A

survey

of

failed

post-retained

restorations

Survival

of

endodontically

treated

post-restored

teeth

depends

on

a

multitude

of

factors

all

of

which

are

practically

impossible

to

include

in

a

randomized

controlled

clinical

study

The

purpose

of

this

survey

was

to

characterize

and

analyze

reported

failures

of

post-retained

restorations

to

identify

factors

critical

to

failure

and

to

type

of

failure

A

questionnaire

was

mailed

to

private

practitioners

in

Denmark

with

a

request

to

complete

the

questionnaire

whenever

a

patient

presented

with

a

failed

post-retained

restoration

Information

was

gathered

on

factors

related

to

the

patient

the

tooth

the

restorative

materials

and

the

techniques

Two-hundred

and

sixty

questionnaires

were

collected

from

practitioners

over

a

period

Functioning

time

until

failure

varied

between

months

and

years

Mean

survival

time

until

failure

was

years

Of

the

failed

restorations

had

functioned

for

years

or

less

Fracture

of

the

tooth

was

the

most

common

type

of

failure

reported

followed

by

loosening

of

the

post

and

fracture

of

the

post

Tapered

posts

implied

an

increased

risk

of

tooth

fracture

compared

to

loosening

or

fracture

of

the

post

and

the

relative

risk

of

tooth

fracture

increased

with

the

functioning

time

until

failure

Fracture

of

the

post

was

more

common

among

male

than

female

patients

On

the

basis

of

this

survey

of

failed

post-retained

restorations

it

was

concluded

that

tapered

posts

were

associated

with

a

higher

risk

of

tooth

fracture

than

were

parallel-sided

posts

Introduction

Endodontically

treated

teeth

have

been

reported

to

have

shorter

survival

time

than

vital

teeth

-LSB-

-RSB-

This

is

believed

to

reflect

that

endodontically

treated

teeth

are

often

structurally

compromised

because

of

a

series

of

events

caries

or

trauma

endodontic

therapy

and

preparation

for

restorative

procedures

-LSB-

-RSB-

Thus

to

minimize

the

negative

influence

on

longevity

the

restorative

procedure

of

endodontically

treated

teeth

calls

for

careful

consideration

of

treatment

alternatives

In

the

case

of

minimal-remaining

tooth

substance

the

retention

of

a

crown

has

traditionally

been

provided

by

a

core

and

the

retention

of

the

core

by

a

post

Two

fundamentally

different

techniques

find

widespread

use

for

the

construction

of

a

post

and

core

-LRB-

-RRB-

a

post

and

core

cast

as

a

single

unit

and

-LRB-

-RRB-

a

prefabricated

post

upon

which

a

core

is

directly

produced

in

e.g.

amalgam

or

resin

composite

The

first

prefabricated

posts

on

the

market

were

made

of

stainless

steel

or

gold-plated

brass

Later

posts

of

titanium

alloys

became

popular

and

recent

developments

have

introduced

posts

made

of

fiber-reinforced

resin

composite

or

ceramic

Simultaneously

adhesive

dentistry

offering

reliable

bonding

to

tooth

substance

and

to

restorative

materials

via

various

pretreatments

has

markedly

progressed

to

widen

the

possibilities

also

for

post

and

core

treatment

As

mentioned

the

post

serves

to

retain

the

restoration

However

the

post

should

also

serve

to

protect

the

remaining

tooth

structure

These

two

functions

of

endodontic

posts

may

be

evaluated

by

determining

the

retentive

ability

of

the

posts

and

the

fracture

resistance

of

endodontically

treated

teeth

provided

with

post-retained

restorations

The

latter

may

also

be

indirectly

determined

by

analysis

of

the

stress-distributing

characteristics

of

the

posts

Post-related

factors

that

have

been

found

to

exert

influence

on

retention

of

posts

and

protection

of

tooth

structure

include

shape

length

diameter

surface

design

and

stiffness

of

the

post

as

well

as

the

type

of

luting

cement

used

As

regard

to

post

shape

parallel-sided

posts

provide

better

retention

and

less

stress

formation

and

increased

fracture

resistance

than

do

tapered

posts

-LSB-

-RSB-

An

increase

in

post

length

has

a

similar

positive

effect

-LSB-

-RSB-

However

some

restrictions

apply

to

post

length

First

the

apical

seal

of

the

endodontic

restoration

must

be

ensured

through

maintenance

of

mm

of

apical

gutta-percha

-LSB-

-RSB-

Second

dentin

in

the

apical

third

of

teeth

is

often

very

thin

This

not

only

increases

the

risk

of

parietal

perforation

but

it

also

weakens

the

tooth

to

a

relatively

high

degree

thereby

increasing

the

risk

of

root

fracture

Little

evidence

is

available

as

regard

to

the

influence

of

post

diameter

Consensus

regarding

retention

seems

to

be

that

an

increase

in

post

diameter

can

not

be

relied

on

as

a

measure

to

improve

retention

-LSB-

-RSB-

A

study

based

on

finite

element

analysis

found

that

stress

formation

in

the

dentin

decreased

when

post

diameter

was

increased

-LSB-

-RSB-

Conversely

another

study

showed

that

thinner

posts

provided

increased

resistance

to

fracture

-LSB-

-RSB-

A

consensus

regarding

fracture

resistance

seems

to

be

that

the

root

canal

should

not

be

uncritically

enlarged

as

decreasing

the

bulk

of

dentin

weakens

the

tooth

and

reduces

the

resistance

to

fracture

-LSB-

-RSB-

As

regard

to

surface

design

serrated

posts

provide

better

retention

than

smooth-sided

posts

and

threaded

posts

provide

even

better

retention

-LSB-

-RSB-

However

the

superior

retention

of

threaded

posts

obtained

by

engaging

in

dentin

simultaneously

is

accompanied

by

increased

stress

formation

within

the

root

-LSB-

-RSB-

and

thus

by

an

increased

risk

of

root

fracture

-LSB-

-RSB-

Post

stiffness

which

is

a

combination

of

post

diameter

and

elastic

modulus

of

the

post

material

is

another

important

characteristic

but

an

unsettled

discussion

prevails

as

to

optimal

stiffness

On

the

one

hand

insufficient

stiffness

will

allow

excessive

distortion

of

the

restoration

at

the

margins

during

function

leading

to

breakdown

of

cement

and

risk

of

secondary

caries

Moreover

a

decrease

in

the

elastic

modulus

of

the

post

material

has

been

found

to

increase

stress

formation

in

the

root

and

to

decrease

fracture

strength

of

the

restored

tooth

-LSB-

-RSB-

Thus

low-modulus

posts

fail

sooner

or

at

lower

stress

values

than

do

high-modulus

posts

On

the

other

hand

several

studies

have

found

that

low-modulus

posts

display

failures

that

cause

little

damage

to

the

remaining

tooth

structure

-LRB-

loss

of

marginal

seal

loss

of

retention

core

fracture

and

post

fracture

-RRB-

while

high-modulus

posts

are

associated

with

a

higher

incidence

of

root

fractures

when

they

finally

fail

that

is

they

cause

more

damage

to

the

remaining

tooth

structure

and

often

result

in

the

extraction

of

the

tooth

involved

-LSB-

-RSB-

Retention

of

posts

in

the

root

canal

is

primarily

sought

through

the

use

of

a

luting

agent

Zinc

phosphate

cement

has

been

the

luting

agent

of

choice

for

many

years

The

progress

within

adhesive

dentistry

and

the

advent

of

ceramic

and

fiber-reinforced

resin

composite

posts

have

prompted

questions

of

the

usefulness

of

resin

cement

for

the

luting

of

posts

Zinc

phosphate

cement

provides

retention

by

mechanical

interlocking

in

irregularities

in

the

surface

of

the

root

canal

on

one

side

and

in

the

surface

of

the

post

on

the

other

After

appropriate

pretreatment

of

the

respective

surfaces

resin

cements

provide

retention

not

only

by

mechanical

interlocking

but

also

by

micromechanical

and

chemical

adhesion

In

vitro

studies

have

found

improved

retention

less

and

more

favorable

stress

formation

and

improved

fracture

resistance

with

resin

cements

than

with

zinc

phosphate

cement

-LSB-

-RSB-

Other

factors

from

those

related

to

the

post

are

of

importance

for

success

of

post-retained

restorations

Thus

researchers

agree

that

the

extent

of

remaining

tooth

structure

is

a

key

issue

for

fracture

prognosis

-LSB-

-RSB-

Another

paramount

factor

is

the

ferrule

effect

Teeth

that

allow

preparation

of

a

ferrule

show

superior

fracture

resistance

-LSB-

-RSB-

The

foregoing

has

shown

that

numerous

factors

influence

the

longevity

of

endodontically

treated

teeth

provided

with

post-retained

restorations

and

that

it

would

be

practically

impossible

to

carry

out

a

randomized

controlled

clinical

trial

that

includes

all

relevant

variables

Previous

studies

have

shown

annual

failure

rates

related

to

post

and

core

treatment

of

up

to

-LSB-

-RSB-

The

purpose

of

this

survey

of

failed

post-retained

restorations

was

to

analyze

reported

failures

in

an

attempt

to

identify

factors

that

are

critical

to

failure

and

to

type

of

failure

Materials

and

methods

A

questionnaire

was

developed

and

piloted

and

in

June

it

was

posted

in

five

copies

to

the

members

of

the

Danish

Dental

Association

who

worked

in

private

practice

-LRB-

practitioners

-RRB-

The

practitioners

were

asked

to

complete

the

questionnaire

in

case

a

patient

presented

with

a

failed

post-retained

restoration

Questions

were

asked

about

age

and

gender

of

the

patient

tooth

involved

number

of

posts

type

design

length

and

diameter

of

the

post

-LRB-

s

-RRB-

type

of

core

type

of

luting

agent

type

of

restoration

height

of

any

ferrule

degree

of

tooth

destruction

functioning

time

until

failure

and

type

of

failure

After

years

questionnaires

had

been

received

and

the

collection

of

questionnaires

was

terminated

Data

were

transferred

to

a

computer

and

analyzed

by

use

of

the

Statistical

Analyzing

System

-LRB-

SAS

-RRB-

Basic

descriptive

statistics

including

frequency

distributions

were

performed

Selected

data

were

analyzed

by

chi-squared

tests

and

Fischer

's

exact

probability

tests

-LRB-

α

-RRB-

Cross

tabulations

were

carried

out

to

further

characterize

the

data

and

to

identify

codeterminants

of

functioning

time

until

failure

and

codeterminants

of

type

of

failure

Results

The

questionnaires

had

been

completed

by

practitioners

The

response

rate

per

question

varied

between

and

Of

the

patients

slightly

more

were

female

-LRB-

-RRB-

than

male

-LRB-

-RRB-

The

age

of

the

patients

varied

between

and

years

and

the

age

distribution

was

statistically

similar

among

female

and

male

patients

The

sample

involved

nearly

three

times

as

many

maxillary

teeth

-LRB-

-RRB-

than

mandibular

teeth

-LRB-

-RRB-

In

the

maxilla

there

were

fewer

molars

-LRB-

-RRB-

than

premolars

-LRB-

-RRB-

and

incisors

and

canines

-LRB-

-RRB-

and

in

the

mandible

there

were

fewer

incisors

and

canines

-LRB-

-RRB-

Table

shows

the

distribution

of

the

types

of

posts

and

cores

Slightly

fewer

teeth

had

been

restored

with

cast

post

and

cores

than

with

prefabricated

posts

and

direct

cores

Of

the

cast

post

and

cores

had

been

cast

in

a

gold

alloy

in

a

silver

palladium

alloy

and

were

unaccounted

for

Of

the

prefabricated

posts

used

in

combination

with

a

direct

core

were

titanium

posts

were

stainless

steel

posts

were

carbon

fiber-reinforced

resin

composite

posts

and

posts

were

either

of

an

alternative

or

unknown

material

Table

of

type

of

post

and

cores

among

failed

restorationsType

of

post

and

coreNumberCast

post

and

post

with

direct

post

with

cast

post

Of

the

failed

post-retained

restorations

comprised

tapered

posts

comprised

parallel-sided

posts

comprised

posts

of

a

combined

tapered-parallel

design

and

comprised

posts

with

a

design

that

was

not

accounted

for

The

majority

of

the

cast

posts

-LRB-

-RRB-

and

of

the

stainless

steel

prefabricated

posts

-LRB-

-RRB-

were

tapered

The

titanium

posts

were

equally

distributed

between

the

three

designs

-LRB-

and

-RRB-

and

the

carbon

fiber-reinforced

resin

composite

posts

were

either

parallel

sided

-LRB-

-RRB-

or

of

a

combined

design

-LRB-

-RRB-

Of

the

failed

post-retained

restorations

that

were

specified

as

regard

to

the

maximum

diameter

of

the

post

comprised

posts

with

a

diameter

equal

to

or

less

than

one

third

of

the

diameter

of

the

root

and

comprised

posts

with

a

diameter

of

more

than

one

third

Of

the

failed

post-retained

restorations

that

were

specified

as

regard

to

length

of

post

comprised

posts

with

a

length

of

more

than

of

the

total

length

of

the

root

Table

shows

the

distribution

of

the

posts

as

regard

to

type

material

and

length

of

posts

For

all

posts

except

those

of

stainless

steel

the

majority

of

the

posts

were

of

a

length

that

was

equal

to

or

more

than

half

the

length

of

the

root

For

the

stainless

steel

posts

were

shorter

than

half

the

length

of

the

root

Table

of

posts

of

failed

restorations

as

regards

post

type

post

material

and

post

length

in

percent

of

root

Of

the

teeth

with

a

failed

restoration

that

had

been

treated

with

a

prefabricated

post

and

a

direct

core

had

a

core

made

in

amalgam

a

core

made

in

resin

composite

a

core

made

in

glass

ionomer

cement

and

a

core

made

in

a

material

that

was

not

accounted

for

Amalgam

had

been

used

more

often

than

resin

composite

for

stainless

steel

posts

-LRB-

vs

-RRB-

whereas

resin

composite

had

been

used

more

often

than

amalgam

for

titanium

posts

-LRB-

vs

-RRB-

and

in

all

cases

for

carbon

fiber-reinforced

resin

composite

posts

Of

the

failed

post-retained

restorations

had

been

luted

with

zinc

phosphate

cement

with

glass

ionomer

cement

with

resin

cement

and

posts

with

an

agent

that

was

not

accounted

for

The

majority

of

the

cast

posts

-LRB-

-RRB-

and

of

the

stainless

steel

prefabricated

posts

-LRB-

-RRB-

had

been

luted

with

zinc

phosphate

cement

Most

titanium

posts

had

been

luted

with

zinc

phosphate

cement

-LRB-

-RRB-

or

glass

ionomer

cement

-LRB-

-RRB-

Of

the

carbon

fiber-reinforced

resin

composite

posts

had

been

luted

with

resin

cement

and

with

glass

ionomer

cement

Of

the

failed

restorations

had

a

ferrule

did

not

have

a

ferrule

and

were

not

accounted

for

As

regards

the

degree

of

tooth

destruction

before

luting

of

the

now

failed

post-retained

restoration

only

restored

teeth

out

of

were

accounted

for

In

cases

the

whole

clinical

crown

had

been

missing

at

the

time

of

restoration

and

in

cases

between

one

half

and

three

fourths

of

the

clinical

crown

had

been

missing

Table

shows

for

how

long

the

restorations

had

functioned

in

the

mouth

at

the

time

of

failure

The

time

of

functioning

of

the

restorations

that

were

accounted

for

varied

between

months

and

years

The

mean

survival

time

until

failure

was

years

and

the

median

survival

time

was

years

Of

the

restorations

had

functioned

years

or

less

at

the

time

of

failure

Of

the

restorations

displayed

fracture

of

the

tooth

displayed

loosening

of

the

post

displayed

fracture

of

the

post

and

displayed

a

type

of

failure

that

was

not

accounted

for

Table

of

failed

post-retained

restorations

as

regards

functioning

time

until

failureFunctioning

time

-LRB-

years

-RRB-

Among

the

post-retained

restorations

that

failed

after

a

relatively

short

functioning

time

-LRB-

years

-RRB-

as

compared

to

a

relatively

long

functioning

time

-LRB-

years

-RRB-

were

-LRB-

-RRB-

significantly

more

parallel-sided

and

combined

tapered-parallel

posts

than

tapered

posts

-LRB-

-RRB-

significantly

more

carbon

fiber-reinforced

resin

composite

prefabricated

posts

than

titanium

and

stainless

steel

prefabricated

posts

-LRB-

-RRB-

significantly

more

cast

silver

palladium

posts

than

cast

gold

posts

-LRB-

-RRB-

significantly

more

resin

composite

cores

than

amalgam

cores

and

finally

-LRB-

-RRB-

significantly

more

posts

luted

with

resin

cement

or

with

glass

ionomer

cement

than

with

zinc

phosphate

cement

These

results

are

unexpected

and

as

the

Discussion

will

argue

the

results

do

not

truly

reflect

factors

critical

to

functioning

time

Several

factors

were

significantly

related

to

type

of

failure

-LRB-

-RRB-

The

frequency

of

tooth

fracture

was

similar

for

female

and

male

patients

-LRB-

vs

-RRB-

as

was

the

frequency

of

loosened

posts

-LRB-

vs

-RRB-

but

the

frequency

of

fractured

posts

was

higher

for

male

than

for

female

patients

-LRB-

vs

-RRB-

-LRB-

-RRB-

The

shorter

the

post

the

lower

the

frequency

of

post

fracture

-LRB-

Table

-RRB-

-LRB-

-RRB-

Tapered

posts

more

often

than

parallel-sided

and

combined

tapered-parallel

posts

displayed

fracture

of

tooth

-LRB-

vs

and

-RRB-

and

less

often

fracture

of

post

-LRB-

vs

and

-RRB-

-LRB-

-RRB-

Stainless

steel

prefabricated

posts

more

often

than

titanium

prefabricated

posts

displayed

fracture

of

the

tooth

-LRB-

vs

-RRB-

-LRB-

-RRB-

Teeth

restored

with

amalgam

cores

displayed

mainly

tooth

fractures

-LRB-

-RRB-

teeth

restored

with

cast

gold

cores

displayed

mainly

tooth

fractures

-LRB-

-RRB-

or

loosening

of

post

-LRB-

-RRB-

and

teeth

restored

with

resin

composite

cores

displayed

all

three

types

of

failure

with

almost

similar

frequency

-LRB-

and

-RRB-

-LRB-

-RRB-

More

teeth

in

which

the

post

had

been

luted

with

zinc

phosphate

cement

displayed

tooth

fracture

than

teeth

for

which

glass

ionomer

cement

or

resin

cement

had

been

used

-LRB-

vs

and

-RRB-

-LRB-

-RRB-

Compared

with

the

frequencies

of

post

fracture

and

loosening

of

the

post

the

frequency

of

tooth

fracture

increased

with

increasing

functioning

time

until

failure

and

correspondingly

restorations

that

failed

early

on

showed

an

equal

frequency

of

the

three

types

of

failure

-LRB-

Table

-RRB-

Table

of

failed

post-retained

restorations

as

regards

length

of

post

and

type

of

failureLength

-LRB-

mm

-RRB-

FailureTotalFracture

of

toothFracture

of

postLoosening

of

of

failed

post-retained

restorations

as

regards

functioning

time

of

restoration

and

type

of

failureFunctioning

time

-LRB-

year

-RRB-

FailureTotalFracture

of

toothFracture

of

postLoosening

of

Discussion

Five

percent

of

the

private

practitioners

that

had

been

invited

to

participate

in

the

survey

did

so

Possible

explanations

for

this

low

response

rate

include

the

open

invitation

design

the

relative

extensive

questionnaire

and

the

fact

that

the

questionnaire

could

not

be

completed

once

and

for

all

but

had

to

be

procured

whenever

a

patient

presented

with

a

failed

post-retained

restoration

Considering

that

prosthodontics

is

not

a

registered

specialty

in

Denmark

there

is

no

reason

to

believe

that

the

treatments

provided

by

the

participating

practitioners

were

of

another

type

or

quality

than

those

provided

by

private

practitioners

in

general

The

fact

that

the

questionnaires

were

filled

out

by

individual

practitioners

in

their

everyday

environment

and

not

by

calibrated

investigators

in

an

optimal

research

facility

explains

that

the

response

rate

to

each

question

was

not

and

also

implies

some

uncertainty

as

to

the

validity

of

the

answers

especially

those

that

included

an

element

of

estimation

e.g.

of

post

diameter

The

abovementioned

factors

call

for

some

caution

when

interpreting

the

results

of

the

survey

As

regards

age

and

gender

the

patient

sample

is

not

only

representative

of

the

private

practice

patient

population

in

Denmark

as

such

but

also

similar

to

patient

samples

in

previously

published

clinical

studies

of

post-retained

restorations

-LSB-

-RSB-

These

clinical

studies

also

supply

explanations

for

the

present

finding

that

more

maxillary

teeth

especially

incisors

and

canines

than

mandibular

teeth

presented

with

failed

post-retained

restorations

Not

only

are

more

maxillary

teeth

than

mandibular

teeth

treated

with

posts

-LSB-

-RSB-

but

post-retained

restorations

in

maxillary

teeth

have

also

been

reported

to

fail

relatively

more

frequently

than

similar

restorations

in

mandibular

teeth

-LSB-

-RSB-

In

particular

post-retained

restorations

in

anterior

maxillary

teeth

have

been

found

to

show

high

failure

rates

presumably

because

of

higher

functional

horizontal

forces

acting

on

the

anterior

teeth

than

on

posterior

teeth

-LSB-

-RSB-

The

types

of

posts

cores

and

cements

used

for

the

failed

restorations

not

only

reflect

that

preferences

vary

among

practitioners

but

also

that

the

age

of

the

restorations

vary

widely

as

did

the

materials

and

treatments

available

at

the

time

of

restoration

Given

the

design

of

this

survey

i.e.

we

have

only

considered

restorations

that

have

failed

we

have

no

knowledge

of

the

entire

population

of

post-retained

restorations

Thus

the

survey

does

not

allow

distinction

between

an

over-representation

of

a

given

material

or

treatment

caused

by

outspread

use

and

over-representation

caused

by

poor

longevity

The

length

of

time

that

the

post-retained

restorations

had

functioned

before

failure

varied

from

months

to

years

with

a

mean

survival

time

until

fracture

of

years

In

agreement

with

the

findings

of

previous

surveys

of

failed

post-retained

crowns

-LSB-

-RSB-

there

was

a

tendency

to

a

relatively

high

initial

failure

rate

However

the

proportion

of

restorations

that

failed

during

the

first

years

was

lower

in

the

present

survey

-LRB-

-RRB-

than

in

the

survey

by

Lewis

and

Smith

-LRB-

-RRB-

-LSB-

-RSB-

In

the

survey

of

Turner

-LSB-

-RSB-

only

had

survived

more

than

years

The

longer

lifespan

found

in

the

present

survey

may

reflect

that

the

samples

of

the

previous

surveys

included

posts

that

were

generally

shorter

and

more

often

smooth

and/or

tapered

than

the

posts

of

the

present

survey

These

features

are

likely

to

have

increased

the

prevalence

of

loosened

posts

at

an

early

stage

A

number

of

specific

types

of

posts

or

materials

were

found

to

have

significantly

high

prevalence

in

the

group

of

post-retained

restorations

that

failed

after

a

relatively

short

functioning

time

carbon

fiber-reinforced

resin

composite

posts

posts

cast

in

silver

palladium

parallel-sided

and

combined

tapered-parallel

posts

resin

composite

cores

and

resin

cement

and

glass

ionomer

cement

Some

of

these

posts

and

materials

are

probably

found

in

this

group

of

restorations

that

failed

after

a

short

functioning

time

not

because

they

in

fact

are

associated

with

short

functioning

time

but

because

they

have

been

on

the

market

for

a

relatively

short

time

compared

with

the

alternatives

and

thus

are

over-represented

in

the

total

group

of

restorations

with

a

short

functioning

time

The

most

commonly

reported

type

of

failure

was

tooth

fracture

followed

by

loosening

of

the

post

and

finally

fracture

of

the

post

This

relative

distribution

is

at

odds

with

that

of

previous

retrospective

studies

which

found

loosening

of

the

post

to

be

the

most

common

type

of

failure

-LSB-

-RSB-

Failures

in

the

form

of

loss

of

retention

are

most

often

restorable

whereas

tooth

fractures

are

most

often

nonrestorable

and

lead

to

extraction

of

the

tooth

It

is

possible

that

it

was

easier

for

the

practitioner

to

remember

to

submit

a

questionnaire

when

a

more

drastic

tooth

fracture

occurred

than

when

a

post

had

lost

retention

and

had

to

be

recemented

Such

a

situation

would

tend

to

underestimate

the

proportion

of

loosened

posts

An

alternative

explanation

is

that

the

distribution

between

types

of

failure

has

indeed

changed

to

reflect

a

decrease

in

the

use

of

smooth

and

tapered

posts

Several

factors

were

found

to

significantly

influence

the

distribution

between

types

of

failures

One

factor

was

gender

of

the

patient

Whereas

there

were

relatively

speaking

equally

many

male

and

female

patients

in

the

group

of

post-retained

restorations

that

displayed

tooth

fracture

or

loosening

of

post

there

were

more

male

patients

in

the

group

of

restorations

that

displayed

post

fracture

It

may

be

that

the

greater

bite

forces

exerted

by

men

-LSB-

-RSB-

not

only

increase

the

failure

rate

of

post-retained

restorations

as

such

-LSB-

-RSB-

but

also

predispose

for

fracture

of

the

post

Another

factor

was

post

length

Failed

shorter

posts

were

associated

with

a

lower

prevalence

of

post

fracture

than

were

longer

posts

This

finding

is

not

readily

explained

on

the

basis

of

stress

analyses

but

may

reflect

that

there

was

an

over-representation

of

stainless

steel

posts

and/or

of

tapered

posts

in

the

group

of

short

posts

Stainless

steel

has

a

higher

elastic

modulus

and

is

a

stronger

material

than

gold

alloys

and

titanium

alloys

-LSB-

-RSB-

and

may

thus

be

expected

to

withstand

higher

stresses

Tapered

posts

were

associated

with

a

higher

prevalence

of

tooth

fracture

than

were

parallel-sided

and

combined

tapered-parallel

posts

and

with

a

correspondingly

lower

prevalence

of

post

fracture

This

significant

tendency

to

fail

because

of

tooth

fracture

confirms

results

of

previous

studies

-LSB-

-RSB-

Likewise

failed

stainless

steel

posts

were

associated

with

a

higher

prevalence

of

tooth

fracture

than

were

titanium

posts

This

could

be

an

indirect

result

of

most

stainless

steel

posts

being

short

having

high

elastic

modulus

and

having

a

tapered

shape

in

contrast

to

most

titanium

posts

being

parallel

sided

Teeth

that

failed

and

had

been

restored

with

amalgam

cores

displayed

significantly

more

tooth

fractures

than

teeth

restored

with

cores

of

resin

composite

and

cast

gold

post

and

cores

Because

of

intermediate

mechanical

properties

e.g.

elastic

modulus

of

amalgam

as

compared

to

resin

composite

and

gold

alloy

mechanical

properties

can

not

easily

explain

the

high

prevalence

of

tooth

fracture

Instead

it

is

likely

to

be

caused

not

by

the

amalgam

core

itself

but

by

other

factors

that

characterize

the

posts

with

which

amalgam

was

used

Amalgam

was

the

preferred

core

material

for

stainless

steel

posts

whereas

resin

composite

was

the

preferred

core

material

for

titanium

posts

As

just

mentioned

stainless

steel

posts

were

more

often

than

other

posts

tapered

a

factor

that

has

also

been

shown

to

predispose

for

tooth

fracture

The

fact

that

zinc

phosphate

cement

also

seemed

to

predispose

for

tooth

fracture

may

similarly

be

explained

not

by

inferiority

of

the

material

itself

but

by

the

posts

with

which

it

was

used

A

relatively

higher

percentage

of

posts

cast

in

silver

palladium

and

of

prefabricated

stainless

steel

posts

had

been

luted

with

zinc

phosphate

cement

than

with

any

other

cement

and

these

two

types

of

posts

were

associated

with

a

high

prevalence

of

tooth

fracture

The

final

factor

that

was

found

to

be

significantly

associated

with

type

of

failure

was

the

length

of

time

that

the

post-retained

restorations

had

functioned

until

failure

The

prevalence

of

tooth

fracture

increased

with

increasing

functioning

time

until

failure

This

finding

is

in

contrast

to

that

of

previous

surveys

of

failed

post-retained

crowns

in

which

no

correlation

between

type

of

failure

and

length

of

functioning

time

was

found

-LSB-

-RSB-

The

discrepancy

can

be

explained

by

the

fact

that

the

present

survey

included

teeth

of

a

much

wider

range

of

functioning

time

until

failure

and

higher

mean

age

at

failure

This

survey

did

not

confirm

the

generally

accepted

paramount

influence

on

fracture

prognosis

of

extent

of

remaining

tooth

structure

and

of

a

ferrule

-LSB-

-RSB-

With

regard

to

extent

of

remaining

tooth

structure

the

reason

is

that

an

exceptionally

low

response

rate

did

not

allow

statistical

analysis

of

this

parameter

on

a

possible

influence

on

functioning

time

and

type

of

failure

The

fact

that

the

survey

also

did

not

find

a

significant

influence

of

the

existence

of

a

ferrule

calls

for

the

following

comments

First

the

fact

that

most

restorations

were

reported

to

have

a

ferrule

reduced

the

probability

that

the

statistical

analysis

would

find

a

significant

effect

Second

it

is

very

positive

to

note

that

clinicians

are

aware

of

the

importance

of

including

a

ferrule

in

the

preparation

Third

the

fact

that

restorations

failed

despite

that

the

crowns

had

a

ferrule

points

to

the

relevance

of

optimizing

other

elements

in

the

restoration

of

an

endodontically

treated

tooth

Conclusions

The

following

are

the

conclusions

derived

from

the

study

Fracture

of

the

tooth

was

the

most

common

type

of

failure

among

the

failed

post-retained

restorations

followed

by

loosening

of

the

post

and

fracture

of

the

post.Compared

with

parallel-sided

posts

tapered

posts

more

often

displayed

fracture

of

the

tooth

and

less

often

fracture

of

the

post.Post-retained

restorations

that

had

functioned

for

a

long

time

before

failure

showed

an

increased

risk

of

tooth

fracture

compared

to

loosening

of

the

post

and

fracture

of

the

post.Fracture

of

the

post

was

more

common

among

male

patients

than

among

female

patients

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B 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 B 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 B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O 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 B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 B 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 B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B O O B 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 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 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 O O O O O O O O O O O O O O O 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 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 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 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 B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 B O O O O O O O O O O O

dowel
fixed prosthodontics
type of failures


Association of antigen processing machinery and HLA class I defects with clinicopathological outcome in cervical carcinoma HLA class I loss is a significant mechanism of immune evasion by cervical carcinoma , interfering with the development of immunotherapies and cancer vaccines . We report the systematic investigation of HLA class I and antigen processing machinery component expression and association with clinical outcome . A tissue microarray containing carcinoma lesions from 109 cervical carcinoma patients was stained for HLA class I heavy chains , β2-microglobulin , LMP2 , LMP7 , LMP10 , TAP1 , TAP2 , ERAP1 , tapasin , calreticulin , calnexin and ERp57 . A novel staining evaluation method was used to ensure optimal accuracy and reliability of expression data , which were correlated with known clinicopathological parameters . Partial HLA class I loss was significantly associated with decreased 5-years overall survival -LRB- 61 % vs. 83 % for normal expression ; P < 0.05 -RRB- and was associated with decreased 5-years disease-free survival -LRB- DFS -RRB- -LRB- 65 % vs. 82 % for normal expression ; P = 0.05 -RRB- . All APM components except LMP10 , calnexin and calreticulin were down-regulated in a substantial number of cases and , except ERAP1 , correlated significantly with HLA class I down-regulation . LMP7 , TAP1 and ERAP1 loss was significantly associated with decreased overall and -LRB- except LMP7 -RRB- DFS -LRB- P < 0.05 and 0.005 , respectively -RRB- . ERAP1 down-regulation was an independent predictor for worse overall and DFS in multivariate analysis -LRB- HR 3.08 ; P < 0.05 and HR 2.84 ; P < 0.05 , respectively -RRB- . HLA class I and APM component down-regulation occur frequently in cervical carcinoma , while peptide repertoire alterations due to ERAP1 loss are a major contributing factor to tumour progression and mortality . Introduction Tumour cells utilize several mechanisms to escape immune-mediated recognition and destruction -LSB- 1 -RSB- . Loss of surface-expressed human leukocyte antigen -LRB- HLA -RRB- class I molecules is particularly important , as this enables tumour cells to evade recognition and lysis by cytotoxic T-lymphocytes -LSB- 2 -- 5 -RSB- . These molecules consist of a glycoprotein heavy chain -LRB- encoded by genes within the HLA regions of chromosome 6p -RRB- and a β2-microglobulin -LRB- β2-m -RRB- light chain -LRB- encoded on chromosome 15q -RRB- -LSB- 6 , 7 -RSB- . Defects in HLA class I expression are caused by multiple mechanisms , including loss of heterozygosity at chromosome 6p ; β2-m or HLA class I mutations ; and defective expression and/or function of components of the antigen processing machinery -LRB- APM -RRB- -LSB- 8 -- 10 -RSB- . The APM is the combination of cellular processes responsible for the presentation of endogenous peptides by HLA class I molecules . These peptides are generated by specialized immunoproteasomes -LRB- composed of LMP2 , LMP7 and LMP10 subunits -RRB- ; and transported from the cytoplasm to the endoplasmic reticulum by TAP -LRB- transporter associated with antigen presentation -RRB- which is composed of TAP1 and TAP2 subunits . Transported peptides undergo length-specific trimming by ERAP1 -LRB- endoplasmic reticulum aminopeptidase associated with antigen presentation 1 -RRB- and are loaded onto empty HLA class I molecules -LRB- with involvement of tapasin and the chaperone molecules calnexin , calreticulin and ERp57 -RRB- -LSB- 11 -- 14 -RSB- . Although various in vitro studies have demonstrated partial or total APM component loss in human malignancies , the extent to which this occurs in vivo is largely unknown . Two recent reports describe down-regulation of several APM components in head and neck squamous cell carcinoma and astrocytoma , and demonstrate an association of these defects with prognosis -LSB- 15 , 16 -RSB- . As HLA class I-restricted processing and presentation of endogenous peptides is crucial to the generation of a virus-specific cytotoxic T-lymphocyte-mediated reaction , APM defects are assumed to contribute significantly to virus-mediated carcinogenesis . Several studies have confirmed interference in various APM processes by viral peptides -LSB- 17 -RSB- . However , the extent to which defective expression of APM components contributes to HPV-induced cervical carcinogenesis remains to be established . One study has assessed the expression of various APM components , although only at the mRNA level -LSB- 18 -RSB- . Other studies of APM component expression in cervical carcinoma were largely limited to TAP1 and TAP2 and encompassed relatively small sample sizes . Moreover , the expression data yielded by these studies are conflicting , with TAP1 loss being reported in 0 -- 50 % of cases -LSB- 18 -- 20 -RSB- . Though TAP1 and HLA class I down-regulation has occasionally been shown to be associated with progressive malignant transformation -LSB- 21 -- 24 -RSB- , a comprehensive study of the association of APM defects with prognostic parameters has yet to be performed . Since information regarding defective APM component expression in cervical cancer can contribute to the development of T cell-based immunotherapies , we have investigated the expression of all known APM components in a large number of cervical cancer specimens using a tissue microarray -LRB- TMA -RRB- - based approach -LSB- 25 , 26 -RSB- . Additionally , we demonstrate for the first time a negative association of APM defects with prognosis and other clinicopathological variables . Materials and methods Clinical characteristics and tissue samples From 109 patients with cervical carcinoma who underwent radical hysterectomy with bilateral pelvic lymphadenectomy -LRB- by the same surgical team -RRB- between 1985 and 1999 , formalin-fixed , paraffin-embedded tissue blocks were retrieved from the archives of the Department of Pathology , Leiden University Medical Centre , The Netherlands . Twenty-six adenocarcinomas and 83 squamous carcinomas were obtained . These cases were not consecutive , but chosen on the basis of availability of sufficient material . The use of clinical material was approved by the institutional review board according to the guidelines of the Dutch Federation of Medical Research Associations . All patients were inhabitants of The Netherlands and had not received pre-operative radiotherapy or chemotherapy . Mean age was 48.5 years , the youngest patient being 24 years and the oldest 87 years at the time of surgery . Follow-up of these patients until 2005 provided information concerning recurrence rate and performance state . Tissue microarray construction The archival slides for all the cases were reviewed ; a slide containing representative tumour was selected , and an area of tumour was encircled on the slide . The corresponding tissue blocks were recovered from the archives and the selected area on the slide was circled on the block for tissue microarray construction . Using a manual tissue microarrayer -LRB- Beecher Instruments , Silver Spring , MD , USA -RRB- , the area of interest in the donor block was cored with a 0.6-mm diameter needle and transferred to a recipient paraffin block . The microarray was constructed with a threefold redundancy -LRB- three spots for each patient -RRB- to increase accuracy . The finalized arrays were then cut into 3 μm-thick sections and mounted on glass slides using an adhesive tape-transfer system -LRB- Instrumedics Inc. , Hackensack , NJ , USA -RRB- with ultraviolet cross-linking . Antibodies The mouse monoclonal antibody -LRB- mAb -RRB- HCA2 which recognizes a determinant expressed on β2-m-free HLA-A -LRB- excluding HLA-A24 -RRB- , HLA-B7301 and HLA-G heavy chains -LSB- 27 , 28 -RSB- , the mAb HC10 which recognizes a determinant on all β2-m-free HLA-B and HLA-C heavy chains and on β2-m-free HLA-A10 , - A28 , - A29 , - A30 , - A31 , - A32 and - A33 heavy chains -LSB- 27 , 29 -RSB- , the LMP2-specific mAb SY1 -LSB- 30 -RSB- , the LMP7-specific mAb HB-2 -LSB- 30 -RSB- , the LMP10-specific mAb TO-7 -LSB- 30 -RSB- , the TAP1-specific mAb NOB-1 -LSB- 31 -RSB- , the TAP2-specific mAb NOB-2 -LSB- 31 -RSB- , the calnexin-specific mAb TO-5 -LSB- 32 -RSB- , the calreticulin-specific mAb TO-11 -LSB- 32 -RSB- , the Erp57-specific mAb TO-2 -LSB- 32 -RSB- and the tapasin-specific mAb TO-3 -LSB- 32 -RSB- were developed and characterized as described . The rabbit polyclonal anti-β2-m-antibody A-139 was purchased from DAKO , Copenhagen , Denmark . The rabbit polyclonal anti-ERAP1 antibody has been described previously and was kindly provided by Dr. M. Tsujimoto , RIKEN , Wako , Saitama , Japan -LSB- 33 -- 35 -RSB- . Immunohistochemical staining The slides were dried for 16 h at 56 °C before being dewaxed with standard xylene and rehydrated through graded alcohols into water . Standard immunohistochemical staining was performed as described previously -LSB- 36 -RSB- . Stromal cells , including infiltrating leukocytes , were used as internal positive controls . Negative control sections of each specimen were processed with omission of the primary antibodies . Evaluation of TMA immunostaining Two researchers -LRB- A.M. and E.J. -RRB- evaluated all patient cores , and scores were averaged to produce a single evaluation with the scorers being blind to the source of the samples . When slides were scored differently , which occurred rarely -LRB- < 5 % -RRB- , a consensus was reached by simultaneous evaluation . Staining was scored semi-quantitatively by the quality control system proposed by Ruiter et al. -LSB- 37 -RSB- . The intensity and percentage of positive cells in the tumour and the stromal cells surrounding the tumour were determined . The intensity of staining was scored as 0 , 1 , 2 , or 3 indicating absent , weak , clear , or strong expression , respectively . The percentage of positive cells was scored as 0 for 0 % ; 1 for 1 -- 5 % ; 2 for 5 -- 25 % ; 3 for 25 -- 50 % ; 4 for 50 -- 75 % and 5 for 75 -- 100 % . The sum of both scores was used to identify three categories of expression : normal expression -LRB- total score 7 -- 8 -RRB- , partial loss -LRB- 3 -- 6 -RRB- and total loss -LRB- 0 -- 2 -RRB- . Immunohistochemical staining demonstrated strong positive expression of all examined markers in stromal tissue and tumour-infiltrating inflammatory cells , thereby providing an internal positive control . Statistical analysis The Chi-square test or , where appropriate , Fisher 's exact test was used for evaluation of associations between expression and clinicopathological parameters . All statistical analyses were performed with the SPSS Version 12 software package . Five-year survival rates were calculated and survival rates were compared according to the Kaplan -- Meier method using the log rank test , while multivariate analysis of survival was performed according to Cox proportional hazard models . Overall survival -LRB- OS -RRB- was defined as survival till death due to cervical carcinoma -LRB- the two patients that dyed from other causes were excluded -RRB- , i.e. disease-specific survival , while disease-free survival -LRB- DFS -RRB- was defined as time to disease recurrence , metastasis , or disease-specific death . All tests were two-sided and the significance level was set to 5 % . Results Clinical features Of the total group of 109 patients , 52 were diagnosed as FIGO stage IB1 , 30 as IB2 , 21 as FIGO IIa and 2 as FIGO IIb . For four patients no FIGO staging was available . Fifty-five patients -LRB- 50 % -RRB- received post-operative radiotherapy due to narrow tumour-free margins of excision , lymph node metastases , vaso-invasive growth , parametrial infiltration , depth of infiltration exceeding 15 mm and/or tumour size exceeding 4 cm . Twenty-seven patients had lymph node metastases . Fifty-one patients -LRB- 58 % -RRB- were HPV16-positive , 21 -LRB- 24 % -RRB- were HPV18-positive , while the remaining patients were positive for HPV31 , HPV33 , HPV45 , HPV52 or HPV68 . At the end of follow-up , 80 patients were alive , 2 suffered from recurrent disease , 3 had a metastasis , 22 had died of the disease and 2 had died of causes unrelated to the primary disease . The median follow-up time was 45 months . HLA class I and APM component expression Representative staining patterns for the HLA class I heavy chains and various APM components are shown in Figs. 1 and 2 . Expression of the HLA class I heavy chains and β2-m is summarized in Table 1 . Of the 109 cases , eight exhibited total β2-m loss ; correspondingly , these cases were also negative for HLA heavy chain cell surface expression . As HLA class I expression , and therefore tumour immunogenicity , is not affected by the APM in a β2-m-negative background these eight cases were excluded from further analysis of APM effects . Furthermore , as HLA class I expression is regulated by the APM in a β2-m-independent manner , the overall HLA class I expression score was based on expression of the heavy chains . If either or all the heavy chains were expressed normally , overall HLA class I expression was considered to be normal . Total loss of all heavy chains was counted as total HLA class I loss , and all other cases were counted as partial loss . Fig. 1Immunohistochemical analysis . Staining patterns of a -- c HLA-A , d -- f HLA-B / - C , g -- i LMP7 and j -- l TAP1 showing a , d , g , j normal expression , b , e , h , k partial loss of expression and c , f , i , l total loss of expressionFig . 2Immunohistochemical staining patterns of ERAP1 . a Normal expression . b Partial loss of expressionTable 1Expression of HLA class I heavy chains , β2-microglobulin and overall HLA class IHLA-AHLA-B / - Cβ2-microglobulinHLA class ITotal lossPartial lossNormal expressionTotal lossPartial lossNormal expressionTotal lossPartial lossNormal expressionTotal lossPartial lossNormal expressionOverall -LRB- n = 109 -RRB- 36 -LRB- 33 -RRB- 22 -LRB- 20 -RRB- 51 -LRB- 47 -RRB- 24 -LRB- 22 -RRB- 29 -LRB- 27 -RRB- 56 -LRB- 51 -RRB- 8 -LRB- 7 -RRB- 28 -LRB- 26 -RRB- 73 -LRB- 67 -RRB- 21 -LRB- 19 -RRB- 24 -LRB- 22 -RRB- 63 -LRB- 59 -RRB- Tumour type AC -LRB- n = 26 -RRB- 6 -LRB- 23 -RRB- 5 -LRB- 19 -RRB- 15 -LRB- 58 -RRB- 4 -LRB- 15 -RRB- 8 -LRB- 31 -RRB- 14 -LRB- 54 -RRB- 1 -LRB- 4 -RRB- 7 -LRB- 27 -RRB- 18 -LRB- 69 -RRB- 4 -LRB- 15 -RRB- 5 -LRB- 19 -RRB- 17 -LRB- 66 -RRB- SCC -LRB- n = 83 -RRB- 30 -LRB- 36 -RRB- 17 -LRB- 21 -RRB- 36 -LRB- 43 -RRB- 20 -LRB- 24 -RRB- 21 -LRB- 25 -RRB- 42 -LRB- 51 -RRB- 7 -LRB- 8 -RRB- 21 -LRB- 25 -RRB- 55 -LRB- 67 -RRB- 17 -LRB- 21 -RRB- 19 -LRB- 23 -RRB- 46 -LRB- 56 -RRB- Data shown as n -LRB- % -RRB- As shown in Table 2 , expression of all APM components except LMP10 , calnexin , and calreticulin was lost in some of the cases . This down-regulation was significantly associated with HLA class I down-regulation for all components , except ERAP1 . Moreover , defects in overall TAP expression -LRB- defined as partial or total loss of either or both of the TAP subunits -RRB- were significantly associated with HLA class I down-regulation -LRB- P < 0.001 -RRB- . Down-regulation of any APM component was significantly associated with HLA class I down-regulation -LRB- P < 0.001 -RRB- . Table 2Overall and histological type-specific expression of APM componentsOverall -LRB- n = 101 -RRB- AC -LRB- n = 25 -RRB- SCC -LRB- n = 76 -RRB- Total lossPartial lossNormal expressionPTotal lossPartial lossNormal expressionPTotal lossPartial lossNormal expressionPLMP214 -LRB- 14 -RRB- 30 -LRB- 30 -RRB- 57 -LRB- 56 -RRB- 0.0145 -LRB- 20 -RRB- 11 -LRB- 44 -RRB- 9 -LRB- 36 -RRB- 0.7729 -LRB- 12 -RRB- 19 -LRB- 25 -RRB- 48 -LRB- 63 -RRB- 0.001 LMP73 -LRB- 3 -RRB- 16 -LRB- 16 -RRB- 82 -LRB- 81 -RRB- < 0.0012 -LRB- 8 -RRB- 9 -LRB- 36 -RRB- 14 -LRB- 56 -RRB- 0.1001 -LRB- 1 -RRB- 7 -LRB- 9 -RRB- 68 -LRB- 90 -RRB- < 0.001 LMP1000101 -LRB- 100 -RRB- TAP14 -LRB- 4 -RRB- 19 -LRB- 19 -RRB- 78 -LRB- 77 -RRB- < 0.0010 -LRB- 0 -RRB- 4 -LRB- 16 -RRB- 21 -LRB- 84 -RRB- 0.2304 -LRB- 5 -RRB- 15 -LRB- 20 -RRB- 57 -LRB- 75 -RRB- 0.001 TAP211 -LRB- 11 -RRB- 26 -LRB- 26 -RRB- 64 -LRB- 63 -RRB- < 0.0014 -LRB- 16 -RRB- 6 -LRB- 24 -RRB- 15 -LRB- 60 -RRB- 0.0057 -LRB- 9 -RRB- 20 -LRB- 26 -RRB- 49 -LRB- 65 -RRB- < 0.001 ERAP1014 -LRB- 15 -RRB- 80 -LRB- 85 -RRB- 0.3140 -LRB- 0 -RRB- 2 -LRB- 9 -RRB- 20 -LRB- 91 -RRB- 0.582012 -LRB- 16 -RRB- 60 -LRB- 84 -RRB- 0.307 Tapasin1 -LRB- 1 -RRB- 24 -LRB- 24 -RRB- 76 -LRB- 75 -RRB- < 0.0010 -LRB- 0 -RRB- 10 -LRB- 40 -RRB- 15 -LRB- 60 -RRB- 0.2841 -LRB- 1 -RRB- 14 -LRB- 18 -RRB- 61 -LRB- 81 -RRB- < 0.001 Calnexin00101 -LRB- 100 -RRB- ERp5712 -LRB- 12 -RRB- 40 -LRB- 40 -RRB- 49 -LRB- 48 -RRB- 0.0171 -LRB- 4 -RRB- 6 -LRB- 24 -RRB- 18 -LRB- 72 -RRB- 0.61511 -LRB- 15 -RRB- 34 -LRB- 45 -RRB- 31 -LRB- 40 -RRB- 0.002 Calreticulin00101 -LRB- 100 -RRB- Data shown as n -LRB- % -RRB- . P values are shown for association with overall HLA class I down-regulation Association with histopathological parameters Depth of invasion exceeding 15 mm and vaso-invasive growth are unfavourable prognostic indicators . Of the cases examined , 51 -LRB- 45 % -RRB- demonstrated depth of invasion greater than 15 mm and 38 -LRB- 33 % -RRB- demonstrated vaso-invasive growth . Total loss of overall HLA class I expression and of any APM component was found to be significantly associated with depth of invasion exceeding 15 mm -LRB- P = 0.034 and 0.045 , respectively -RRB- , while no significant association was found for defective expression of any marker and vaso-invasion . None of the markers was found to be significantly associated with FIGO stage , corresponding to the lack of a significant association with histopathological parameters . No association of any of the markers with specific HPV status of the patients was detected . Interestingly , partial LMP2 and/or LMP7 loss was significantly associated with an absence of detectable lymph node metastases -LRB- P = 0.023 and 0.021 , respectively -RRB- . Association with overall and disease-free survival As shown in Table 3 and Figs. 3 and 4 , partial loss of overall HLA class I expression was significantly associated with decreased OS , and exhibited a trend towards a significant association with DFS , whereas total loss was not significantly associated with survival . Moreover , partial HLA-A loss -LRB- evaluated by HCA2 reactivity -RRB- was significantly associated with decreased OS and DFS , whereas total loss was not significantly associated with survival . Partial , but not total HLA-B / - C loss -LRB- assessed by HC10 reactivity -RRB- showed a trend towards significant association with decreased survival . Table 3HLA class I and APM component expression and 5-years survival ratesOverall survivalDisease-free survivalNormal expressionPartial lossTotal lossNormal expressionPartial lossTotal lossHLA-A42 -LRB- 83 -RRB- 12 -LRB- 55 -RRB- 29 -LRB- 81 -RRB- 42 -LRB- 83 -RRB- 13 -LRB- 60 -RRB- 28 -LRB- 77 -RRB- HLA-B / - C45 -LRB- 81 -RRB- 19 -LRB- 64 -RRB- 18 -LRB- 75 -RRB- 46 -LRB- 82 -RRB- 19 -LRB- 67 -RRB- 18 -LRB- 73 -RRB- HLA class I52 -LRB- 83 -RRB- 15 -LRB- 61 -RRB- 16 -LRB- 77 -RRB- 52 -LRB- 82 -RRB- 16 -LRB- 65 -RRB- 15 -LRB- 75 -RRB- LMP724 -LRB- 74 -RRB- 17 -LRB- 87 -RRB- 1 -LRB- 33 -RRB- 62 -LRB- 76 -RRB- 13 -LRB- 80 -RRB- 2 -LRB- 50 -RRB- TAP161 -LRB- 78 -RRB- 8 -LRB- 70 -RRB- 1 -LRB- 33 -RRB- 63 -LRB- 81 -RRB- 13 -LRB- 67 -RRB- 1 -LRB- 25 -RRB- ERAP166 -LRB- 82 -RRB- 5 -LRB- 38 -RRB- -- 65 -LRB- 81 -RRB- 7 -LRB- 50 -RRB- -- Data shown as n -LRB- % -RRB- Fig. 3Kaplan -- Meier curves for overall survival . Patients with normal expression , partial loss , and total loss of a HLA-A , b HLA-B / - C , c overall HLA class I , d LMP7 , e TAP1 and f ERAP1 expression . P values are shown for comparison with normal expressionFig . 4Kaplan -- Meier curves for disease-free survival . Patients with normal expression , partial loss , and total loss of a HLA-A , b HLA-B / - C , c overall HLA class I , d LMP7 , e TAP1 and f ERAP1 expression . P values are shown for comparison with normal expression Decreased OS and DFS was significantly associated with total TAP1 loss , but not with normal expression and partial loss . A similar pattern was observed for partial LMP7 loss , though this was not significantly associated with DFS . ERAP1 loss was significantly associated with decreased OS and DFS as compared to normal ERAP1 expression . Multivariate analysis As expected , well known prognostic parameters such as depth of infiltration exceeding 15 mm , presence of vaso-invasive growth , and presence of lymph node metastases were significantly associated with a shorter survival , while FIGO-stages of IB2 or more showed a significant association with shorter DFS -LRB- Table 4 -RRB- . HPV type was not significantly associated with survival -LRB- data not shown -RRB- . Subsequently , multivariate analysis was performed on overall HLA class I , TAP1 , LMP7 -LRB- for OS only -RRB- and ERAP1 expression , combined with the aforementioned prognostic factors . ERAP1 down-regulation was demonstrated to be an independent prognostic parameter for shorter OS -LRB- hazard ratio -LRB- HR -RRB- 3.08 ; 95 % CI 1.07 -- 8.90 ; P = 0.037 -RRB- and shorter DFS -LRB- HR 2.84 ; 95 % CI 1.01 -- 8.03 ; P = 0.049 -RRB- , as was presence of lymph node metastases -LRB- HR 7.02 ; 95 % CI 2.55 -- 19.32 ; P < 0.001 for OS and HR 6.64 ; 95 % CI 2.53 -- 17.44 ; P < 0.001 for DFS -RRB- . Total TAP1 loss was also an independent predictor for shorter DFS -LRB- HR 6.72 ; 95 % CI 1.29 -- 35.06 ; P = 0.024 -RRB- . Table 4Histopathological parameters and 5-years survival ratesOverall survivalDisease-free survival % P % PDepth of infiltration < 15 mm -LRB- n = 59 -RRB- 8890 ≥ 15 mm -LRB- n = 51 -RRB- 60 < 0.00157 < 0.001Vaso-invasion Absent -LRB- n = 68 -RRB- 8185 Present -LRB- n = 38 -RRB- 590.031540.002 Lymph nodes Negative -LRB- n = 80 -RRB- 8686 Positive -LRB- n = 27 -RRB- 43 < 0.00139 < 0.001 Histopathological class SCC -LRB- n = 84 -RRB- 7576 AC -LRB- n = 26 -RRB- 770.924740.662 FIGO stage ≤ IB1 -LRB- n = 52 -RRB- 8291 ≥ IB2 -LRB- n = 53 -RRB- 690.060570.002 Discussion In the present study we have investigated HLA class I and APM component expression in cervical carcinoma patients and have evaluated the relationship of these factors with survival and other clinicopathological parameters . Our results show that HLA class I down-regulation occurs frequently -LRB- ∼ 40 % of cases -RRB- in cervical carcinoma , corresponding to previous reports -LSB- 19 , 21 -RSB- . Synchronous partial or total loss of various APM components was found in a substantial number of cases and was demonstrated to be significantly associated with HLA class I down-regulation . We observed partial HLA class I loss to be significantly associated with decreased OS . Of the APM components , TAP1 , LMP7 and ERAP1 down-regulation exhibited significant associations with decreased survival . ERAP1 down-regulation was shown to be an independent , significant predictor of shorter survival . Though defective APM component expression is hypothesized to be an important mechanism of HLA class I down-regulation and therefore immune evasion by tumour cells , information regarding APM down-regulation in carcinomas is limited . This is partly due to the scarcity of reliable antibodies for most APM components other than the TAP subunits . Furthermore , most previous studies of APM expression in cervical cancer were limited by small sample sizes . Lastly , the use of various scoring methods for evaluation of immunohistochemical staining has rendered proper interpretation of findings difficult , leading to conflicting data , e.g. regarding the frequency of TAP1 loss -LSB- 9 , 18 -- 20 -RSB- . In this study , a panel of recently reported monoclonal antibodies was used to investigate expression of all known APM components in a large set of cervical carcinoma lesions using a TMA-based approach -LSB- 30 -- 33 -RSB- . Staining was evaluated according to a previously reported quality control system -LSB- 37 -RSB- , which is more stringent in defining total and partial expression loss than other evaluation methods , which consider ≤ 25 % positive cells as total loss . The system used here defines total loss as complete or near-complete negativity and is therefore more reliable . Moreover , by excluding β2-m-negative cases from the analysis , all outcome parameters can be reliably correlated to expression levels of the various APM components , as any influence of defective APM expression will only be detectable in a β2-m-positive setting . As an APM component , ERAP1 has been shown to be an important determinant of the repertoire of HLA class I-presented peptides -LSB- 14 , 34 , 38 -- 41 -RSB- . However , to date no systematic investigation of its potential role in HLA class I down-regulation in tumours has been performed . To our knowledge , this is the first description of ERAP1 down-regulation in a human carcinoma . Additionally , we have identified ERAP1 loss as an independent predictor of shorter survival , in the absence of associated HLA class I down-regulation . A possible explanation for this observation is the role of ERAP1 in shaping the antigen repertoire : ERAP1 down-regulation may lead to the preferential loading and presentation of non-tumour-associated peptides , thereby yielding a less immunogenic phenotype and facilitating tumour growth and progression . This hypothesis is supported by previous reports on the disrupted nature of presented peptide repertoire in ERAP1-deficient mice -LSB- 41 , 42 -RSB- . Also , the effect of down-regulation of several APM components on the presentation of specific HPV 16 E6-derived epitopes has been firmly established -LSB- 43 -- 45 -RSB- . Although we found that total TAP1 loss was an independent predictor for shorter DFS , this finding should be interpreted with caution as only four patients exhibited this loss . The decreased significance of ERAP1 as an independent predictor for shorter DFS can be explained by the interdependency of ERAP1-mediated peptide trimming and TAP-mediated peptide transport . An interesting finding in this study is the observed association of LMP2 and LMP7 down-regulation with an absence of lymph node metastasis ; this finding is seemingly corroborated by reports that certain immunogenic peptides in melanoma and renal-cell carcinoma are not generated by immunoproteasomes and that presentation of these peptides is reduced upon up-regulation of the immunoproteasomal subunits -LSB- 46 -RSB- . Though altered proteasomal cleavage of endogenous proteins can lead to an alternative , less immunogenic antigen repertoire , an adequate mechanism for the association of LMP down-regulation with an absence of lymph node metastases remains to be postulated . Interestingly , we have recently observed that genetic variation in various APM components is significantly associated with increased cervical carcinoma risk . Although the underlying mechanisms of APM component loss are largely unknown , the occurrence of single nucleotide polymorphisms in the various APM genes could form a genetic basis predisposing to either mutations , or regulatory down-regulation -LSB- 47 , 48 -RSB- . Partial HLA class I loss was observed to be significantly associated with decreased OS ; correspondingly , reports in colon cancer have shown partial , but not total HLA class I loss to be associated with worse OS -LSB- 49 -RSB- . This finding can be explained by the ability of low levels of HLA class I molecules to provide tumour cells with an escape not only from cytotoxic T lymphocyte-mediated , but also from natural killer -LRB- NK -RRB- cell-mediated cytotoxicity -LSB- 2 -RSB- . This hypothesis is further supported by the association of decreased survival with total TAP1 loss , which has been shown previously to lead to partial HLA class I loss -LSB- 2 , 50 -- 52 -RSB- . In the present study , an association of loss or down-regulation of expression of the various APM molecules with aberrant HLA expression was observed . Moreover , the partial HLA loss observed is most likely an underestimation of the extent of HLA alterations present as immunohistochemical staining on paraffin sections does not reveal loss of specific HLA alleles -LSB- 8 -RSB- . However , there are also other aberrations known to lead to HLA class I alterations and it is plausible to hypothesize that all these mechanisms can coexist in the same tumour sample . In conclusion , we have shown here that defective expression of HLA class I and of various APM components occurs frequently in cervical carcinoma . Partial HLA class I loss and total TAP1 loss are associated with decreased survival , while down-regulation of immunoproteasomal subunits is associated with decreased risk of lymphogenic metastasis . Moreover , we report the first description of ERAP1 down-regulation in a human carcinoma , and have found that this is an independent prognostic parameter for decreased survival . A complete understanding of the mechanisms and relevance of HLA class I and APM component down-regulation and immune evasion may contribute to the rational design of tumour vaccines and T-cell-based immunotherapies .

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

antigen processing machinery
erap1
cervical carcinoma