buffer

mM

pH

or

a

membrane

preparation

of

bacteria

-LRB-

containing

μg

of

protein

-RRB-

was

used

After

isolation

and

classification

of

the

bacteria

from

contaminated

buffer

solutions

experiments

were

performed

with

intact

bacteria

-LRB-

approximately

bacteria/sample

which

roughly

equals

μg

of

total

protein/sample

-RRB-

unless

otherwise

indicated

using

the

standard

procedure

-LRB-

see

above

-RRB-

The

cell

number

was

estimated

from

the

optical

density

-LRB-

OD

-RRB-

of

the

overnight

culture

suspension

and

the

assumption

that

-LSB-

-RSB-

Data

analysis

Data

were

analyzed

using

Prism

-LRB-

Graph

Pad

San

Diego

CA

USA

-RRB-

values

were

determined

by

fitting

data

to

sigmoidal

concentration-inhibition

curves

Results

are

presented

as

means

standard

error

of

the

mean

-LRB-

SEM

-RRB-

from

the

number

of

observations

Isolation

of

microorganisms

from

contaminated

buffer

solutions

and

classification

Microorganisms

were

isolated

from

Tris-HCl

buffer

pH

by

plating

μl

of

the

buffer

on

LB

agar

plates

Single

colonies

were

isolated

and

incubated

overnight

in

LB

medium

at

°C

with

constant

shaking

at

rpm

Further

separation

was

achieved

by

plating

bacteria

from

overnight

cultures

on

plate

count

-LRB-

Merck

Darmstadt

-RRB-

and

blood

agar

-LRB-

Oxoid

Wesel

-RRB-

Morphology

of

colonies

was

visually

examined

Conventional

physiological

and

biochemical

characterization

assays

including

gram

staining

catalase

and

oxidase

activity

motility

and

oxidation/fermentation

-LRB-

O/F

-RRB-

test

were

carried

out

and

analyzed

according

to

Bergey

's

Manual

of

Systematic

Bacteriology

using

reagents

from

Merck

-LRB-

Darmstadt

-RRB-

-LSB-

-RSB-

In

addition

the

following

kits

and

appliances

were

used

BBL

OXI/FERM

Tube

II

-LRB-

Schwarz

Pharma

GmbH

Germany

-RRB-

API

NE

strips

and

apiweb

software

-LRB-

Biomerieux

Nürtingen

Germany

-RRB-

and

VITEK

fully

automated

system

-LRB-

Biomerieux

Nürtingen

Germany

-RRB-

Results

Protein

dependence

of

-LSB-

-RSB-

adenine

binding

As

a

first

step

we

reevaluated

-LRB-

unpublished

-RRB-

data

obtained

in

initial

studies

that

had

been

performed

to

investigate

the

suitability

of

-LSB-

-RSB-

adenine

as

a

radioligand

for

labeling

adenine

receptors

Figure

shows

-LSB-

-RSB-

adenine

binding

to

different

membrane

preparations

from

-LRB-

a

-RRB-

rat

brain

striatum

-LRB-

b

-RRB-

rat

brain

cortex

and

-LRB-

c

-RRB-

cells

and

cells

For

each

membrane

preparation

different

amounts

of

protein

were

investigated

-LRB-

and

μg

for

striatum

and

μg

for

cortex

and

μg

for

HEK

and

CHO

cells

-RRB-

In

all

cases

we

found

a

large

approximately

linear

increase

in

specific

binding

with

increasing

protein

concentration

whereas

the

increase

in

nonspecific

binding

was

small

No

specific

binding

was

detected

in

the

absence

of

protein

-LRB-

see

Fig.

-RRB-

Fig.

dependence

of

-LSB-

-RSB-

adenine

binding

to

adenine

receptors

in

rat

brain

striatal

membranes

-LRB-

a

-RRB-

in

rat

brain

cortical

membranes

-LRB-

b

-RRB-

and

in

human

embryonic

kidney

-LRB-

-RRB-

cell

membranes

and

in

Chinese

hamster

ovary

cell

-LRB-

CHO

-RRB-

membranes

-LRB-

c

-RRB-

Different

amounts

of

protein

were

incubated

for

min

with

nM

of

-LSB-

-RSB-

adenine

in

Tris-HCl

buffer

pH

-LRB-

n

-RRB-

Nonspecific

binding

was

determined

in

the

presence

of

μM

adenine

Microbial

contaminations

in

buffer

solutions

As

a

next

step

we

investigated

whether

microbial

contaminations

present

in

incubation

buffers

that

were

not

freshly

prepared

might

be

responsible

for

high

counts

occasionally

observed

in

-LSB-

-RSB-

adenine

binding

studies

in

our

laboratory

We

performed

a

systematic

analysis

of

buffer

solutions

-LRB-

mM

Tris-HCl

pH

-RRB-

stored

at

different

conditions

-LRB-

different

periods

of

time

and

temperatures

-RRB-

in

different

containers

-LRB-

plastic

glass

different

sizes

-RRB-

used

in

our

laboratory

Samples

of

buffer

solutions

were

taken

and

radioligand

binding

studies

were

performed

with

-LSB-

-RSB-

adenine

-LRB-

nM

-RRB-

using

the

same

procedure

as

for

the

labeling

of

adenine

receptors

except

that

no

tissue

or

cell

membrane

preparation

was

added

Most

investigated

buffer

solutions

did

not

show

any

specific

-LSB-

-RSB-

adenine

binding

However

one

sample

of

Tris

buffer

that

had

been

taken

from

a

plastic

container

with

a

small

orifice

stored

at

°C

exhibited

high

affinity

binding

of

-LSB-

-RSB-

adenine

-LRB-

data

not

shown

-RRB-

In

contrast

radioligands

used

for

the

labeling

of

adenosine

receptors

-LRB-

-LSB-

-RSB-

-LSB-

-RSB-

cyclopentyladenosine

-LRB-

CCPA

-RRB-

-LRB-

-RRB-

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

-LRB-

-RRB-

-LRB-

m-methoxystyryl

-RRB-

-LRB-

MSX

-RRB-

-LRB-

-RRB-

-LSB-

-RSB-

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

-LRB-

PSB

-RRB-

-LRB-

-RRB-

-LSB-

-RSB-

-RRB-

or

receptors

-LRB-

-LSB-

-RSB-

adenylic

acid

-LRB-

1,1-dichloro-1-phosphonomethyl-1-phosphonyl

-RRB-

anhydride

-LRB-

PSB

-RRB-

-RRB-

-LSB-

-RSB-

did

not

show

any

specific

binding

to

that

buffer

solution

Figure

shows

-LSB-

-RSB-

adenine

binding

determined

in

differently

treated

Tris-buffer

solutions

-LRB-

in

the

absence

of

added

protein

-RRB-

Freshly

prepared

buffer

did

not

show

any

specific

-LSB-

-RSB-

adenine

binding

After

storing

the

buffer

for

day

at

°C

a

small

degree

of

-LSB-

-RSB-

adenine

binding

could

be

observed

and

after

weeks

-LSB-

-RSB-

adenine

binding

was

significant

-LRB-

cpm

specific

binding

-RRB-

There

was

a

large

exponential

increase

in

specific

-LSB-

-RSB-

adenine

binding

with

time

and

after

weeks

at

°C

approximately

cpm

-LRB-

specific

binding

-RRB-

were

measured

After

the

buffer

was

filtered

through

μm

filters

-LSB-

-RSB-

adenine

binding

was

completely

abolished

Heating

of

the

buffer

for

min

at

°C

dramatically

reduced

-LSB-

-RSB-

adenine

binding

whereas

heating

for

min

at

°C

or

heating

at

°C

for

min

in

an

autoclave

led

to

complete

loss

of

specific

-LSB-

-RSB-

adenine

binding

Fig.

-LSB-

-RSB-

Adenine

binding

in

the

absence

of

added

protein

-LRB-

cells

or

cell

membranes

-RRB-

Incubation

buffer

-LRB-

Tris-HCl

pH

-RRB-

was

kept

in

a

plastic

container

at

°C

for

up

to

weeks

Differently

treated

buffer

samples

-LRB-

μl

-RRB-

were

tested

for

-LSB-

-RSB-

adenine

binding

The

results

shown

represent

means

of

three

independent

experiments

standard

error

of

the

mean

Isolation

of

microbial

buffer

contaminants

As

our

results

indicated

that

adenine

binding

was

due

to

microbial

contaminations

growing

in

the

incubation

buffer

we

decided

to

isolate

the

contaminants

in

order

to

characterize

and

eventually

identify

them

The

microorganisms

were

isolated

by

plating

contaminated

incubation

buffer

on

agar

plates

This

led

to

identification

of

three

bacterial

strains

differing

in

the

morphology

of

the

formed

colonies

The

three

strains

were

then

separately

amplified

in

medium

over

night

and

membrane

preparations

were

obtained

to

perform

homologous

competition

binding

assays

using

-LSB-

-RSB-

adenine

-LRB-

Fig.

-RRB-

Fig.

curves

for

adenine

versus

nM

-LSB-

-RSB-

adenine

obtained

with

membrane

preparations

from

rat

brain

cortex

and

isolated

microorganisms

-LRB-

contaminants

-RRB-

-LRB-

a

-RRB-

and

intact

bacteria

-LRB-

b

-RRB-

values

-LRB-

a

-RRB-

contaminant

nM

-LRB-

n

-RRB-

values

-LRB-

b

-RRB-

Membrane

preparations

of

contaminant

showed

a

very

high

affinity

for

adenine

-LRB-

n

-RRB-

whereas

membrane

preparations

of

the

other

two

contaminants

appeared

to

have

considerably

lower

affinities

-LRB-

contaminant

n

contaminant

nM

n

-RRB-

For

comparison

the

binding

curve

for

adenine

at

rat

brain

cortical

adenine

receptors

-LRB-

-RRB-

is

shown

-LRB-

Fig.

-RRB-

Using

amounts

of

membrane

preparations

of

contaminant

that

contained

more

than

μg

protein/assay

tube

led

to

depletion

of

the

radioligand

-LRB-

more

than

of

the

added

radioligand

was

bound

to

the

protein

-RRB-

-LRB-

data

not

shown

-RRB-

Identification

of

microbial

contaminants

In

order

to

identify

the

contaminants

standard

procedures

and

classification

kits

were

used

-LRB-

see

Table

-RRB-

Standard

tests

including

catalase

oxidase

fermentation

motility

test

and

gram

staining

indicated

that

contaminant

is

Pseudomonas

spp.

or

a

strain

of

Achromobacter

spp.

contaminant

is

Achromobacter

spp.

and

contaminant

Acinetobacter

spp.

-LSB-

-RSB-

All

three

bacteria

are

strictly

aerobic

gram-negative

rods

For

further

classification

a

standardized

system

-LRB-

Api

NE

-RRB-

for

classification

of

bacteria

was

used

which

combines

conventional

and

assimilation

tests

for

identification

of

gram-negative

rods

not

belonging

to

the

Enterobacteriaceae

It

was

found

that

contaminant

was

positive

for

oxidase

nitrate

reduction

glucose

degradation

gluconate

caprate

adipate

maltose

citrate

and

phenylacetate

whereas

contaminant

was

negative

for

glucose

and

caprate

and

contaminant

only

showed

positive

results

for

caprate

maltose

and

phenylacetate

Those

results

confirmed

contaminant

already

presumed

to

belong

to

the

species

Acinetobacter

as

A.

lwoffii

with

a

probability

of

and

led

to

the

identification

of

contaminant

as

A.

xylosoxidans

-LRB-

probability

-RRB-

and

of

contaminant

as

A.

denitrificans

-LRB-

probability

-RRB-

-LRB-

see

Table

-RRB-

Identification

of

the

two

Achromobacter

strains

with

the

VITEK

fully

automated

system

for

rapid

bacterial

identification

and

antibiotic

susceptibility

confirmed

both

as

members

of

the

Achromobacter

spp.

with

a

probability

of

each

however

differences

were

found

in

the

results

for

phosphate

citrate

and

proline

assimilation

-LRB-

for

details

see

Table

-RRB-

Table

summarizes

selected

test

results

which

were

positive

for

at

least

one

of

the

strains

Table

of

classification

test

results

using

standard

procedures

and

kitsTestContaminant

of

colonies

FormRoundRoundRound

mm

ColorGreyBeigeWhite

BrimSmoothSmoothSmooth

SurfaceShinyShinyShiny

Cross

sectionRaisedRaisedRaised

Gram

stainNegativeNegativeNegativeBiochemistry

Catalase

Oxidase

O/F

test

Motility

Lysine/BBL

Ox/Ferm

Tube

II

n.d.

ResultAchromobacter

spp.

or

Pseudomonas

spp.Achromobacter

spp.Acinetobacter

spp.Api

NE

nitrate

reduction

GLU

glucose

GNT

gluconate

CAP

caprate

ADI

adipate

MLT

maltose

CIT

citrate

PAC

phenylacetate

OX

oxidase

ResultA

xylosoxidans

-LRB-

-RRB-

A.

denitrificans

-LRB-

-RRB-

A.

lwoffii

-LRB-

-RRB-

VITEK

N-LGGH

γ-L-glutamyl

-LRB-

nutrient

assimilation

-RRB-

n.d.

P-BPHO

bis

-LRB-

p-nitrophenyl

-RRB-

phosphate

n.d.

C-CIT

citrate

n.d.

C-KGA

α-acetoglutaryc

acid

n.d.

N-LPROT

L-proline-NA

n.d.

ResultAchromobacter

spp.

-LRB-

-RRB-

Achromobacter

spp.

-LRB-

-RRB-

n.d.

n.d.

not

determined

-LSB-

-RSB-

Adenine

binding

assays

with

isolated

intact

bacteria

Adenine

competition

binding

studies

were

performed

using

isolated

intact

bacteria

-LRB-

Fig.

-RRB-

The

value

obtained

with

whole

bacterial

cells

of

A.

xylosoxidans

-LRB-

-RRB-

was

in

the

same

concentration

range

as

that

obtained

with

membrane

preparations

of

the

same

bacteria

previously

designated

contaminant

-LRB-

-RRB-

For

the

other

two

bacteria

A.

denitrificans

and

A.

lwoffii

values

were

to

lower

when

determined

in

whole

bacterial

cells

compared

with

membrane

preparations

From

the

homologous

competition

experiments

KD

and

Bmax

values

were

estimated

for

A.

xylosoxidans

-LRB-

membranes

and

intact

cells

-RRB-

For

bacterial

membrane

preparations

a

KD

value

of

nM

and

a

Bmax

value

of

pmol/mg

protein

was

calculated

-LRB-

n

-RRB-

For

the

intact

bacteria

a

KD

value

of

nM

and

a

Bmax

value

of

sites/cell

-LRB-

n

-RRB-

was

obtained

Structure

activity

relationships

Affinities

for

selected

compounds

at

adenine

binding

sites

of

A.

xylosoxidans

were

determined

in

competition

assays

using

whole

bacterial

cells

and

compared

with

data

obtained

in

binding

studies

at

the

rat

brain

adenine

receptor

-LSB-

-RSB-

Figure

shows

competition

curves

for

selected

compounds

which

exhibited

high

affinity

i.e.

adenine

-LRB-

-RRB-

hypoxanthine

-LRB-

-RRB-

and

-LRB-

-RRB-

The

results

for

all

compounds

tested

are

summarized

in

Table

Whereas

the

affinity

of

adenine

for

the

binding

sites

of

A.

xylosoxidans

was

in

the

same

range

as

for

the

rat

adenine

receptor

the

affinities

for

hypoxanthine

and

differed

substantially

from

those

determined

for

the

rat

adenine

receptor

Hypoxanthine

showed

very

low

affinity

for

the

rat

adenine

receptor

-LRB-

Ki

nM

-RRB-

but

high

affinity

for

the

bacterial

-LSB-

-RSB-

adenine

binding

sites

with

an

value

in

the

low

nanomolar

range

-LRB-

nM

-RRB-

For

the

opposite

was

true

the

Ki

value

for

rat

brain

cortical

adenine

receptor

was

nM

-LSB-

-RSB-

whereas

the

value

for

the

A.

xylosoxidans

binding

site

was

in

the

micromolar

range

-LRB-

nM

-RRB-

Fig.

curves

for

adenine

hypoxanthine

and

versus

nM

-LSB-

-RSB-

adenine

obtained

with

Achromobacter

xylosoxidans

-LRB-

intact

bacteria

-RRB-

-LRB-

Table

of

affinities

of

adenine

and

selected

compounds

for

the

rat

adenine

receptor

and

bacterial

binding

sites

determined

in

radioligand

binding

studiesCompoundAchromobacter

xylosoxidans

-LRB-

intact

bacteria

-RRB-

Rat

brain

cortical

SEM

-LSB-

μM

-RSB-

a

-LRB-

inhibition

SEM

at

μM

-RRB-

Ki

SEM

-LSB-

μM

-RSB-

a

-LRB-

inhibition

SEM

at

μM

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Uracil

-LRB-

-RRB-

-LRB-

-RRB-

Xanthine

-LRB-

-RRB-

-LRB-

-RRB-

Uridine

-LRB-

-RRB-

d

-LRB-

-RRB-

Adenosine

-LRB-

-RRB-

caResults

are

from

three

independent

experiments

performed

in

triplicate

unless

otherwise

notedbResults

are

from

eight

independent

experiments

performed

in

triplicatecKi

values

and

inhibition

values

are

from

-LSB-

-RSB-

dResults

from

two

independent

experiments

performed

in

triplicate

Discussion

-LSB-

-RSB-

Adenine

has

been

successfully

used

by

us

-LSB-

-RSB-

and

other

laboratories

-LSB-

-RSB-

to

label

the

recently

discovered

rat

and

mouse

adenine

receptors

However

problems

with

-LSB-

-RSB-

adenine

binding

assays

have

been

reported

by

two

laboratories

-LSB-

-RSB-

These

have

led

to

the

suggestion

that

-LSB-

-RSB-

adenine

was

not

a

suitable

radioligand

for

the

labeling

of

G

protein-coupled

adenine

receptors

-LSB-

-RSB-

IJzerman

and

coworkers

-LSB-

-RSB-

reported

that

-LSB-

-RSB-

adenine

binding

was

not

protein

dependent

and

that

it

bound

with

nanomolar

affinity

to

glass

fiber

filters

in

the

absence

of

added

protein

These

results

which

were

contradictory

to

our

own

data

prompted

us

to

reexamine

the

-LSB-

-RSB-

adenine

binding

results

that

we

had

obtained

during

the

past

years

trying

to

find

an

explanation

for

the

discrepancies

In

contrast

to

the

results

reported

by

IJzerman

and

coworkers

-LSB-

-RSB-

binding

of

-LSB-

-RSB-

adenine

to

various

membrane

preparations

was

strictly

protein

dependent

in

our

hands

as

expected

-LRB-

Fig.

-RRB-

Specific

binding

linearly

increased

with

increased

amounts

of

protein

and

thus

increased

numbers

of

adenine

receptors

Nonspecific

binding

was

generally

low

for

-LSB-

-RSB-

adenine

and

there

was

only

a

minor

increase

in

nonspecific

binding

with

increasing

protein

concentrations

In

the

absence

of

protein

no

specific

binding

was

observed

-LRB-

Fig.

-RRB-

These

results

indicated

that

-LSB-

-RSB-

adenine

was

a

suitable

radioligand

for

labeling

adenine

receptors

In

fact

we

could

recently

perform

-LSB-

-RSB-

adenine

binding

assays

on

a

background

for

the

first

time

namely

at

the

mouse

adenine

receptor

heterologously

expressed

in

insect

cells

Whereas

membrane

preparations

of

the

nontransfected

cells

did

not

exhibit

any

specific

-LSB-

-RSB-

adenine

binding

cell

membranes

prepared

from

cells

infected

with

recombinant

baculoviruses

bound

-LSB-

-RSB-

adenine

with

high

affinity

-LSB-

-RSB-

However

when

we

looked

carefully

at

all

of

our

previous

-LSB-

-RSB-

adenine

binding

data

we

found

a

few

-LSB-

-RSB-

adenine

binding

experiments

that

could

not

be

evaluated

due

to

unusually

high

radioactivity

counts

These

occasional

problems

had

been

solved

by

carefully

controlling

the

experimental

conditions

e.g.

by

using

freshly

prepared

buffer

solutions

Stimulated

by

the

experiences

reported

by

the

IJzerman

group

-LSB-

-RSB-

we

decided

to

perform

a

systematic

study

to

find

out

the

reasons

for

those

problems

which

might

also

be

causative

for

erroneous

-LSB-

-RSB-

adenine

binding

results

in

other

laboratories

-LSB-

-RSB-

High

affinity

binding

of

-LSB-

-RSB-

adenine

to

filter

paper

as

suggested

by

IJzerman

and

coworkers

-LSB-

-RSB-

could

be

excluded

by

our

experiments

as

buffer

solutions

that

were

freshly

prepared

did

not

show

any

specific

-LSB-

-RSB-

adenine

binding

in

filtration

assays

using

glass

fiber

GF/B

filters

the

same

filters

that

had

been

used

by

the

IJzerman

group

-LSB-

-RSB-

On

the

contrary

we

discovered

that

bacterial

contaminations

which

can

be

present

in

buffer

solutions

express

high-affinity

binding

sites

for

-LSB-

-RSB-

adenine

and

therefore

impede

adenine

receptor

binding

assays

When

we

examined

different

buffer

solutions

stored

in

our

laboratory

we

discovered

high

-LSB-

-RSB-

adenine

binding

in

Tris-HCl

buffer

solution

-LRB-

pH

-RRB-

stored

at

°C

in

a

plastic

container

That

container

had

only

a

small

orifice

and

was

therefore

difficult

to

purify

Thus

microbial

contamination

in

this

container

was

carried

over

when

new

buffer

solution

was

prepared

When

the

contaminated

buffer

was

filtered

through

a

bacteria-tight

filter

-LRB-

μm

-RRB-

or

heated

in

order

to

denature

proteins

specific

-LSB-

-RSB-

adenine

binding

was

abolished

strongly

indicating

that

microbial

proteins

were

responsible

for

the

high

affinity

binding

of

adenine

A

further

indication

that

a

living

organism

was

involved

was

the

fact

that

adenine

binding

increased

exponentially

with

time

Interestingly

the

microorganisms

grew

better

at

°C

than

at

room

temperature

Our

first

presumption

that

the

contaminants

might

belong

to

yeast

could

not

be

proven

Saccharomyces

cerevisiae

was

used

as

a

control

organism

for

binding

studies

but

showed

no

-LSB-

-RSB-

adenine

binding

-LRB-

data

not

shown

-RRB-

Three

microorganisms

were

isolated

from

the

contaminated

Tris-buffer

solution

and

identified

using

standard

procedures

and

kits

Two

of

these

contaminants

were

assigned

to

the

genera

Achromobacter

and

one

to

Acinetobacter

Both

bacteria

species

are

gram-negative

rods

and

are

strictly

aerobic

They

are

commonly

found

in

soil

and

water

-LSB-

-RSB-

For

healthy

humans

or

animals

they

are

not

pathogenic

but

especially

A.

xylosoxidans

and

A.

lwoffii

have

gained

increasing

importance

due

to

their

ability

to

cause

nosocomial

infections

-LSB-

-RSB-

These

bacteria

are

able

to

grow

under

nonoptimal

conditions

such

as

low

temperature

and

restricted

nutrient

supply

-LSB-

-RSB-

They

are

able

to

metabolize

a

wide

variety

of

organic

substances

such

as

chemical

pollutants

in

the

environment

and

can

therefore

be

used

as

bioreporters

and

for

the

degradation

of

pollutants

-LSB-

-RSB-

This

is

consistent

with

the

fact

that

these

bacteria

are

able

to

grow

in

simple

Tris-HCl

buffer

at

low

temperature

Both

A.

lwoffii

and

Achromobacter

spp.

exhibit

specific

binding

sites

for

adenine

with

values

as

low

as

nM

for

A.

xylosoxidans

Thus

the

detected

adenine

binding

site

in

A.

xylosoxidans

exhibits

an

even

higher

affinity

than

the

rat

-LRB-

nM

-LSB-

-RSB-

nM

-LSB-

-RSB-

-RRB-

or

mouse

-LRB-

nM

-LSB-

-RSB-

-RRB-

adenine

receptor

The

binding

affinities

for

A.

lwoffii

and

A.

denitrificans

were

more

that

lower

with

affinities

in

the

low

micromolar

range

-LRB-

μM

and

μM

respectively

-RRB-

when

membrane

preparations

were

analyzed

and

about

lower

when

intact

cells

were

investigated

for

binding

-LRB-

nM

and

nM

respectively

-RRB-

For

A.

xylosoxidans

only

a

threefold

difference

was

found

when

binding

affinities

for

membranes

were

compared

with

those

with

intact

cells

-LRB-

Fig.

-RRB-

The

specific

high-affinity

-LSB-

-RSB-

adenine

binding

site

in

A.

xylosoxidans

appeared

to

be

expressed

in

extraordinarily

high

density

as

amounts

of

membrane

preparations

that

contained

more

than

μg

of

protein/assay

tube

led

to

depletion

of

the

radioligand

-LRB-

i.e.

more

than

of

the

added

radioligand

was

bound

to

the

protein

-RRB-

Rough

estimations

of

receptor

densities

by

homologous

competition

assays

confirmed

the

high

expression

levels

So

far

the

nature

of

these

high-affinity

adenine

binding

sites

in

bacteria

is

not

known

However

bacteria

express

a

large

number

of

transporters

including

nucleobase

transporters

in

order

to

secure

their

nutrient

supply

-LRB-

for

review

see

-LSB-

-RSB-

-RRB-

Nucleobase

transport

in

bacteria

has

been

mainly

studied

in

Escherichia

coli

and

Bacillus

spp.

as

well

as

in

the

fungi

Aspergillus

nidulans

and

Neurospora

crassa

-LSB-

-RSB-

Usually

these

transporters

fulfill

two

main

functions

Firstly

purines

can

serve

as

preformed

bases

for

nucleotide

biosynthesis

and

secondly

they

serve

as

nitrogen

sources

-LSB-

-RSB-

Distinct

adenine

uptake

systems

have

been

identified

e.g.

in

E.

coli

-LSB-

-RSB-

The

high

density

of

the

detected

-LSB-

-RSB-

adenine

binding

sites

in

A.

xylosoxidans

would

be

consistent

with

its

function

as

a

purine

transporter

and

points

to

an

important

role

of

this

protein

which

appears

to

be

upregulated

when

the

bacteria

are

transferred

to

Tris

buffer

-LRB-

unpublished

observation

-RRB-

a

medium

poor

in

nutrients

Such

an

effect

has

been

described

for

protozoa

-LSB-

-RSB-

purine

salvage

enzymes

and

transporters

can

be

dramatically

up

or

down-regulated

according

to

growth

stage

and

availability

of

purine

sources

-LSB-

-RSB-

Examples

are

the

high-affinity

hypoxanthine

transporter

in

Trypanosoma

brucei

brucei

which

shows

a

increased

transport

rate

after

h

of

purine

deprivation

and

the

adenine

transporter

in

Crithidia

luciliae

which

shows

a

increase

of

adenine

uptake

after

purine

starvation

-LSB-

-RSB-

For

E.

coli

as

well

as

for

B.

subtilis

two

adenine

transport

systems

have

been

described

a

low

and

a

high-affinity

transport

system

-LSB-

-RSB-

The

latter

system

is

important

when

the

concentration

of

adenine

is

low

-LSB-

-RSB-

Differences

in

adenine-binding

affinity

observed

in

our

studies

when

intact

cells

were

compared

with

membrane

preparations

-LRB-

Fig.

-RRB-

might

be

explained

by

the

existence

of

different

transporters

in

the

bacteria

-LRB-

Achromobacter

and

Acinetobacter

-RRB-

Whereas

bacterial

membrane

preparations

were

obtained

directly

from

an

overnight

culture

grown

in

complete

medium

binding

studies

at

whole

bacteria

were

performed

after

growing

them

in

Tris-HCl

buffer

a

nutrient-poor

medium

in

which

they

may

have

upregulated

the

high-affinity

transporters

-LSB-

-RSB-

In

order

to

investigate

the

structure

activity

relationships

of

the

high-affinity

adenine

binding

site

in

A.

xylosoxidans

a

series

of

compounds

including

adenine

derivatives

other

purines

and

pyrimidines

-LRB-

uracil

xanthine

hypoxanthine

-RRB-

and

nucleosides

-LRB-

uridine

adenosine

-RRB-

were

investigated

in

binding

studies

and

the

results

were

compared

with

those

obtained

at

the

rat

adenine

receptor

As

expected

structure

activity

relationships

at

the

rat

adenine

receptor

were

very

different

from

those

at

the

bacterial

adenine

binding

site

Hypoxanthine

for

example

which

exhibits

a

low

affinity

for

the

rat

adenine

receptor

-LRB-

nM

-RRB-

bound

to

the

bacterial

site

with

higher

affinity

-LRB-

nM

-RRB-

and

which

showed

a

high

affinity

for

murine

adenine

receptors

-LRB-

nM

-RRB-

bound

with

a

lower

affinity

-LRB-

nM

-RRB-

to

the

bacterial

site

Several

compounds

that

had

shown

affinity

in

the

micromolar

range

at

rat

adenine

receptors

-LRB-

adenosine

-RRB-

were

completely

inactive

at

Achromobacter

binding

sites

Alexander

had

previously

reported

that

hypoxanthine

at

a

concentration

of

μM

completely

blocked

-LSB-

-RSB-

adenine

binding

to

rat

brain

membranes

in

his

experiments

the

results

of

which

were

not

consistent

with

the

labeling

of

a

G

protein-coupled

adenine

receptor

-LSB-

-RSB-

It

might

be

speculated

that

he

actually

labeled

a

bacterial

adenine

binding

site

rather

than

the

rat

adenine

receptor

which

would

explain

the

discrepant

results

The

limited

number

of

compounds

-LRB-

five

-RRB-

investigated

by

IJzerman

and

coworkers

-LSB-

-RSB-

do

not

allow

a

full

comparison

of

the

structure

activity

relationships

but

large

differences

were

observed

for

two

compounds

deoxyadenosine

-LRB-

nM

-LSB-

-RSB-

vs.

nM

-LRB-

rat

adenine

receptor

-RRB-

-LSB-

-RSB-

-RRB-

and

-LRB-

nM

-LSB-

-RSB-

nM

-LRB-

rat

adenine

receptor

-RRB-

-LSB-

-RSB-

-RRB-

indicating

the

labeling

of

a

very

different

presumably

a

bacterial

binding

site

by

the

authors

-LSB-

-RSB-

The

fact

that

hypoxanthine

exhibits

high

affinity

for

the

-LSB-

-RSB-

adenine

binding

site

in

A.

xylosoxidans

in

the

same

concentration

range

as

adenine

itself

is

another

indication

that

the

labeled

protein

may

be

a

purine

transporter

Nucleobase

transporters

have

been

identified

in

bacteria

fungi

protozoa

algae

plants

and

mammals

but

only

few

have

been

cloned

and

analyzed

in

detail

-LSB-

-RSB-

Five

basic

families

of

nucleobase

transporters

have

been

described

the

nucleobase-ascorbate

transporters

-LRB-

NAT

-RRB-

which

include

members

from

archaea

eubacteria

fungi

plants

and

metazoa

the

purine-related

transporters

-LRB-

PRP

-RRB-

which

are

restricted

to

procaryotes

and

fungi

the

purine

permeases

-LRB-

PUP

-RRB-

which

are

purine

transporters

exclusively

found

in

plants

and

the

equilibrative

-LRB-

ENT

-RRB-

and

concentrative

-LRB-

CNT

-RRB-

nucleoside

transporters

which

not

only

transport

nucleosides

but

may

also

transport

nucleobases

-LSB-

-RSB-

Bacteria

have

developed

different

transport

systems

for

related

compounds

which

allow

them

to

independently

absorb

those

compounds

This

is

an

advantage

when

growing

under

nutritional

deprivation

-LSB-

-RSB-

For

C.

luciliae

a

nucleobase

transporter

that

recognizes

adenine

and

hypoxanthine

equally

well

has

been

described

-LSB-

-RSB-

In

E.

coli

adenine

and

uracil

have

different

specific

transport

systems

as

do

xanthine

and

guanine

whereas

hypoxanthine

might

utilize

the

guanine

transporter

-LSB-

-RSB-

For

B.

subtilis

specific

transport

systems

for

guanine

and

hypoxanthine

for

guanosine

and

inosine

as

well

as

three

independent

uptake

systems

for

adenine

adenosine

and

uracil

have

been

identified

-LSB-

-RSB-

From

the

described

observations

we

conclude

that

the

high-affinity

binding

sites

found

in

bacteria

isolated

from

Tris-buffer

solutions

have

not

much

in

common

with

the

adenine

receptors

found

in

mammals

The

genomes

of

the

bacteria

A.

baumannii

and

of

several

closely

related

bacteria

such

as

Pseudomonas

spp.

are

known

Therefore

we

performed

a

search

to

identify

potential

sequences

with

homology

to

the

rat

and

mouse

adenine

receptors

which

however

yielded

no

hits

It

appears

likely

that

-LSB-

-RSB-

adenine

labels

a

high-affinity

nucleobase

transporter

for

adenine

and

hypoxanthine

in

Achromobacter

spp.

a

bacterium

isolated

as

a

contaminant

from

Tris-HCl

buffer

The

high

affinity

in

the

low

nanomolar

range

is

in

fact

unusual

therefore

it

may

be

speculated

that

the

labeled

protein

might

belong

to

a

new

type

of

high-affinity

bacterial

nucleobase

transporter

Conclusions

In

conclusion

we

have

demonstrated

that

-LSB-

-RSB-

adenine

is

a

well-suited

radioligand

for

the

labeling

of

G

protein-coupled

adenine

receptors

but

precaution

is

advised

for

preparing

and

storing

buffers

used

for

the

assays

to

avoid

bacterial

contaminations

After

systematically

analyzing

occasionally

encountered

irregularities

in

-LSB-

-RSB-

adenine

binding

assays

we

were

able

to

isolate

three

bacteria

commonly

found

in

soil

and

water

from

Tris-HCl

buffer

They

were

identified

as

A.

lwoffii

A.

xylosoxidans

and

A.

denitrificans

and

revealed

high-affinity

binding

sites

for

-LSB-

-RSB-

adenine

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

achromobacter
acinetobacter
adenine receptor
purine transporter
radioligand binding studies

bacterial buffer contamination
g protein-coupled receptor
nosocomial infection
purinergic receptor
[3h]adenine


Validation

of

tissue

microarray

technology

in

squamous

cell

carcinoma

of

the

esophagus

Tissue

microarray

-LRB-

TMA

-RRB-

technology

has

been

developed

to

facilitate

high-throughput

immunohistochemical

and

in

situ

hybridization

analysis

of

tissues

by

inserting

small

tissue

biopsy

cores

into

a

single

paraffin

block

Several

studies

have

revealed

novel

prognostic

biomarkers

in

esophageal

squamous

cell

carcinoma

-LRB-

ESCC

-RRB-

by

means

of

TMA

technology

although

this

technique

has

not

yet

been

validated

for

these

tumors

Because

representativeness

of

the

donor

tissue

cores

may

be

a

disadvantage

compared

to

full

sections

the

aim

of

this

study

was

to

assess

if

TMA

technology

provides

representative

immunohistochemical

results

in

ESCC

A

TMA

was

constructed

containing

triplicate

cores

of

formalin-fixed

paraffin-embedded

squamous

cell

carcinomas

of

the

esophagus

The

agreement

in

the

differentiation

grade

and

immunohistochemical

staining

scores

of

E-cadherin

and

between

TMA

cores

and

a

subset

of

randomly

selected

donor

paraffin

blocks

was

determined

using

kappa

statistics

The

concurrence

between

TMA

cores

and

donor

blocks

was

moderate

for

-LRB-

κ

-RRB-

and

E-cadherin

-LRB-

κ

-RRB-

substantial

for

differentiation

grade

-LRB-

κ

-RRB-

and

-LRB-

κ

-RRB-

and

almost

perfect

for

-LRB-

κ

-RRB-

and

-LRB-

κ

-RRB-

TMA

technology

appears

to

be

a

valid

method

for

immunohistochemical

analysis

of

molecular

markers

in

ESCC

provided

that

the

staining

pattern

in

the

tumor

is

homogeneous

Introduction

Esophageal

carcinoma

is

the

eighth

most

common

type

of

cancer

in

the

world

-LSB-

-RSB-

Although

the

recent

rise

in

incidence

of

esophageal

cancer

has

predominantly

been

caused

by

an

increase

in

adenocarcinomas

the

majority

of

esophageal

cancer

cases

globally

are

squamous

cell

carcinomas

-LSB-

-RSB-

For

both

histological

types

radical

en

bloc

esophagectomy

with

an

extensive

lymph

node

dissection

offers

the

best

chance

for

cure

leading

to

an

overall

survival

rate

of

around

-LSB-

-RSB-

Well-known

histopathological

factors

for

prognostication

of

esophageal

cancer

include

the

TNM

stage

the

number

of

positive

lymph

nodes

and

the

presence

of

extracapsular

lymph

node

involvement

-LSB-

-RSB-

Recently

there

has

been

a

growing

interest

in

the

prognostic

value

of

molecular

markers

in

-LRB-

esophageal

-RRB-

cancer

-LSB-

-RSB-

The

expression

of

such

markers

is

often

studied

by

immunohistochemistry

on

formalin-fixed

paraffin-embedded

tumor

slides

Tissue

microarray

-LRB-

TMA

-RRB-

technology

has

been

developed

to

enable

high-throughput

immunohistochemical

analyses

-LSB-

-RSB-

By

inserting

small

-LRB-

diameter

e.g.

mm

-RRB-

donor

tissue

core

biopsies

into

a

single

recipient

paraffin

block

this

technique

allows

for

rapid

analysis

of

large

numbers

of

tissues

under

standardized

laboratory

and

evaluation

conditions

without

significantly

damaging

the

patient

's

tissue

In

addition

TMA

technology

leads

to

a

significant

reduction

of

the

amount

of

consumables

used

and

time

needed

for

interpretation

increasing

cost-effectiveness

A

potential

disadvantage

compared

to

full

tissue

sections

is

that

the

donor

cores

may

not

be

representative

for

the

whole

tumor

particularly

in

case

of

heterogeneous

tumors

and

heterogeneously

expressed

molecular

markers

Hence

some

validation

studies

have

been

performed

in

various

cancers

using

different

kinds

of

antibodies

-LSB-

-RSB-

Although

several

studies

have

revealed

novel

prognostic

biomarkers

in

esophageal

squamous

cell

cancer

-LRB-

ESCC

-RRB-

by

means

of

TMA

technology

-LSB-

-RSB-

this

technique

has

not

yet

been

validated

for

these

tumors

The

aim

of

the

present

study

was

therefore

to

validate

TMA

technology

in

ESCC

by

assessing

the

concurrence

of

immunohistochemical

staining

scores

of

established

molecular

markers

with

various

expression

patterns

between

triplicate

mm

core

biopsies

of

the

TMA

and

their

whole

tissue

section

counterparts

Materials

and

methods

TMA

construction

Formalin-fixed

paraffin-embedded

tissues

from

thoracic

ESCCs

of

consecutive

patients

having

undergone

esophagolymphadenectomy

at

the

authors

institute

between

and

were

retrieved

from

the

archives

of

the

Department

of

Pathology

Patients

who

received

neoadjuvant

therapy

were

excluded

from

this

study

The

study

was

carried

out

in

accordance

with

the

ethical

guidelines

of

our

institution

concerning

informed

consent

about

the

use

of

patient

's

materials

after

surgical

procedures

By

an

experienced

pathologist

-LRB-

FtK

-RRB-

three

representative

tumor

regions

were

marked

on

one

selected

hematoxylin

and

eosin

-LRB-

H&E

-RRB-

stained

section

of

each

tumor

avoiding

areas

of

necrosis

From

these

three

tumor

regions

a

tissue

cylinder

with

a

diameter

of

mm

was

punched

out

of

the

corresponding

paraffin

block

-LRB-

donor

block

-RRB-

and

placed

into

the

TMA

paraffin

block

using

a

manual

tissue

arrayer

-LRB-

MTA-I

Beecher

Instruments

Sun

Prairie

USA

-RRB-

which

was

guided

by

the

MTABooster

-LRB-

Alphelys

Plaisir

France

-RRB-

The

distribution

and

position

of

the

cores

was

determined

in

advance

with

the

TMA-designer

Software

-LRB-

Alphelys-TMA

Designer

Version

Plaisir

France

-RRB-

Cores

of

normal

esophageal

mucosa

lymph

node

kidney

liver

spleen

and

prostate

were

incorporated

in

the

tissue

array

block

as

internal

controls

Immunohistochemistry

For

each

marker

a

slide

of

the

TMA

and

one

of

every

selected

donor

paraffin

block

were

immunohistochemically

stained

Table

shows

the

details

of

all

antibodies

dilutions

incubation

times

and

antigen

retrieval

methods

applied

in

this

study

Table

of

antibodies

used

and

details

of

tissue

processingPrimary

antibodyStaining

patternSourceaClone

and

codeAntigen

retrievalDilutionIncubation

time

-LRB-

min/room

temperature

-RRB-

DetectionbPositive

pH

Strept

pH

autoclave

pH

-LRB-

-RRB-

pH

Strept

pH

Strept

ABCSerous

adenocarcinoma

of

the

endometriumAutostaineraBiogenex

San

Ramon

CA

USA

Chemicon

Chemicon

International

Temecula

CA

USA

Dako

DakoCytomation

Glostrup

Denmark

Neomarkers

Fremont

USA

Zymed

Zymed

Laboratories

San

Francisco

CA

USA.bStrept

ABC

is

biotinylated

horse

antimouse

Vector

diluted

in

PBS

followed

by

streptavidin

biotin

complex

diluted

Powervision

ready

to

use

-LRB-

Poly-HRP-antiMs/Rb/RtIgG

biotin-free

ImmunoVision

Technologies

Norwell

CA

USA

-RRB-

For

all

stainings

sections

were

deparaffinized

in

xylene

for

min

followed

by

dehydration

through

graded

alcohols

Endogenous

peroxidase

activity

was

blocked

for

min

in

a

buffer

solution

of

pH

-LRB-

containing

g

citric

acid

g

disodium

hydrogen

phosphate

g

sodium

azide

in

l

of

water

-RRB-

with

hydrogen

peroxide

-LRB-

-RRB-

After

antigen

retrieval

for

min

a

cooling

down

period

of

min

was

followed

by

incubation

with

the

primary

antibody

Depending

on

the

antibody

used

slides

were

incubated

with

the

secondary

antibody

followed

by

the

streptavidin

biotin

complex

or

slides

were

directly

incubated

with

Powervision

-LRB-

details

of

both

products

shown

in

the

legend

of

Table

-RRB-

Then

the

peroxidase

reactivity

was

developed

by

diaminobenzidine

for

min

and

slides

were

counterstained

with

Mayer

's

hematoxylin

In

between

steps

slides

were

washed

with

phosphate-buffered

saline

-LRB-

pH

-RRB-

Immunohistochemical

scoring

By

two

observers

-LRB-

FtK

and

JB

-RRB-

conjointly

the

degree

of

differentiation

and

the

percentage

of

immunohistochemically

stained

tumor

cells

were

determined

in

all

TMA

cores

and

in

the

full

sections

of

the

selected

donor

blocks

Histologic

grade

was

scored

as

well-differentiated

-LRB-

-RRB-

moderately

differentiated

-LRB-

-RRB-

or

poorly

differentiated

-LRB-

-RRB-

-LSB-

-RSB-

Staining

of

and

were

marked

as

negative

-LRB-

of

tumor

nuclei

stained

-RRB-

weakly

positive

-LRB-

-RRB-

or

strongly

positive

-LRB-

-RRB-

-LSB-

-RSB-

Cytokeratin

-LRB-

CK

-RRB-

and

staining

were

scored

as

negative

-LRB-

of

tumor

cell

cytoplasms

stained

-RRB-

weakly

positive

-LRB-

-RRB-

or

strongly

positive

-LRB-

-RRB-

E-cadherin

expression

was

regarded

negative

when

of

tumor

cell

membranes

stained

and

positive

when

stained

-LSB-

-RSB-

Cores

were

considered

lost

if

of

cells

contained

tumor

-LRB-

sampling

error

-RRB-

or

when

of

tissue

was

present

-LRB-

absent

core

-RRB-

Cases

were

excluded

if

two

out

of

three

cores

were

lost

When

the

scores

between

the

cores

of

a

particular

case

differed

the

most

frequent

score

determined

the

overall

score

In

case

of

three

different

scores

in

one

case

the

middle

score

was

chosen

When

only

two

cores

were

available

with

both

a

different

score

the

case

was

excluded

from

further

analysis

-LSB-

-RSB-

Statistical

analysis

Statistical

analyses

were

performed

using

the

SPSS

software

for

Windows

-LRB-

Version

SPSS

Chicago

IL

USA

-RRB-

Sixty-four

donor

blocks

-LRB-

of

the

tumors

incorporated

in

the

TMA

-RRB-

were

randomly

chosen

by

means

of

a

random

selection

function

of

SPSS

To

determine

the

chance-corrected

agreement

between

the

immunohistochemical

staining

scores

of

TMA

cores

and

large

sections

the

Cohen

's

weighted

kappa

statistic

was

calculated

Chance-corrected

agreement

was

considered

poor

if

κ

slight

if

κ

fair

if

κ

moderate

if

κ

substantial

if

κ

and

almost

perfect

if

κ

-LSB-

-RSB-

The

overall

agreement

was

defined

as

the

percentage

of

correct

agreement

between

the

TMA

and

the

donor

blocks

from

the

total

number

of

cases

-LSB-

-RSB-

Results

Of

the

-LRB-

-RRB-

tumor

tissue

cores

that

were

transferred

into

the

TMA

paraffin

block

a

median

of

-LRB-

-RRB-

was

available

for

immunohistochemical

scoring

on

the

TMA

slides

used

in

this

study

-LRB-

Table

-RRB-

Of

the

randomly

selected

cases

a

median

of

-LRB-

-RRB-

of

cores

-LRB-

-RRB-

was

evaluable

on

the

TMA

slides

Table

of

the

amount

of

cores

that

were

evaluable

absent

or

contained

too

little

tumor

in

all

cases

and

in

the

randomly

selected

cases

on

the

TMA

slides

H&ECK

TMA

cases

-LRB-

n

-RRB-

No

of

evaluable

No

of

absent

No

of

cores

without

selected

TMA

cases

-LRB-

n

-RRB-

No

of

evaluable

No

of

absent

No

of

cores

without

E

hematoxylin

and

eosin

On

the

H&E

stained

TMA

slide

-LRB-

-RRB-

of

the

randomly

chosen

cases

were

represented

by

cores

-LRB-

-RRB-

by

cores

One

-LRB-

-RRB-

case

was

excluded

from

further

analysis

because

only

a

single

core

was

available

The

agreement

in

the

scores

for

the

grade

of

differentiation

between

the

TMA

cores

and

the

full

sections

is

shown

in

Table

The

weighted

kappa

score

was

Table

in

the

degree

of

differentiation

between

TMA

cores

and

full

sections

Full

well-differentiated

moderately

differentiated

poorly

differentiated

Fifty-nine

-LRB-

-RRB-

of

the

randomly

selected

cases

stained

for

were

represented

by

cores

-LRB-

Fig.

-RRB-

the

remaining

cases

by

cores

The

immunohistochemical

scores

of

the

TMA

and

the

donor

blocks

are

shown

in

Table

Overall

agreement

in

scores

between

the

TMA

and

the

donor

blocks

was

with

a

kappa

of

Fig.

of

strong

staining

in

TMA

cores

and

the

corresponding

full

section

a

Three

TMA

cores

representing

one

tumor

magnification

b

Enlargement

of

the

middle

TMA

core

depicted

in

a

magnification

c

Part

of

the

slide

of

the

donor

block

of

the

same

tumor

magnification

in

immunohistochemical

scores

between

TMA

cores

and

full

slides

stained

for

and

Full

sections

For

two

cases

were

excluded

because

only

one

tumor

core

was

left

and

three

cases

were

also

excluded

because

the

two

available

cores

had

discrepant

immunohistochemical

scores

Fifty

-LRB-

-RRB-

of

cases

had

complete

agreement

-LRB-

Table

-RRB-

Four

cases

were

scored

one

class

higher

on

TMA

when

compared

with

the

full

sections

Conversely

five

other

cases

were

classified

lower

on

TMA

with

one

case

two

classes

lower

Kappa

score

was

Regarding

E-cadherin

staining

three

assessable

cores

were

present

in

of

the

cases

two

cores

in

Overall

agreement

in

E-cadherin

staining

scores

was

accomplished

in

of

cases

-LRB-

Table

-RRB-

In

one

case

a

higher

score

was

found

on

the

TMA

compared

to

the

full

section

In

cases

the

expression

of

E-cadherin

was

scored

lower

on

TMA

than

on

the

full

sections

The

observed

kappa

was

Table

in

immunohistochemical

scores

between

TMA

cores

and

full

slides

stained

for

E-cadherin

Full

sectionsE-cadherin

TotalκTMA

Three-core

analysis

of

staining

could

be

performed

in

of

selected

cases

and

two-core

analysis

in

Two

cases

were

represented

by

a

single

tumor

core

and

were

therefore

excluded

from

further

analysis

staining

was

scored

as

moderate

on

both

TMA

and

full

sections

in

-LRB-

-RRB-

of

selected

cases

In

of

cases

the

scores

of

the

TMA

were

similar

to

that

of

the

full

sections

Thirteen

cases

were

discordant

-LRB-

Table

-RRB-

kappa

was

Table

in

immunohistochemical

scores

between

TMA

cores

and

full

slides

stained

for

and

Full

sections

With

regard

to

staining

two

cores

were

present

in

-LRB-

-RRB-

cases

and

cores

were

available

in

-LRB-

-RRB-

cases

One

case

was

excluded

as

it

was

represented

by

only

one

TMA

core

Complete

agreement

was

achieved

in

of

the

selected

tumors

-LRB-

Table

-RRB-

In

the

eight

nonconcordant

cases

the

difference

was

one

class

resulting

in

a

kappa

of

Discussion

After

its

introduction

in

TMA

technology

has

been

applied

in

the

immunohistochemical

analysis

of

various

malignancies

including

squamous

cell

carcinomas

and

adenocarcinomas

of

the

esophagus

-LSB-

-RSB-

Although

it

seems

a

very

attractive

method

for

high-throughput

analysis

of

hundreds

of

tissues

simultaneously

it

may

have

limitations

as

the

evaluation

of

the

marker

expression

is

reduced

from

full-section

analysis

to

a

few

tissue

cores

of

only

mm

in

diameter

especially

for

proteins

that

are

heterogeneously

expressed

or

that

are

cell

cycle-dependent

-LSB-

-RSB-

It

is

therefore

essential

to

assess

in

each

type

of

cancer

individually

and

for

every

molecular

marker

whether

TMA

technology

is

feasible

and

valid

-LSB-

-RSB-

To

our

knowledge

this

has

not

been

done

in

esophageal

cancer

In

our

TMA

containing

triplicate

cores

of

ESCCs

a

median

of

of

cores

was

uninformative

-LRB-

lost

during

tissue

processing

and

containing

too

little

tumor

-RRB-

which

is

comparable

to

the

results

reported

in

other

studies

-LSB-

-RSB-

Improper

selection

of

representative

tumor

areas

on

the

donor

block

's

H&E

slide

by

the

pathologist

or

incorrect

punching

of

these

representative

areas

out

of

the

donor

block

can

cause

tissue

cores

that

contain

too

little

tumor

Possible

causes

of

absent

cores

are

the

size

and

fragility

of

the

tumor

tissue

used

and

the

aggressiveness

of

tissue

processing

applied

-LSB-

-RSB-

Moreover

the

number

of

available

cores

on

the

TMA

slide

depends

on

the

level

at

which

the

TMA

paraffin

block

has

been

sectioned

The

slides

stained

for

H&E

and

were

one

of

the

first

slides

that

were

cut

from

our

TMA

block

whereas

sections

stained

for

and

E-cadherin

were

taken

slightly

deeper

On

these

latter

sections

a

lower

number

of

absent

cores

was

observed

-LRB-

Table

-RRB-

showing

that

not

all

cores

were

placed

at

the

exact

same

level

in

the

TMA

block

during

TMA

construction

mainly

due

to

dissimilar

thicknesses

of

the

donor

paraffin

blocks

that

were

used

to

construct

the

TMA

-LSB-

-RSB-

The

agreement

in

immunohistochemical

results

of

the

markers

between

our

TMA

and

the

full

sections

varied

from

moderate

to

almost

perfect

-LRB-

κ

to

-RRB-

which

is

consistent

with

the

results

reported

in

other

TMA

validation

studies

-LSB-

-RSB-

The

observed

variation

in

agreement

could

be

due

to

tumor

heterogeneity

topographical

variation

in

the

expression

pattern

of

the

molecular

marker

or

to

the

scoring

criteria

used

-LSB-

-RSB-

Regarding

tumor

heterogeneity

the

optimal

amount

of

tissue

cores

incorporated

in

the

TMA

has

been

a

matter

of

debate

Several

validation

studies

have

shown

that

three

cores

are

highly

representative

for

the

full

section

-LSB-

-RSB-

The

addition

of

a

fourth

core

did

not

add

to

the

percentage

of

agreement

in

a

colorectal

cancer

TMA

-LSB-

-RSB-

Moreover

the

more

cores

punched

per

case

the

fewer

cases

can

be

placed

into

the

TMA

reducing

throughput

Adding

a

fourth

core

may

nevertheless

be

worthwhile

in

tissues

prone

to

uninformative

cores

due

to

small

lesions

such

as

dysplasias

or

carcinomas

in

situ

-LSB-

-RSB-

In

our

TMA

the

amount

of

uninformative

cores

was

low

-LRB-

-RRB-

probably

because

ESCCs

have

a

large

diameter

thereby

increasing

the

chance

of

obtaining

a

core

containing

tumor

tissue

Taken

together

we

consider

it

justified

to

utilize

three

biopsy

cores

in

ESCCs

Nonetheless

using

such

a

low

amount

of

cores

requires

careful

selection

of

the

tumor

regions

by

an

experienced

pathologist

to

deal

with

the

heterogeneity

of

the

tumor

in

the

TMA

-LSB-

-RSB-

The

agreement

between

TMA

and

full

sections

was

substantial

to

almost

perfect

for

and

Because

of

cases

have

shown

a

very

strong

expression

of

and

only

of

cases

showed

negative

staining

this

molecular

marker

does

not

subdivide

ESCCs

and

consequently

will

not

be

a

prognostic

marker

for

this

malignancy

was

more

evenly

distributed

over

the

three

scoring

groups

but

because

one

case

was

scored

two

classes

lower

on

TMA

when

compared

to

the

full

section

kappa

was

lower

when

compared

to

The

relatively

moderate

concordance

in

case

of

may

be

explained

by

the

fact

that

almost

of

cases

were

situated

in

one

category

-LRB-

staining

of

of

tumor

cells

-RRB-

with

discordant

cases

deviating

from

this

category

E-cadherin

also

had

a

moderate

concordance

mainly

because

the

relatively

faint

staining

intensity

of

this

molecular

maker

made

its

assessment

in

our

TMA

very

difficult

-LRB-

Fig.

-RRB-

Fig.

example

of

E-cadherin

staining

in

TMA

cores

and

the

corresponding

full

section

a

Three

TMA

cores

representing

one

tumor

magnification

b

Enlargement

of

the

right

TMA

core

depicted

in

a

magnification

c

Part

of

the

slide

of

the

donor

block

of

the

same

tumor

magnification

TMA

technology

was

also

found

to

be

valid

for

determining

the

histologic

grade

of

differentiation

in

ESCC

Complete

agreement

between

TMA

and

full

sections

occurred

in

-LRB-

out

of

κ

-RRB-

of

selected

cases

which

is

high

when

compared

to

the

agreement

achieved

in

a

TMA

of

bladder

cancer

-LSB-

-RSB-

Due

to

its

homogeneous

staining

pattern

showed

excellent

concordance

-LRB-

κ

-RRB-

in

our

microarray

-LRB-

Fig.

-RRB-

Fig.

example

of

staining

in

TMA

cores

and

the

corresponding

full

section

a

Three

TMA

cores

representing

one

tumor

magnification

b

Enlargement

of

the

left

TMA

core

depicted

in

a

magnification

c

Part

of

the

slide

of

the

donor

block

of

the

same

tumor

magnification

The

concurrence

between

the

TMA

and

the

full

sections

is

affected

by

the

cut-off

values

of

the

immunohistochemical

scoring

system

of

the

stainings

as

well

-LSB-

-RSB-

The

application

of

a

two-class

scoring

system

in

an

endometrial

cancer

TMA

improved

κ

to

compared

to

with

a

three-class

system

-LSB-

-RSB-

In

our

study

the

two-class

scoring

system

did

not

substantially

affect

the

kappa

-LRB-

data

not

shown

-RRB-

Because

the

E-cadherin

expression

had

a

very

low

intensity

in

our

ESCCs

we

have

chosen

to

apply

a

two-class

system

In

addition

the

cut-off

values

indicating

a

strong

immunohistochemical

expression

were

set

higher

in

the

cytokeratins

-LRB-

-RRB-

than

in

the

other

molecular

markers

-LRB-

cut-off

value

-RRB-

because

otherwise

practically

all

tumors

would

be

designated

having

a

strong

expression

of

cytokeratins

Now

that

our

esophageal

cancer

TMA

has

been

validated

it

will

be

used

to

correlate

the

expression

of

various

molecular

pathways

with

clinicopathologic

data

aiming

at

detecting

markers

of

prognostic

significance

and

molecular

targets

for

new

therapies

Because

the

agreement

between

TMA

slides

and

full

sections

depended

on

the

molecular

marker

stained

for

it

should

be

considered

to

assess

the

expression

pattern

of

a

marker

on

a

full

section

first

before

staining

a

TMA

slide

When

a

focal

or

heterogeneous

expression

pattern

is

noticed

it

might

be

more

valuable

to

assess

marker

expression

by

means

of

full

sections

instead

of

TMA

On

the

other

hand

when

a

marker

shows

a

homogeneously

diffuse

expression

pattern

staining

a

TMA

slide

does

allow

for

high-throughput

screening

of

tumors

When

a

prognostic

molecular

marker

has

been

identified

by

means

of

TMA

technology

it

is

recommended

to

verify

the

results

by

full-section

analysis

In

conclusion

this

study

has

demonstrated

TMA

technology

to

be

a

valid

method

for

immunohistochemical

analysis

in

ESCC

with

agreement

levels

for

well-known

molecular

markers

with

different

staining

potential

between

TMA

and

full

sections

ranging

from

moderate

to

almost

perfect

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

squamous cell carcinoma
validation studies

biological markers
esophageal neoplasms
protein microarray analysis