DNA-PK

contributes

to

the

phosphorylation

of

AIRE

Importance

in

transcriptional

activity

The

autoimmune

regulator

-LRB-

AIRE

-RRB-

protein

is

a

key

mediator

of

the

central

tolerance

for

tissue

specific

antigens

and

is

involved

in

transcriptional

control

of

many

antigens

in

thymic

medullary

epithelial

cells

-LRB-

mTEC

-RRB-

Mutations

in

the

AIRE

gene

cause

a

rare

disease

named

autoimmune

dystrophy

-LRB-

APECED

-RRB-

Here

we

report

using

GST

pull-down

assay

mass-spectrometry

and

co-immunoprecipitation

that

a

heterotrimeric

complex

of

DNA-Dependent

Protein

Kinase

-LRB-

DNA-PK

-RRB-

consisting

of

and

DNA-PK

catalytic

subunit

-LRB-

DNA-PKcs

-RRB-

is

a

novel

interaction

partner

for

AIRE

In

vitro

phosphorylation

assays

show

that

the

residues

and

are

DNA-PK

phosphorylation

sites

in

AIRE

In

addition

we

demonstrate

that

DNA-PKcs

is

expressed

in

AIRE

positive

mTEC

cell

population

and

that

introduction

of

mutations

into

the

AIRE

phosphorylation

sites

decrease

the

capacity

of

AIRE

to

activate

transcription

from

reporter

promoters

In

conclusion

our

results

suggest

that

phosphorylation

of

the

AIRE

protein

at

and

by

DNA-PK

influences

AIRE

transactivation

ability

and

might

have

impact

on

other

aspects

of

the

functional

regulation

of

the

AIRE

protein

Introduction

AIRE

-LRB-

autoimmune

regulator

-RRB-

is

a

defective

gene

in

APECED

-LRB-

autoimmune-polyendocrinopathy-candidiasis-ectodermal

dystrophy

OMIM

-RRB-

the

genetic

autoimmune

disease

which

manifests

as

autoimmunity

to

multiple

endocrine

glands

In

human

tissues

AIRE

is

expressed

in

the

thymus

spleen

and

lymph

nodes

-LSB-

-RSB-

The

AIRE

protein

-LRB-

kDa

-RRB-

has

several

features

which

indicate

that

it

might

function

in

transcriptional

regulation

AIRE

shares

similar

domain

architecture

with

the

family

proteins

-LRB-

and

-RRB-

which

all

have

HSR

-LRB-

for

homogenously

staining

region

-RRB-

SAND

-LRB-

for

and

-RRB-

and

PHD

-LRB-

for

plant

homeodomain

-RRB-

domains

HSR

is

central

for

AIRE

di

or

oligomerization

-LSB-

-RSB-

The

SAND

domain

is

a

DNA

binding

domain

found

in

several

mammalian

nuclear

proteins

NMR

spectroscopy

studies

of

the

SAND

domain

have

shown

that

a

conserved

amino

acid

motif

KDWK

is

essential

for

DNA

recognition

-LSB-

-RSB-

Although

AIRE

lacks

the

KDWK

motif

its

SAND

domain

has

also

been

shown

to

bind

DNA

in

vitro

-LSB-

-RSB-

The

PHD

fingers

are

found

in

nuclear

proteins

that

are

often

involved

in

chromatin-mediated

transcriptional

regulation

and

appear

to

mediate

protein

protein

interactions

The

zinc-binding

cysteine-rich

regions

of

the

PHD

finger

form

two

flexible

loops

and

two

protein

ligands

have

been

proposed

to

bind

one

PHD

finger

simultaneously

-LSB-

-RSB-

It

has

been

suggested

that

one

of

the

ligands

can

be

specific

for

each

PHD

For

example

the

PHD

finger

of

can

bind

phosphoinositides

whereas

PHD

of

the

repressor

binds

to

a

component

of

the

NuRD

chromatin

remodelling

complex

-LSB-

-RSB-

Another

ligand

which

might

be

common

for

all

PHD

fingers

has

been

long

suggested

to

be

chromatin

and

recently

PHD

domains

of

and

BPTF

proteins

were

reported

to

bind

tri-methylated

lysine

of

histone

-LSB-

-RSB-

However

despite

the

determination

of

its

structure

no

conclusive

ligand

of

the

AIRE

PHD

finger

has

yet

been

identified

-LSB-

-RSB-

We

and

others

have

shown

that

AIRE

can

activate

reporter

genes

from

the

interferon

beta

promoter

or

in

a

system

where

AIRE

is

tethered

via

heterologous

DNA

binding

domain

to

the

reporter

-LSB-

-RSB-

The

transcriptional

activation

region

of

AIRE

has

been

initially

mapped

to

the

two

C-terminal

PHD

fingers

as

the

mutations

in

these

domains

severely

decreased

the

transactivation

capacity

-LSB-

-RSB-

More

recent

reports

have

shown

that

several

other

APECED-causing

mutations

in

the

HSR

or

SAND

domain

also

decrease

the

AIRE

transactivation

ability

-LSB-

-RSB-

So

far

the

only

known

protein

partner

for

AIRE

is

a

general

transcriptional

co-regulator

and

histone

acetyltransferase

CREBP

binding

protein

-LRB-

CBP

-RRB-

CBP

has

been

reported

to

bind

directly

to

AIRE

in

vitro

it

colocalizes

with

AIRE

into

the

nuclear

bodies

in

monocytic

cell

cultures

and

it

enhances

the

transcription

of

reporter

genes

in

collaboration

with

AIRE

-LSB-

-RSB-

The

mechanisms

how

AIRE

in

collaboration

with

CBP

activates

its

target

genes

are

largely

unknown

DNA-dependent

protein

kinase

-LRB-

DNA-PK

-RRB-

is

a

Ser/Thr

kinase

that

belongs

to

the

phosphatidylinositol

-LRB-

-RRB-

family

It

is

formed

as

holoenzyme

in

the

presence

of

DNA

by

two

Ku

regulatory

subunits

-LRB-

kDa

-RRB-

and

-LRB-

kDa

-RRB-

and

a

large

catalytic

subunit

DNA-PKcs

-LRB-

kDa

-RRB-

With

very

few

exceptions

DNA-PKcs

is

active

only

when

it

is

in

hetero-trimeric

complex

with

Ku

proteins

and

in

interaction

with

DNA

or

RNA

-LSB-

-RSB-

The

main

function

of

DNA-PK

is

to

recognize

double

stranded

DNA

breaks

and

to

catalyze

a

repair

process

known

as

non-homologous

end

joining

-LRB-

NHEJ

-RRB-

In

a

similar

way

DNA-PK

is

crucial

for

V

-LRB-

D

-RRB-

J

recombination

in

developing

T

and

B

cells

Concordantly

DNA-PKcs

or

Ku

deficient

mice

are

severely

immunodeficient

with

elevated

radiosensitivity

and

susceptible

for

tumor

development

-LSB-

-RSB-

In

addition

to

the

role

of

DNA-PK

in

chromatin

repair

DNA-PK

has

been

shown

to

phosphorylate

many

proteins

involved

in

cell

cycling

and

transcriptional

regulation

such

as

Myc

Fos

Jun

TBP

TFIIB

RNA

Polymerase

II

-LSB-

-RSB-

and

several

nuclear

receptors

for

example

the

glucocorticoid

-LSB-

-RSB-

progesterone

-LSB-

-RSB-

and

androgen

receptors

-LSB-

-RSB-

DNA-PK

can

positively

and

negatively

modulate

transcription

As

a

repressor

DNA-PK

binds

directly

to

a

specific

DNA

sequence

element

within

the

mouse

mammary

tumor

virus

-LRB-

MMTV

-RRB-

promoter

-LSB-

-RSB-

Similarly

the

whole

DNA-PK

complex

has

been

shown

to

bind

to

the

E-box/TATA

like

elements

and

to

suppress

the

human

xanthine

oxidoreductase

-LRB-

hXOR

-RRB-

gene

expression

-LSB-

-RSB-

At

the

same

time

the

Ku

proteins

have

been

described

as

transcriptional

recycling

co-activators

of

androgen

receptor

-LSB-

-RSB-

In

addition

to

these

examples

DNA-PK

can

modulate

gene

expression

via

RNA-dependent

phosphorylation

of

hnRNP

proteins

-LSB-

-RSB-

In

order

to

further

define

molecular

mechanisms

of

AIRE

functioning

we

searched

for

novel

protein

partners

of

AIRE

using

the

GST

pull-down

approach

combined

with

mass

spectrometry

We

found

that

AIRE

interacts

with

DNA-PK

complex

proteins

DNA-PKcs

and

Further

in

vitro

phosphorylation

assays

demonstrated

that

DNA-PK

phosphorylates

the

AIRE

protein

in

vitro

at

the

residues

and

In

addition

we

show

that

the

mutation

of

either

of

the

phosphorylation

sites

significantly

decreases

AIRE

transcriptional

activity

Materials

and

methods

Plasmids

Plasmid

was

generated

by

insertion

of

PCR-amplified

sequence

encoding

human

amino

acids

into

the

NcoI

and

KpnI

restriction

sites

of

the

vector

-LRB-

EMBL-Heidelberg

protein

expression

and

purification

laboratory

-RRB-

To

generate

pGST-AIRE-SPP

-LRB-

-RRB-

the

sequence

encoding

AIRE

amino

acids

was

cloned

into

EcoRI

and

XhoI

sites

of

vector

-LRB-

a

gift

from

K.

Saksela

University

of

Tampere

Finland

-RRB-

pGST

and

mutation

constructs

were

generated

by

altering

amino

acid

or

to

alanines

and

to

leucine

in

pGST-AIRE

-LSB-

-RSB-

constructs

by

PCR-based

site-directed

mutagenesis

The

control

plasmid

was

a

gift

from

T.

Punga

-LRB-

Uppsala

University

Sweden

-RRB-

and

were

prepared

by

further

cloning

the

mutated

AIRE

fragments

from

and

into

EcoRI

and

SalI

sites

of

pSI

vector

-LRB-

Promega

-RRB-

To

generate

luciferase

reporter

plasmids

pBL-INV

and

pBL-LOR

the

promoter

areas

of

involucrin

-LRB-

nt

-RRB-

and

loricrin

-LRB-

nt

-RRB-

genes

were

cut

out

from

the

plasmids

pTZhINV-nlbgal

and

-LRB-

gifts

from

A.

Männik

FitBiotech

Estonia

-RRB-

and

cloned

into

the

HindIII

site

of

pBL-KS

-LRB-

a

gift

from

K.

Saksela

University

of

Tampere

Finland

-RRB-

The

cloned

plasmids

were

verified

by

sequencing

Expression

and

purification

of

GST

fusion

proteins

The

GST-tagged

proteins

were

expressed

either

from

Section

or

previously

described

-LSB-

-RSB-

AIRE

GST-fusion

constructs

pGST-AIRE

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

pAIRE-SAND

-LRB-

-RRB-

in

Escherichia

coli

strain

by

induction

with

mM

IPTG

for

h

at

room

temperature

Full-length

GST-AIRE

and

PHD-containing

fusion

proteins

were

expressed

in

the

presence

of

mM

The

proteins

were

purified

using

Glutathione

Sepharose

-LRB-

AmershamBiosciences

-RRB-

according

to

the

manufacturer

's

instructions

except

that

N-laurylsarcosine

and

Triton

were

added

to

increase

solubility

of

the

proteins

and

μM

was

included

when

GST-AIRE

and

PHD-containing

fusion

proteins

were

purified

The

purified

proteins

were

verified

on

SDS-PAGE

and

Coomassie

Blue

staining

GST

pull-down

and

mass

spectrometry

μg

of

nuclear

extract

prepared

from

human

monocyte

cell

line

according

to

-LSB-

-RSB-

was

incubated

with

μg

of

GST-fusion

proteins

bound

to

μl

of

packed

sepharose

beads

in

buffer

-LRB-

mM

HEPES

pH

mM

NaCl

mM

glycerol

mM

EDTA

μM

mM

DTT

and

proteinase

inhibitor

mix

-RRB-

over-night

at

°C

The

beads

were

washed

intensively

with

buffer

the

bound

proteins

were

eluted

and

separated

on

SDS-PAGE

The

specific

protein

bands

were

cut

out

and

analyzed

with

mass

spectrometry

Mass

mapping

of

the

peptides

generated

was

performed

with

an

Ultraflex

MALDI-TOF/TOF

mass

spectrometer

-LRB-

Bruker-Daltonics

Bremen

Germany

-RRB-

equipped

with

a

nitrogen

laser

in

a

positive

ion

reflector

mode

using

acid

as

the

matrix

The

MALDI

spectra

were

externally

calibrated

with

the

standard

peptide

mixture

from

Bruker-Daltonics

-LRB-

Bremen

Germany

-RRB-

In

case

of

a

peptide

fragmentation

analysis

a

peptide

from

above

mass

mapping

analysis

was

selected

as

a

precursor

ion

and

subjected

for

further

MS/MS

fragmentation

in

the

MALDI-TOF/TOF

lift-mode

Database

searches

were

carried

out

by

either

Mascot

peptide

map

fingerprint

or

Mascot

MS/MS

ions

search

-LRB-

-RRB-

Transfections

and

immunoprecipitations

The

transfections

were

carried

out

with

in

vitro

reagent

-LRB-

Fermentas

-RRB-

according

to

the

manufacturer

's

protocol

For

immunoprecipitations

approximately

cells

were

transfected

with

μg

of

pcAIRE

-LSB-

-RSB-

or

either

his

-LRB-

Invitrogen

-RRB-

or

-LRB-

Clontech

-RRB-

vector

After

h

a

whole-cell

extract

was

prepared

by

lysis

with

ml

of

lysis

buffer

-LRB-

mM

Tris

pH

M

NaCl

mM

mM

mM

DTT

mM

EDTA

glycerol

protease

inhibitors

-RRB-

for

min

on

ice

The

lysates

were

treated

five

times

with

the

syringe

to

shear

genomic

DNA

centrifuged

at

rpm

min

at

°C

The

supernatants

were

diluted

times

with

the

lysis

buffer

lacking

and

immunoprecipitations

were

performed

with

mouse

monoclonal

anti-DNA-PK

-LRB-

BD

Pharmingen

-RRB-

anti-AIRE

-LSB-

-RSB-

anti-HuR

-LRB-

a

gift

from

J.

Steitz

Yale

University

USA

-RRB-

anti-HA

-LRB-

SantaCruzBiotech

-RRB-

anti-GAPDH

-LRB-

Ambion

-RRB-

anti-c-myc

-LRB-

Sigma

-RRB-

and

rabbit

polyclonal

anti-GST

-LRB-

SantaCruzBiotech

-RRB-

antibodies

μg

of

each

antibody

was

added

to

μg

of

whole-cell

extracts

and

incubated

for

h

at

°C

then

μl

of

packed

protein

G-Sepharose

beads

-LRB-

Amersham

Pharmacia

Biotech

-RRB-

were

included

and

incubation

was

continued

for

h

at

°C

The

immunoprecipitates

were

washed

times

with

the

IP

buffer

-LRB-

mM

Tris

pH

M

NaCl

mM

mM

mM

DTT

mM

EDTA

glycerol

protease

inhibitors

-RRB-

and

eluted

from

the

beads

with

SDS

loading

buffer

separated

on

SDS-PAGE

and

subjected

to

Western

blot

with

anti-AIRE

or

DNA-PK

antibody

-LRB-

-RRB-

and

ECL

detection

Transfer

DNA-PK

was

carried

out

with

M

CAPS

buffer

pH

supplemented

with

methanol

in

semi-dry

blotting

system

for

min

at

Where

indicated

RNaseA/RNaseT

mix

-LRB-

Fermentas

-RRB-

with

final

concentrations

μg/ml

and

u/ml

respectively

was

added

Ethidium

bromide

-LRB-

EtBr

-RRB-

and

micrococcal

nuclease

-LRB-

MNase

-RRB-

treatments

were

performed

as

demonstrated

earlier

-LSB-

-RSB-

Either

mg/ml

EtBr

was

added

to

the

extract

or

u

of

MNase

-LRB-

Fermentas

-RRB-

in

μl

of

digestion

buffer

-LRB-

mM

HEPES

pH

mM

mM

NaCl

mM

-RRB-

was

used

Digestion

efficiency

of

MNase

was

tested

in

the

separate

control

experiments

using

the

same

conditions

On

agarose

gels

only

the

mononucleosomal

fraction

of

DNA

was

detected

when

whole

nuclei

were

treated

and

no

DNA

was

left

when

free

DNA

was

digested

Computer

prediction

of

AIRE

phosphorylation

Scansite

program

at

-LSB-

-RSB-

and

NetPhos

program

at

-LSB-

-RSB-

were

used

to

predict

AIRE

phosphorylation

sites

Phosphorylation

assays

The

kinase

assays

were

performed

with

SignaTECT

DNA-PK

Kinase

Assay

System

kit

-LRB-

Promega

-RRB-

essentially

according

to

the

manufacturer

's

protocol

The

phosphorylation

reactions

were

carried

out

in

the

presence

of

for

min

at

°C

and

either

μg

of

peptide

-LRB-

EPPLSQEAFADLWKK

Promega

-RRB-

or

μg

of

GST

fusion

proteins

were

used

as

the

substrates

Efficiencies

of

labeling

of

the

substrates

were

measured

by

radioactivity

counter

-LRB-

Guardian

Wallac

-RRB-

Nuclear

extracts

and

immunoprecipitations

were

made

as

described

in

Sections

and

Either

μg

-LRB-

Fig.

-RRB-

or

μg

-LRB-

all

other

figures

-RRB-

of

nuclear

extracts

were

used

per

reaction

Alternatively

U

of

purified

DNA-PK

complex

-LRB-

Promega

-RRB-

in

the

presence

of

ng

of

calf

thymus

DNA

was

used

μM

hydrochloride

-LRB-

Sigma

-RRB-

was

added

when

indicated

In

assays

using

GST

fusion

proteins

as

the

substrates

the

GST

proteins

bound

to

μl

of

packed

glutathione

beads

were

pre-washed

with

the

kinase

reaction

buffer

-LRB-

SignaTECT

kit

buffer

supplemented

with

mM

and

mM

-RRB-

After

the

kinase

reaction

the

beads

were

washed

three

times

with

ice

cold

washing

buffer

-LRB-

mM

HEPES

pH

mM

NaCl

BSA

mM

and

mM

-RRB-

before

measuring

the

radioactivity

Mice

thymic

stromal

cell

isolation

cell

sorting

and

RT-PCR

background

wild

type

mice

were

maintained

at

the

mouse

facility

of

the

Institute

of

Molecular

and

Cell

Biology

Tartu

University

Thymuses

from

to

mice

were

used

Thymic

stromal

cell

isolation

cell

sorting

and

RT-PCR

were

carried

out

as

described

previously

-LSB-

-RSB-

Luciferase

reporter

assays

Luciferase

assays

were

performed

using

Luciferase

Assay

System

kit

-LRB-

Promega

-RRB-

according

to

the

manufacturer

's

protocol

cells

in

plates

were

transfected

with

μg

of

INV-pBL

or

LOR-pBL

and

μg

pSI-AIRE

or

for

h

In

Western

blot

anti-AIRE

antibody

was

use

and

μg

of

pSI-AIRE

or

were

transfected

for

h.

Results

DNA-PK

complex

proteins

co-purify

with

AIRE

PHD

domain

In

order

to

find

new

AIRE

interacting

partners

we

expressed

the

first

PHD

domain

-LRB-

-RRB-

of

AIRE

as

a

GST-fusion

protein

-LRB-

Fig.

-RRB-

and

performed

the

GST

pull-down

using

the

nuclear

extract

prepared

from

monocytic

cells

The

interacting

proteins

were

separated

on

SDS-PAGE

and

visualized

by

Coomassie

staining

Three

protein

bands

with

molecular

weight

of

about

and

over

kDa

were

consistently

seen

after

the

SDS-PAGE

analyses

-LRB-

Fig.

-RRB-

Next

the

protein

bands

were

excised

from

the

gel

and

analyzed

by

mass

spectrometry

The

MALDI-TOF

mass

map

analysis

demonstrated

that

the

two

bands

with

molecular

masses

of

about

and

kDa

belong

to

and

respectively

whereas

the

protein

with

the

mass

over

kDa

was

presumed

to

be

the

catalytic

subunit

of

DNA-PK

-LRB-

DNA-PKcs

-RRB-

-LRB-

Table

-RRB-

Mass

map

identifications

with

low

sequence

coverage

were

further

verified

by

MALDI-TOF/TOF

fragmentation

of

three

tryptic

peptides

-LRB-

Table

-RRB-

Thus

these

results

identified

a

trimeric

complex

of

DNA-PK

as

a

candidate

AIRE

interacting

partner

Full-length

AIRE

interacts

with

DNA-PK

To

further

confirm

the

pull-down

results

and

to

demonstrate

that

the

full-length

AIRE

protein

is

able

to

interact

with

DNA-PK

we

carried

out

co-immunoprecipitation

experiments

So

far

no

AIRE

endogenous

expression

on

protein

level

has

been

detected

in

immortalized

cell-lines

As

subcellular

localization

of

transfected

AIRE

in

immortalized

cell

lines

is

similar

to

the

AIRE

localization

in

thymus

in

vivo

as

well

as

AIRE

is

able

to

activate

transcription

in

those

cells

we

transfected

cells

with

the

plasmid

expressing

full-length

AIRE

or

empty

vector

as

a

negative

control

The

whole-cell

extracts

were

prepared

from

transfected

cells

and

used

in

co-immunoprecipitations

with

anti-DNA-PKcs

anti-AIRE

or

various

control

antibodies

The

precipitates

were

separated

on

SDS-PAGE

and

immunoblotted

with

anti-AIRE

antibody

As

seen

in

Fig.

anti-DNA-PKcs

and

anti-AIRE

antibodies

-LRB-

as

a

positive

control

-RRB-

were

able

to

precipitate

AIRE

protein

whereas

control

antibodies

against

GST

GAPDH

and

HuR

-LRB-

a

nuclear

mRNA-binding

protein

-RRB-

could

not

Similarly

AIRE

and

DNA-PKcs

interaction

was

detectable

when

the

immunoprecipitation

with

anti-AIRE

antibody

followed

by

the

Western

with

anti-DNA-PKcs

antibody

was

carried

out

-LRB-

Fig.

-RRB-

Since

the

whole-cell

extracts

used

in

immunoprecipitations

contained

remarkable

amounts

of

chromatin

and

RNA

-LRB-

data

not

shown

-RRB-

we

next

wanted

to

test

whether

the

interaction

between

AIRE

and

DNA-PK

is

DNA

and

RNA

dependent

or

independent

Previously

a

DNA

intercalator

ethidium

bromide

-LRB-

EtBr

-RRB-

has

been

shown

to

efficiently

disrupt

the

binding

of

Ku

proteins

to

and

DNA-PK

-LSB-

-RSB-

whereas

the

micrococcal

nuclease

has

been

widely

used

for

the

disruption

of

chromatin

as

well

as

for

the

digestion

of

contaminating

DNA

from

cell

lysates

and

immunoprecipitates

-LSB-

-RSB-

Consequently

we

transfected

the

cells

with

the

plasmid

expressing

full-length

myc-tagged

AIRE

or

empty

vector

as

a

negative

control

and

performed

co-immunoprecipitations

with

anti-DNA-PKcs

antibodies

in

the

presence

of

EtBr

RNaseA/T

mix

or

treated

the

immunoprecipitates

with

the

micrococcal

nuclease

-LRB-

Fig.

top

panel

-RRB-

As

a

positive

control

to

show

that

the

same

level

of

the

AIRE

protein

was

present

in

each

lysate

the

immunoprecipitations

were

also

performed

with

the

anti-myc

antibodies

-LRB-

Fig.

bottom

panel

-RRB-

Fig.

top

panel

reveals

that

DNA-PKcs

still

co-immunoprecipitates

AIRE

from

the

cell

extracts

treated

with

EtBr

or

RNaseA

which

indicates

that

AIRE

does

not

require

DNA

or

RNA

to

interact

with

DNA-PKcs

However

when

we

treated

the

co-immunoprecipitates

with

micrococcal

nuclease

we

observed

a

weaker

interaction

between

AIRE

and

DNA-PKcs

-LRB-

Fig.

top

panel

-RRB-

which

suggests

that

in

the

cell

lysates

containing

DNA

and

chromatin

a

fraction

of

AIRE

binds

DNA-PK

through

the

DNA

and/or

chromatin

In

conclusion

the

co-immunoprecipitation

results

together

confirm

that

the

AIRE

protein

interacts

with

DNA-PK

DNA-PK

phosphorylates

the

AIRE

protein

DNA-PK

is

a

nuclear

serine/threonine

protein

kinase

that

has

been

reported

to

phosphorylate

many

nuclear

targets

To

study

whether

the

AIRE

protein

is

one

of

the

targets

of

DNA-PK

we

used

several

different

assays

which

enabled

quantitation

of

DNA-PK

kinase

activity

We

first

studied

whether

the

AIRE

protein

co-immunoprecipitates

DNA-PK

kinase

activity

Co-immunoprecipitations

from

nuclear

extracts

prepared

from

cells

transfected

with

myc-tagged

AIRE

were

carried

out

with

antibodies

recognizing

DNA-PKcs

myc

tag

and

GAPDH

-LRB-

as

a

negative

control

-RRB-

The

co-immunoprecipitates

were

further

incubated

with

biotinylated

derived

peptide

as

a

DNA-PK

specific

substrate

-LSB-

-RSB-

in

the

presence

of

DNA-PKcs

activating

dsDNA

and

As

a

positive

control

nuclear

extract

was

used

instead

of

the

co-immunoprecipitates

Fig.

shows

that

a

similar

amount

of

was

incorporated

into

the

peptide

when

AIRE

and

DNA-PKcs

co-immunoprecipitates

were

applied

whereas

no

activity

was

detectable

with

GAPDH

co-immunoprecipitate

The

relatively

high

phosphorylation

efficiency

achieved

with

AIRE

co-immunoprecipitate

was

most

likely

due

to

the

reason

that

the

co-immunoprecipitation

with

anti-myc

antibody

is

more

efficient

than

that

of

with

DNA-PK

antibody

Since

the

peptide

used

in

the

assay

is

a

DNA-PK

specific

substrate

-LSB-

-RSB-

these

data

show

that

the

AIRE/DNA-PK

complex

has

kinase

activity

and

suggest

that

AIRE

too

might

be

phosphorylated

by

DNA-PK

To

further

investigate

whether

AIRE

can

be

phosphorylated

by

DNA-PK

we

carried

out

three

different

in

vitro

kinase

assays

where

recombinant

GST-AIRE

and

GST

alone

were

used

as

the

substrates

First

we

performed

the

experiments

using

the

nuclear

extract

from

cells

as

a

source

of

kinases

in

the

absence

or

presence

of

a

specific

inhibitor

for

family

kinases

-LRB-

Fig.

-RRB-

Second

we

did

the

kinase

assay

using

the

nuclear

extracts

either

from

DNA-PKcs

positive

-LRB-

-RRB-

or

DNA-PKcs

negative

-LRB-

-RRB-

cell

lines

-LRB-

Fig.

-RRB-

Third

we

carried

out

kinase

reactions

using

a

purified

HeLa

DNA-PK

complex

again

in

the

absence

or

presence

of

-LRB-

Fig.

-RRB-

As

a

result

we

observed

that

the

phosphorylation

level

of

the

AIRE

protein

reached

up

to

of

that

of

the

protein

in

all

three

experimental

approaches

whereas

only

background

phosphorylation

of

GST

alone

was

detected

-LRB-

Fig.

D

-RRB-

In

concordance

the

kinase

activity

was

decreased

although

moderately

when

inhibitor

was

added

-LRB-

Fig.

-RRB-

or

when

the

nuclear

extract

from

DNA-PKcs

negative

cells

was

used

-LRB-

Fig.

-RRB-

indicating

that

besides

DNA-PKcs

other

kinases

can

phosphorylate

the

AIRE

protein

In

order

to

identify

which

region

of

the

AIRE

protein

is

phosphorylated

we

expressed

different

AIRE

domains

as

GST

fusion

proteins

and

tested

their

phosphorylation

using

the

nuclear

extracts

as

a

source

of

kinases

The

domain

structure

of

the

AIRE

protein

is

given

in

Fig.

Fig.

shows

that

the

nuclear

extract

phosphorylated

more

efficiently

the

GST-fusions

of

N-terminal

AIRE

containing

the

first

-LRB-

-RRB-

-LRB-

-RRB-

or

-LRB-

-RRB-

amino

acids

-LRB-

Fig.

-RRB-

while

about

times

less

phosphorylation

was

seen

with

the

GST-fusions

of

AIRE

C-terminal

fragments

containing

SAND

-LRB-

-RRB-

or

-LRB-

-RRB-

regions

A

similar

pattern

of

AIRE

phosphorylation

was

observed

when

the

phosphorylation

assay

was

performed

with

DNA-PKcs

competent

or

DNA-PKcs

deficient

cell

extracts

-LRB-

Fig.

-RRB-

Thus

although

in

pull-down

experiments

the

first

PHD

of

AIRE

alone

was

able

to

interact

with

DNA-PK

the

main

DNA-PK

target

seems

to

be

the

N-terminal

part

of

AIRE

indicating

that

AIRE

and

DNA-PK

make

multiple

contacts

Taken

together

these

results

demonstrate

that

DNA-PK

can

phosphorylate

AIRE

in

vitro

and

that

the

N-terminal

region

of

AIRE

is

the

predominant

target

of

DNA-PK

and

are

the

phosphorylation

sites

on

the

AIRE

protein

The

N-terminal

region

of

AIRE

contains

the

HSR

domain

that

mediates

di

or

tetramerization

of

the

protein

-LSB-

-RSB-

Interestingly

previous

in

vitro

experiments

have

indicated

that

AIRE

dimerization

may

occur

as

a

result

of

phosphorylation

-LSB-

-RSB-

We

next

aimed

to

find

the

DNA-PK

specific

phosphorylation

sites

on

AIRE

Using

the

Scansite

and

NetPhos

programs

we

found

two

putative

DNA-PK

phosphorylation

sites

within

and

close

to

the

HSR

domain

of

the

AIRE

protein

-LRB-

Figs.

and

-RRB-

To

verify

the

prediction

we

changed

either

or

to

alanines

both

in

wild

type

GST-AIRE

and

truncated

GST-AIRE

-LRB-

-RRB-

constructs

and

tested

the

influence

of

these

mutations

on

the

phosphorylation

efficiency

Fig.

demonstrates

that

compared

to

GST-AIRE

the

phosphorylation

levels

of

the

mutant

proteins

were

decreased

about

when

the

cell

extracts

from

either

or

cells

were

used

as

source

of

cellular

kinases

Finally

we

performed

the

phosphorylation

assays

using

purified

DNA-PK

complex

-LRB-

Fig.

-RRB-

In

agreement

with

the

previous

experiments

a

reduction

of

about

in

the

phosphorylation

levels

of

the

and

mutants

was

detected

whereas

the

APECED

patient

mutation

did

not

negatively

affect

the

AIRE

phosphorylation

level

Thus

we

conclude

that

amino

acids

and

of

the

AIRE

protein

are

the

phosphorylation

sites

of

DNA-PK

DNA-PK

is

expressed

in

thymic

medullary

epithelial

cells

-LRB-

mTEC

-RRB-

DNA-PK

is

a

ubiquitously

expressed

protein

which

levels

are

enhanced

by

induction

of

DNA

double

strand

brakes

-LSB-

-RSB-

To

study

whether

the

DNA-PK

and

AIRE

interaction

might

occur

in

vivo

we

studied

whether

DNA-PK

is

also

present

in

thymic

medullary

epithelial

cells

-LRB-

mTEC

-RRB-

the

only

subset

of

cells

where

the

AIRE

protein

expression

is

detected

thus

far

-LSB-

-RSB-

Thus

we

purified

the

thymic

mTEC

based

on

the

cell

surface

marker

EpCAM

-LSB-

-RSB-

and

analyzed

the

expression

of

Aire

DNA-PK

and

two

AIRE

target

genes

involucrin

and

loricrin

The

involucrin

and

loricrin

genes

are

highly

expressed

during

the

terminal

differentiation

of

epidermal

keratinocytes

-LSB-

-RSB-

and

are

downregulated

in

mTEC

of

Aire

knock-out

mouse

-LSB-

-RSB-

Fig.

right

panel

demonstrates

that

DNA-PKcs

mRNA

is

highly

expressed

in

thymic

epithelial

cells

including

the

AIRE-enriched

mTEC

fraction

As

characteristic

to

Aire-regulated

genes

in

the

thymus

the

expression

levels

of

involucrin

and

loricrin

were

relatively

low

remarkable

lower

than

those

of

DNA-PKcs

and

Aire

in

mTEC

subpopulation

-LRB-

Fig.

left

panel

-RRB-

and

mutations

downregulate

the

transactivation

activity

of

AIRE

Previously

it

has

been

demonstrated

that

several

missense

mutations

in

the

HSR

domain

affect

the

dimerization

cellular

localization

and

the

transactivation

ability

of

AIRE

-LSB-

-RSB-

Thus

we

next

tested

whether

the

mutation

of

AIRE

phosphorylation

sites

influences

the

AIRE

transactivation

activity

We

cloned

AIRE

with

and

mutations

into

mammalian

expression

vector

and

performed

luciferase

assays

using

the

lysates

from

cells

transfected

with

wild

type

AIRE

or

the

mutant

constructs

As

AIRE

target

genes

human

loricrin

and

involucrin

gene

promoters

fused

to

the

luciferase

gene

were

used

Fig.

demonstrates

that

when

the

cells

were

transfected

with

AIRE

and

AIRE

mutation

constructs

significantly

lower

transactivation

activity

compared

to

the

wild

type

AIRE

was

seen

Western

blot

of

the

transfected

lysates

with

anti-AIRE

antibody

shows

that

both

mutants

are

expressed

even

in

higher

level

than

the

wild

type

AIRE

construct

-LRB-

Fig.

-RRB-

In

conclusion

the

luciferase

assays

suggest

that

the

phosphorylation

status

of

AIRE

at

and

is

important

for

the

transactivation

activity

Discussion

In

this

study

we

demonstrate

that

AIRE

interacts

with

and

is

phosphorylated

by

the

DNA-PK

complex

The

best

DNA-PK

substrates

are

the

DNA

associated

proteins

and

the

most

effective

phosphorylation

occurs

when

DNA-PK

is

colocalized

with

the

same

DNA

molecule

as

its

target

proteins

-LSB-

-RSB-

Binding

to

the

Ku

proteins

improves

the

affinity

of

DNA-PKcs

for

DNA

ends

about

-LSB-

-RSB-

This

is

in

keeping

with

the

finding

showing

that

AIRE

is

able

to

bind

DNA

via

its

SAND

domain

-LSB-

-RSB-

In

addition

to

the

double

strand

brakes

the

DNA-PK

and/or

the

Ku

proteins

are

known

to

bind

telomeres

and

sequence-specific

promoter

elements

-LSB-

-RSB-

single

stranded

DNA

-LSB-

-RSB-

nucleosomes

-LSB-

-RSB-

RNA

-LSB-

-RSB-

and

base

unpairing

regions

-LRB-

BURs

-RRB-

which

typically

are

found

in

nuclear

matrix

attachment

regions

-LRB-

MARs

-RRB-

-LSB-

-RSB-

and

also

colocalize

to

RNA

polymerase

II

elongation

sites

-LSB-

-RSB-

Interestingly

AIRE

has

been

reported

to

associate

with

nuclear

matrix

-LSB-

-RSB-

and

has

been

proposed

to

regulate

gene

clusters

-LSB-

-RSB-

In

addition

the

AIRE

protein

contains

PHD

domains

which

recently

have

been

shown

to

recognize

specifically

modified

histones

within

the

chromatin

-LSB-

-RSB-

Thus

AIRE

appears

to

be

a

chromatin

and

DNA-binding

protein

In

this

study

we

observed

that

although

AIRE

is

able

to

bind

DNA-PK

without

DNA

most

likely

the

DNA

or

chromatin

is

contributing

to

the

AIRE

and

DNA-PK

interaction

-LRB-

Fig.

-RRB-

This

is

in

concordance

with

DNA-PK

ability

to

phosphorylate

its

substrates

more

efficiently

at

the

presence

of

DNA

-LSB-

-RSB-

At

least

two

possibilities

can

be

considered

how

DNA-PK

AIRE

and

DNA

or

chromatin

may

interact

First

DNA

or

chromatin

or

even

certain

DNA

sequence

elements

or

specifically

modified

chromatin

may

increase

the

affinity

of

AIRE

for

DNA-PK

Second

posttranslationally

modified

AIRE

subpopulations

may

bind

DNA-PK

in

different

conditions

As

we

can

not

exclude

that

this

type

of

experiments

can

be

influenced

by

the

overexpression

of

AIRE

further

studies

are

needed

to

find

out

the

role

of

chromatin

in

AIRE

and

DNA-PK

interaction

in

vivo

especially

in

the

thymic

environment

Phosphorylation

of

AIRE

by

cAMP

dependent

protein

kinases

A

-LRB-

PKA

-RRB-

and

C

-LRB-

PKC

-RRB-

has

been

reported

earlier

-LSB-

-RSB-

though

the

phosphorylated

domain

of

AIRE

remained

unknown

We

here

demonstrate

that

in

vitro

the

AIRE

protein

is

mainly

phosphorylated

at

the

N-terminal

region

and

define

two

residues

and

as

DNA-PK

specific

target

sites

resides

within

and

is

located

not

far

from

the

HSR

domain

Both

these

phosphorylation

sites

are

located

outside

the

finger

the

domain

of

AIRE

that

was

initially

shown

to

pull-down

DNA-PK

indicating

that

multiple

different

contacts

can

occur

between

AIRE

and

the

DNA-PK

complex

However

as

previously

reported

-LSB-

-RSB-

our

in

vitro

phosphorylation

studies

also

indicate

that

other

kinases

may

phosphorylate

the

AIRE

protein

and

that

phosphorylation

sites

other

than

and

should

exist

within

AIRE

The

HSR

domain

of

AIRE

has

been

shown

to

be

responsible

for

the

dimerization

and

tetramerization

of

the

protein

-LSB-

-RSB-

and

several

but

not

all

APECED

mutations

within

the

HSR

decrease

AIRE

transactivation

activity

and

influence

AIRE

cellular

localization

-LSB-

-RSB-

In

addition

previous

in

vitro

experiments

have

indicated

that

AIRE

dimerization

may

occur

as

a

result

of

phosphorylation

-LSB-

-RSB-

As

shown

in

this

study

conversion

of

and

amino

acids

to

alanines

suppresses

AIRE

transactivation

activity

However

we

did

not

observe

any

influence

of

these

mutations

to

AIRE

dimerization

and

cellular

localization

-LRB-

data

not

shown

-RRB-

Thus

since

the

mutations

that

do

not

impact

AIRE

dimerization

and

the

dot

formation

ability

can

downregulate

AIRE

transactivation

activity

the

N-terminal

part

of

AIRE

appears

to

be

involved

in

transcriptional

regulation

not

only

via

its

dimerization

activity

It

is

possible

that

status

of

AIRE

phosphorylation

at

N-terminus

influences

AIRE

interactions

with

other

protein

partners

or

with

DNA

In

response

to

ionizing

radiation

DNA-PK

can

have

indirect

effects

on

transcriptional

regulation

It

has

been

shown

that

phosphorylation

of

interferon

regulated

factor

-LRB-

-RRB-

and

pancreatic

duodenal

-LRB-

-RRB-

protein

by

DNA-PK

in

response

to

ionizing

radiation

directs

these

proteins

to

degradation

by

the

proteasome

and

therefore

downregulates

and

target

genes

-LSB-

-RSB-

Alternatively

phosphorylation

of

transcription

factor

by

DNA-PK

in

response

to

ionizing

radiation

leads

to

stabilization

but

disrupts

transactivation

activity

of

As

a

result

the

expression

of

target

genes

and

Histone

is

downregulated

-LSB-

-RSB-

We

also

studied

whether

UV

irradiation

changes

AIRE

cellular

localization

and

expression

level

in

various

cell

cultures

but

we

could

not

detect

remarkable

differences

-LRB-

data

not

shown

-RRB-

Thus

it

seems

that

DNA-PK

phosphorylates

AIRE

independently

of

its

function

in

DNA

repair

Based

on

research

on

AIRE

deficient

mice

it

is

evident

that

AIRE

upregulates

the

expression

level

of

many

genes

in

thymus

-LSB-

-RSB-

Studies

in

tissue

culture

cells

confirm

that

AIRE

indeed

activates

several

promoters

-LSB-

-RSB-

Thus

although

in

vivo

the

AIRE

protein

expression

is

only

detected

in

thymic

medullary

epithelial

cells

-LSB-

-RSB-

the

transfected

AIRE

protein

has

similar

transactivating

properties

and

nuclear

localization

pattern

as

AIRE

in

the

thymus

-LSB-

-RSB-

Despite

these

studies

the

mechanisms

of

how

AIRE

regulates

its

target

genes

are

still

unclear

Posttranscriptional

modifications

like

phosphorylation

acetylation

and

ubiquitination

are

widely

used

to

regulate

activity

of

transcription

factors

and

regulators

In

this

study

we

first

describe

the

phosphorylation

sites

of

AIRE

and

propose

that

its

phosphorylation

status

is

important

for

its

transactivation

activity

However

several

questions

remain

including

what

are

the

signals

that

trigger

AIRE

phosphorylation

and

how

AIRE

phosphorylation

is

connected

with

its

function

in

the

thymus

The

differentiation

of

AIRE

positive

thymic

epithelial

cells

was

recently

shown

to

be

dependent

on

RANK-mediated

signaling

-LSB-

-RSB-

which

is

known

to

activate

several

protein

kinases

-LSB-

-RSB-

As

we

showed

in

this

study

DNA-PK

is

expressed

in

medullary

epithelial

cells

-LRB-

Fig.

-RRB-

Whether

the

thymic

epithelial

differentiation

process

correlates

with

the

activation

of

DNA-PK

and

other

possible

AIRE-specific

kinases

needs

further

studies

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

apeced
hsr domain
phd finger
phosphorylation


The Microanatomic Location of Metastatic Breast Cancer in Sentinel Lymph Nodes Predicts Nonsentinel Lymph Node Involvement Background The majority of sentinel node -LRB- SN -RRB- positive breast cancer patients do not have additional non-SN involvement and may not benefit from axillary lymph node dissection -LRB- ALND -RRB- . Previous studies in melanoma have suggested that microanatomic localization of SN metastases may predict non-SN involvement . The present study was designed to assess whether these criteria might also be used to be more restrictive in selecting breast cancer patients who would benefit from an ALND . Axillary nodal status is among the most important prognostic factors in breast cancer patients . SN biopsy with an intensive pathological assessment of selectively removed lymph nodes is currently a highly accurate , minimally invasive technique to assess nodal status .1,2 It reduces the morbidity of breast cancer surgery by avoiding unnecessary axillary lymph node dissection -LRB- ALND -RRB- in patients with negative sentinel nodes -LRB- SNs -RRB- .3 The optimal treatment of patients with a positive SN is however less clear . The few SNs can be cost-effectively analyzed by multiple-level evaluation and immunohistochemistry -LRB- IHC -RRB- which increases the likelihood of detecting small metastases .4 The decision to proceed with an ALND in patients with macrometastatic SN involvement does not pose a major clinical dilemma . The need for routine ALND in patients with minimal SN involvement however continues to be debated since only a minority of these patients -LRB- 10 -- 15 % -RRB- show non-SN involvement .5 Predicting the chance of involvement of the non-SN would facilitate the selection of patients with a potential therapeutic benefit of ALND . Several features of the primary tumor and the involved SNs have been investigated as potential predictors for non-SN involvement . Primary tumor size , palpability , presence of peritumoral lymphovascular invasion , number of tumor-involved SNs , size of the SN metastases , and extracapsular extension -LRB- ECE -RRB- correlate with non-SN status .6 -- 10 However , none of these factors are sensitive and reproducible enough to reliably identify a subgroup of patients who might be spared ALND . Several melanoma studies have reported that the microanatomic pattern of SN involvement and the penetrative depth -LRB- defined as the maximum distance of cancer cells from the inner margin of the SN capsule -RRB- predict non-SN involvement .11 -- 14 In breast cancer , this has been studied less extensively .15 We therefore set out to study the predictive value of microanatomic location and penetrative depth of SN metastatic deposits for non-SN metastases , accurately assessed by morphometry , in a large series of SN-positive breast cancer patients . Patients And Methods Patients A retrospective database was analyzed , including patients with invasive breast cancer and a tumor positive axillary SN followed by ALND , treated at the University Medical Center Utrecht or the St Antonius Hospital in Nieuwegein from January 2000 to May 2007 -LRB- n = 357 -RRB- , including patients from our previous study .16 Exclusion criteria were multicentric tumors , neoadjuvant chemotherapy , and a total of fewer than six lymph nodes examined . SN Biopsy Technique The technical aspects used for the SN procedure are described in detail elsewhere .16 Briefly , before surgery , SN identification was performed by peritumoral injection of 120 MBq 99mTc-Nanocolloid -LRB- Amersham Cygne , Eindhoven , The Netherlands -RRB- in a maximal volume of 0.5 mL . Dynamic and static scintigraphic images were subsequently obtained . On the same day , immediately preoperatively 0.5 mL Patent blue dye -LRB- Guerbet , Aulnay-sous-Bois , France -RRB- was injected intradermally and intra/peritumorally . The SN was identified after careful dissection of blue lymphatic channels and detection of radioactivity with a handheld gamma ray detection probe . Palpation of the open axilla was performed to detect enlarged non-SN metastases . Histopathological Evaluation The SNs were processed according to the protocols described previously .16 -- 18 SNs were lamellated according to their size , fixed in neutral buffered formaldehyde , and completely embedded . Step sections 5 μm thick were cut at five levels with 250 μm intervals for staining with haematoxylin and eosin -LRB- H&E -RRB- . In the absence of apparent metastases by H&E examination , immunohistochemistry was performed with CAM 5.2 -LRB- Beckton Dickinson , Franklin Lakes , New Jersey , USA -RRB- or CK AE1/3 -LRB- Dako , Glostrup , Denmark -RRB- at each level . All non-SNs were identified visually or by palpation , dissected , processed routinely , and examined at one level with H&E staining . All SNs and non-SNs were examined initially by multiple pathologists at the two institutions , reviewed histologically , and reclassified according to the current 6th edition of the American Joint Committee on Cancer -LRB- AJCC -RRB- staging system by one observer -LRB- CHMvD -RRB- . All cases were evaluated without knowledge of non-SN involvement . Clinicopathological Features Clinicopathological features recorded included age , pT -LRB- TNM system of the AJCC -RRB- , histological subtype -LRB- according to the WHO -RRB- , histological grade -LRB- defined according to the Nottingham modified Bloom -- Richardson score based on the percentage of tubule formation , nuclear pleomorphism and mitotic activity -RRB- , mitotic activity index -LRB- MAI -RRB- ,19 steroid receptor and HER-2 / neu status -LRB- not routinely determined before 2005 -RRB- . SN and Non-SN Characteristics SN characteristics included metastatic size according to the 6th edition of the AJCC staging system -LSB- isolated tumor cells -LRB- ITC -RRB- -LRB- ≤ 0.2 mm -RRB- , micrometastases -LRB- > 0.2 mm and ≤ 2 mm -RRB- , macrometastases -LRB- > 2 mm -RRB- -RSB- , the number of SNs , ECE , maximal diameter of the largest metastases , microanatomic location of the metastatic deposit , and the penetrative depth . If multiple but distinct deposits were identified in the same SN , the largest metastasis was recorded . If single tumor cells , cluster of nests were continuous , or separated by a few cells distance , they were measured as one focus .20 If more than one SN was involved in an individual patient , the most extensive and/or deepest metastatic deposit was recorded . In case both axillary and internal mammary SNs were involved , the features were measured in the axillary SN . The microanatomic location of metastatic deposits within each SN was classified as subcapsular , combined subcapsular and parenchymal , parenchymal or extensive . Extensive SN involvement , as defined in the study of Ruiter et al. ,21 was a deposit > 5 mm in diameter -LRB- Fig. 1 -RRB- . The centripetal depth was , according to Starz et al. ,11 defined as the maximal depth at which tumor cells have infiltrated the SN , as measured from the inner margin of the capsule -LRB- Fig. 2 -RRB- , further denoted tumor penetrative depth according to the proposal of Scolyer et al. 12 Multifocality was defined as two or more separated metastatic deposits at some distance from each other . All measurements were calculated microscopically in the plane of the tissue sections using interactive video morphometry systems -LRB- Q-PRODIT , Leica , Cambridge , UK or Research Video Assistant , Baarn , The Netherlands -RRB- . FIG. 1 . Pattern of distribution of metastatic breast cancer deposits in SNs.FIG . 2 . The tumor penetrative depth of metastases was defined as the maximal distance of breast cancer cells from the inner margin of the SN capsule -LRB- arrow -RRB- -LRB- H&E , original magnification × 10 -RRB- . Non-SN characteristics included the total number of non-SNs , maximal tumor diameter , AJCC classification , and ECE . If more than one non-SN was involved , the largest diameter was recorded . Statistical Analysis Statistical analysis was performed using SPSS 13.0 for Windows . Patients were divided into groups with and without non-SN involvement . The Pearson chi-square test was used to determine the relationship between categorical variables -LRB- histological type and grade , steroid receptor and HER-2 / neu status , number of SNs , AJCC classification , ECE -RRB- on the one hand and non-SN status on the other . Continuous data -LRB- age , diameter primary tumor , MAI , SN tumor diameter -RRB- were analyzed using the Mann -- Whitney U-test . P-values < 0.05 were considered significant . All relevant variables that were associated with the presence of positive non-SNs were included in a multivariate logistic regression model . SN metastatic characteristics -LRB- diameter and penetrative depth -RRB- were further compared by receiver operating characteristic -LRB- ROC -RRB- analysis , calculating the area under curve -LRB- AUC -RRB- as a measure of discriminative value . Results The median age was 53 years -LRB- range 22 -- 86 years -RRB- and the mean histological invasive tumor size was 2.4 cm -LRB- range 0.3 -- 9.0 cm -RRB- . Overall , 571 SNs were obtained -LRB- mean 1.6 SN per patient -RRB- of the 357 patients with a positive SN as well as 4939 non-SNs -LRB- mean 14 per patient -RRB- . The metastatic deposits were subcapsular in 167 patients -LRB- 47 % -RRB- , combined subcapsular and parenchymal in 40 patients -LRB- 11 % -RRB- , parenchymal in 11 patients -LRB- 3 % -RRB- , and extensive in 139 patients -LRB- 39 % -RRB- -LRB- Fig. 1 -RRB- . Of the 357 patients , 24 -LRB- 7 % -RRB- patients had ITC only in the SN , whereas 112 -LRB- 31 % -RRB- had micrometastases and 221 -LRB- 62 % -RRB- had macrometastases . Localisation of ITCs was subcapsular -LRB- 96 % -RRB- or parenchymal -LRB- 4 % -RRB- . SN micrometastases were located subcapsularly -LRB- 81 % -RRB- , parenchymally -LRB- 5 % -RRB- or had combined localisation -LRB- 13 % -RRB- . The overall prevalence of non-SN involvement was 38 % -LRB- 136/357 patients -RRB- . Other descriptive characteristics of the study population are listed in Tables 1 and 2 . TABLE 1 . Clinicopathological characteristics of 357 invasive breast cancer patients with a positive SN and subsequent axillary lymph node dissectionFeatureNo . % Mean age -LRB- range -RRB- 54 y -LRB- 22 -- 86 -RRB- Primary tumorMean tumor size -LRB- range -RRB- 2.4 cm -LRB- 0.3 -- 9.0 -RRB- pT118752 pT215042 pT3195 pTx10Histological subtype Invasive ductal cancer29884 Invasive lobular cancer3610 Others236Histological grade -LRB- B&R -RRB- 17421 216145 312234MAI , mean/2 mm2 -LRB- range -RRB- 13 -LRB- 0 -- 102 -RRB- Steroid receptor status ER -- positive ‡ 32290 ER -- negative3410 ER -- unknown 1 PR -- positive ‡ 28480 PR -- negative7020 PR -- unknown3HER-2 / neu status Positive2615 Negative15085 Unknown181 ‡ ≥ 10 % immunoreactive neoplastic cells.TABLE 2 . SN and non-SN characteristics of 357 invasive breast cancer patients with a positive SN and subsequent axillary lymph node dissectionFeature No . % SNTotal number of SNs571Mean number of SNs 1.6 Total number of positive SNs419Mean diameter SN metastases 4.7 mmAJCC classification of SN metastatic size ITC247 Micrometastasis11231 Macrometastasis22162Extracapsular extension No25772 Yes10028Microanatomic location Subcapsular16747 Parenchymal 113 Combined4011 Extensive13939Mean penetrative depth2 .9 mmNon-SNsTotal number of non-SNs4939Mean number of non-SNs -LRB- range -RRB- 14 -LRB- 5 -- 38 -RRB- Total number of positive non-SNs474Total number of negative non-SNs4465Size of non-SN metastases ITC32 Micrometastases3324 Macrometastases10074 The following factors were significant predictors of non-SN metastases by univariate analysis : primary tumor size , number of involved SNs , ECE , AJCC classification , diameter , and penetrative depth of the SN metastatic deposit -LRB- Tables 3 and 4 -RRB- . None of the other classic variables -LRB- age , histological subtype and grade , steroid receptor and HER-2 / neu status , MAI -RRB- of the primary tumor correlated significantly with non-SN involvement . TABLE 3 . Comparison of categorical clinicopathological and SN characteristics in invasive breast cancer patients without and with non-SN metastases by Pearson chi-square testWithout non-SN metastasesWith non-SN metastasesP valueFeatureNo -LRB- % -RRB- No -LRB- % -RRB- Total 221 -LRB- 62 -RRB- 136 -LRB- 38 -RRB- Histological subtype0 .914 Ductal183 -LRB- 51 -RRB- 115 -LRB- 32 -RRB- Lobular22 -LRB- 6 -RRB- 14 -LRB- 4 -RRB- Others16 -LRB- 5 -RRB- 7 -LRB- 2 -RRB- Histological grade -LRB- B&R -RRB- 0.798 ER positive198 -LRB- 56 -RRB- 124 -LRB- 35 -RRB- 0.715 Negative22 -LRB- 6 -RRB- 12 -LRB- 3 -RRB- PR positive179 -LRB- 50 -RRB- 106 -LRB- 30 -RRB- 0.589 Negative41 -LRB- 12 -RRB- 29 -LRB- 8 -RRB- HER-2 / neu positive16 -LRB- 9 -RRB- 10 -LRB- 6 -RRB- 0.670 Negative83 -LRB- 47 -RRB- 67 -LRB- 38 -RRB- No . of SN0 .246 1131 -LRB- 37 -RRB- 89 -LRB- 25 -RRB- > 190 -LRB- 25 -RRB- 47 -LRB- 13 -RRB- No . of involved SN 0.050 1198 -LRB- 55 -RRB- 112 -LRB- 31 -RRB- > 123 -LRB- 6 -RRB- 24 -LRB- 7 -RRB- AJCC SN metastases < 0.001 ITC21 -LRB- 6 -RRB- 3 -LRB- 1 -RRB- Micrometastasis86 -LRB- 24 -RRB- 26 -LRB- 7 -RRB- Macrometastasis114 -LRB- 32 -RRB- 107 -LRB- 30 -RRB- Extracapsular extension < 0.001 No175 -LRB- 49 -RRB- 82 -LRB- 23 -RRB- Yes46 -LRB- 13 -RRB- 54 -LRB- 15 -RRB- Microanatomic location SN metastases < 0.001 Subcapsular126 -LRB- 35 -RRB- 41 -LRB- 11 -RRB- Combined23 -LRB- 6 -RRB- 17 -LRB- 5 -RRB- Parenchymal8 -LRB- 2 -RRB- 3 -LRB- 1 -RRB- Extensive64 -LRB- 18 -RRB- 75 -LRB- 21 -RRB- Multifocality0 .087 No176 -LRB- 49 -RRB- 118 -LRB- 33 -RRB- Yes45 -LRB- 13 -RRB- 18 -LRB- 5 -RRB- TABLE 4 . Comparison of continuous clinicopathological and SN characteristics in invasive breast cancer patients without and with non-SN metastases by Mann -- Whitney U-testWithout non-SN metastasesWith non-SN metastasesP valueFeatureTotal no . -LRB- % -RRB- 221 -LRB- 62 -RRB- 136 -LRB- 38 -RRB- Age -LRB- years , mean -RRB- 54530.249 Primary tumor diameter , cm -LRB- mean -RRB- 2.32.50.019 MAI , 2mm2 -LRB- mean -RRB- 13120.693 SN tumor diameter , mm -LRB- mean -RRB- 3.66.5 < 0.001 SN tumor penetrative depth , mm -LRB- mean -RRB- 2.23.9 < 0.001 The primary tumor features histological grade and diameter were associated with multiple SN tumor deposits -LRB- P = 0.03 and 0.05 , respectively -RRB- . Frequency of non-SN metastases in patients with SN ITC -LRB- N = 24 -RRB- , micro - -LRB- N = 112 -RRB- and macrometastases -LRB- N = 221 -RRB- was 12.5 % , 23 % , and 48 % , respectively -LRB- P < 0.001 , Fig. 3 -RRB- . Of those three patients with SN ITC and involved non-SNs , two had a non-SN micro - and one had a non-SN macrometastasis . One of these ITC was located in the parenchyma , the other two patients had a subcapsular location . Two of these three patients with SN ITC and non-SN involvement showed multiple small cell clusters and single cells in the SN . FIG. 3 . Flow chart showing distribution of SN AJCC classification according to non-SN involvement . The microanatomic localization of SN metastatic deposits correlated with non-SN involvement . Patients with subcapsular -LRB- N = 167 -RRB- , combined subcapsular and parenchymal -LRB- N = 40 -RRB- , parenchymal -LRB- N = 11 -RRB- and extensive -LRB- N = 139 -RRB- tumor deposits showed non-SN involvement in 25 % , 42 % , 27 % , and 54 % of cases , respectively -LRB- Table 3 -RRB- . Morphometrically assessed penetrative depth of SN metastases was also associated with non-SN involvement . Frequency of non-SN metastases in patients with a SN tumor penetrative depth < 0.155 mm -LRB- N = 29 -RRB- , 0.155 -- 2.7 mm -LRB- N = 181 -RRB- and > 2.7 mm -LRB- N = 147 -RRB- was 10 % , 28 % , and 56 % , respectively -LRB- P < 0.001 , Table 5 -RRB- . In ROC analysis , diameter -LRB- AUC = 0.686 -RRB- and the penetrative depth -LRB- AUC = 0.680 -RRB- of the SN tumor deposit had comparable discriminative value -LRB- Fig. 4 -RRB- . TABLE 5 . Predictive value of penetrative depth of SN metastases for non-SN involvement in patients with invasive breast cancerSN penetrative depth -LRB- mm -RRB- N No . of patients with non-SN involvement -LRB- % -RRB- < 0.155293 -LRB- 10 -RRB- 0.155 -- 2.718151 -LRB- 28 -RRB- > 2.714782 -LRB- 56 -RRB- FIG. 4 . ROC curves showing the sensitivity and specificity of SN tumor diameter and penetrative depth as predictors of non-SN involvement . The larger the area below the curve , the more accurate the prediction of non-SN involvement -LRB- P < 0.001 -RRB- . In multivariate analysis , SN tumor diameter -LRB- P = 0.032 -RRB- and SN tumor penetrative depth -LRB- < 0.155 mm versus 0.155 -- 2.7 mm versus > 2.7 mm -RRB- -LRB- P = 0.015 -RRB- were significant in predicting non-SN involvement . Cutoff values were interactively statistically as those best discriminating between low and high risk of non-SN metastases . Risk stratification by combining these features identified a low-risk group , an intermediate-risk group , and a high risk group for non-SN metastases with frequency of non-SN metastases in 11 % , 29 % , and 56 % of patients , respectively -LRB- Table 6 -RRB- . Frequencies of non-SN involvement in patients with SN micrometastases and a subcapsular -LRB- N = 91 -RRB- , combined subcapsular and parenchymal -LRB- N = 15 -RRB- , and parenchymal location -LRB- N = 6 -RRB- were 21 % , 40 % , and 17 % , respectively . Frequencies of non-SN involvement in patients with SN macrometastases and a subcapsular -LRB- N = 54 -RRB- , combined subcapsular and parenchymal -LRB- N = 25 -RRB- , parenchymal -LRB- N = 4 -RRB- and extensive location -LRB- N = 138 -RRB- were 39 % , 44 % , 25 % , and 54 % , respectively . TABLE 6 . Predictive value of a combination of AJCC classification of SN metastases and penetrative depth of SN metastases for non-SN involvement in patients with invasive breast cancerNNo . of patients with non-SN involvement -LRB- % -RRB- ITC/SN penetrative depth < 2.7 mm or Micrometastases/penetrative depth < 0.155 mm384 -LRB- 11 -RRB- Micro - or macrometastases/penetrative depth 0.155 -- 2.7 mm17250 -LRB- 29 -RRB- Micro - or macrometastases/penetrative depth > 2.7 mm14782 -LRB- 56 -RRB- Discussion The lymphatic spread of breast cancer cells has been shown to follow an orderly progression via the SN to non-SNs , which implies that the risk of spread of tumor from the SN to the non-SN may depend on the extent of SN involvement . While many studies have focused on the maximal diameter of the metastatic tumor deposit22 , 23 we investigated the microanatomic location and the penetrative depth of the metastatic deposits as a putative predictor of non-SN involvement . This was based on the concept that within the SN , tumor cells also follow an orderly route , arriving in the subcapsular sinuses through an afferent lymph vessel . Later , there is subcapsular outgrowth of malignant cells in the marginal sinuses and into the cortical parenchyma . Finally , these cells extend to the deeper zones of the lymph node parenchyma , frequently following the medullary sinuses to efferent lymph vessels .24 Consistent with this concept we found that the microanatomic location , the size , and the penetrative depth of SN tumor deposits were correlated significantly with non-SN involvement . This finding is consistent with recent melanoma studies . Startz et al. 11 proposed a micromorphometric classification , based on the depth of the metastasis from the capsule and the number of 1-mm slices containing melanoma . This classification was a highly significant predictor for distant metastases and overall survival . Dewar et al. 13 also recorded that the microanatomic location of melanoma SN metastases predicts non-SN involvement , and proposed that it would be possible to safely avoid a lymph node dissection in patients with subcapsular deposits only . Indeed , subcapsular location and small tumor penetrative depth correlated with less non-SN involvement in our breast cancer study , although no subgroup of patients could be selected without non-SN involvement . The measurement of the SN tumor penetrative depth was difficult in many cases , especially in case of extensive tumor deposits . In these cases the tumor deposits frequently extended beyond the center of the lymph node , making it difficult to determine which edge of the SN capsule should be used to measure the tumor penetrative depth . Similar difficulties were encountered when the SN had a lobulated outline . Further studies which more rigorously define the tumor penetrative depth may strengthen its predictive power and reproducibility . In conclusion , patients with breast carcinoma and SN involvement can be stratified into subgroups at significantly different risk for non-SN involvement , according to microanatomic localization of the SN metastatic deposits and penetrative depth into the SN . However , based on these features no subgroup of patients could be selected without non-SN involvement .

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