Expression

microarray

analysis

of

papillary

thyroid

carcinoma

and

benign

thyroid

tissue

emphasis

on

the

follicular

variant

and

potential

markers

of

malignancy

The

most

common

sub-variant

of

papillary

thyroid

carcinoma

-LRB-

PTC

-RRB-

is

the

so-called

follicular

variant

-LRB-

FVPTC

-RRB-

which

is

a

particularly

problematic

lesion

and

can

be

challenging

from

a

diagnostic

viewpoint

even

in

resected

lesions

Although

fine

needle

aspiration

cytology

is

very

useful

in

the

diagnosis

of

PTC

its

accuracy

and

utility

would

be

greatly

facilitated

by

the

development

of

specific

markers

for

PTC

and

its

common

variants

We

used

the

recently

developed

Applied

Biosystems

microarray

system

to

interrogate

a

series

of

benign

thyroid

lesions

and

conditions

and

samples

of

PTC

-LRB-

six

with

classic

morphology

and

eight

with

follicular

variant

morphology

-RRB-

TaqMan

reverse

transcriptase-polymerase

chain

reaction

was

used

to

validate

the

expression

portfolios

of

selected

transcripts

Our

data

corroborates

potential

biomarkers

previously

identified

in

the

literature

such

as

LYN

BAX

and

cluster

of

differentiation

-LRB-

-RRB-

However

we

have

also

identified

numerous

transcripts

never

previously

implicated

in

thyroid

carcinogenesis

and

many

of

which

are

not

represented

on

other

microarray

platforms

Diminished

expression

of

metallothioneins

featured

strongly

among

these

and

suggests

a

possible

role

for

this

family

as

tumour

suppressors

in

PTC

Fifteen

transcripts

were

significantly

associated

with

FVPTC

morphology

Surprisingly

these

genes

were

associated

with

an

extremely

narrow

repertoire

of

functions

including

the

major

histocompatibility

complex

and

cathepsin

families

Introduction

Papillary

thyroid

carcinoma

-LRB-

PTC

-RRB-

is

the

most

common

endocrine

malignancy

and

encompasses

a

variety

of

morphological/architectural

variants

all

of

which

are

characterized

by

a

distinctive

nuclear

appearance

In

recent

years

PTC

has

become

an

important

paradigm

of

solid

tumour

molecular

pathogenesis

principally

arising

from

intensive

investigation

prompted

by

the

Chernobyl

accident

The

discovery

of

ret

rearrangements

-LSB-

-RSB-

and

their

association

with

radiation

-LSB-

-RSB-

was

followed

by

the

demonstration

of

the

BRAF

mutation

-LSB-

-RSB-

found

more

commonly

associated

with

sporadic

PTC

in

non-radiation

exposed

populations

-LSB-

-RSB-

In

the

past

our

group

and

others

have

noted

an

association

between

classic

morphology

and

the

BRAF

mutation

and

between

variant

morphology

and

ret

rearrangements

particularly

ret/PTC

-LSB-

-RSB-

Similarly

ret/PTC

appears

to

strongly

correlate

with

the

solid/follicular

variant

seen

commonly

in

children

exposed

to

the

Chernobyl

fallout

-LSB-

-RSB-

Nevertheless

the

utility

of

these

genetic

lesions

to

diagnostic

pathology

and

clinical

practice

has

remained

negligible

Recently

gene

expression

microarray

technology

has

been

used

to

attempt

to

identify

clinically

relevant

biomarkers

of

malignancy

related

to

the

thyroid

-LSB-

-RSB-

The

discovery

of

such

a

biomarker

or

panel

of

biomarkers

allied

to

the

gold

standard

triage

method

of

fine

needle

aspiration

cytology

-LRB-

FNAC

-RRB-

would

represent

a

significant

advancement

in

the

treatment

of

the

solitary

thyroid

nodule

An

intriguing

but

commonly

occurring

variant

of

PTC

is

known

as

follicular

variant

-LRB-

FVPTC

-RRB-

This

lesion

which

by

definition

retains

the

classic

nuclear

features

of

PTC

shows

no

evidence

of

the

architectural

papillae

FVPTC

may

be

a

controversial

lesion

due

to

interobserver

variation

in

its

pathological

diagnosis

-LSB-

-RSB-

Further

the

occurrence

of

follicular

patterned

lesions

with

poorly

or

incompletely

developed

nuclear

features

may

occur

which

are

easily

dismissed

as

benign

thyroid

nodules

This

has

led

to

the

controversial

designation

well-differentiated

tumour

of

uncertain

malignant

potential

for

tumours

of

this

type

-LSB-

-RSB-

It

is

clear

that

this

is

a

complex

and

contentious

area

and

that

further

work

needs

to

be

done

to

ascertain

the

underlying

molecular

biology

of

this

particular

variant

Recently

inroads

into

elucidation

of

molecular

pathways

underpinning

PTC

have

been

carried

out

using

microarray

studies

The

overriding

objective

of

these

investigations

was

to

identify

clinically

useful

biomarkers

However

the

majority

of

these

studies

have

analysed

PTC

as

though

it

were

a

homogenous

singular

entity

without

deference

in

a

detailed

manner

to

sub-variants

and

in

particular

the

most

common

variant

-LRB-

FVPTC

-RRB-

The

identification

of

specific

biomarkers

of

FVPTC

and

a

deeper

understanding

of

its

origins

are

clearly

warranted

The

aim

of

this

expression

microarray

study

using

a

novel

microarray

platform

was

twofold

to

identify

markers

that

distinguish

PTC

from

benign

thyroid

tissue

and

lesions

and

secondly

to

identify

potential

markers

and

further

explore

the

molecular

pathology

of

FVPTC

Materials

and

methods

Patients

and

tissue

samples

Tissue

from

thyroid

resections

was

collected

prospectively

from

patients

undergoing

partial

or

total

thyroidectomy

for

a

variety

of

reasons

at

St.

James

's

Hospital

Dublin

The

study

had

approval

of

the

local

ethics

committee

and

informed

consent

was

obtained

from

each

patient

by

the

clinical

team

before

surgery

Small

samples

-LRB-

cm

-RRB-

were

divided

and

immediately

snap-frozen

in

liquid

nitrogen

for

storage

at

°C

until

use

Histopathological

examination

of

formalin-fixed

paraffin-embedded

sections

was

performed

by

two

pathologists

-LRB-

SF

and

MT

-RRB-

for

diagnostic

categorisation

Classification

of

neoplastic

tissue

was

made

according

to

a

recognised

system

-LSB-

-RSB-

The

cohort

comprised

benign

lesions

or

conditions

including

follicular

adenoma

nodular

goitre

normal

thyroid

tissue

and

Graves

's

thyroiditis

The

remaining

samples

were

diagnosed

as

PTC

and

comprised

six

classical

morphology

PTC

and

eight

FVPTC

-LRB-

see

Table

-RRB-

Immediately

before

RNA

extraction

frozen

sections

were

cut

and

stained

to

confirm

the

presence

of

representative

lesional

tissue

with

morphology

corresponding

to

that

noted

in

the

diagnostic

formalin-fixed

paraffin-embedded

sections

Table

thyroid

thyroid

hyperplasia

with

focal

lymphocytic

's

classic

classic

classic

classic

classic

classic

morphologyList

of

the

benign

and

malignant

lesions

that

were

used

in

this

study.FV

follicular

variant

PTC

papillary

thyroid

carcinoma

RNA

isolation

and

characterization

Samples

were

ground

in

liquid

nitrogen

and

homogenised

in

RLT

buffer

-LRB-

Qiagen

UK

-RRB-

RNA

was

then

extracted

using

the

RNeasy

Mini

Kit

with

optional

on-column

RNase-free

DNase

digestion

-LRB-

Qiagen

-RRB-

according

to

the

manufacturer

's

instructions

RNA

quantity

was

determined

using

UV

spectrophotometry

RNA

quality

was

assessed

using

the

RNA

Nano

LabChip

Kit

in

conjunction

with

the

Agilent

Bioanalyzer

-LRB-

Agilent

Technologies

Waldbronn

Germany

-RRB-

Microarray

analysis

Applied

Biosystems

Human

Genome

Survey

Arrays

were

used

to

analyse

the

transcriptional

profiles

of

thyroid

RNA

samples

in

this

study

Digoxigenin-UTP-labelled

cRNA

was

generated

and

linearly

amplified

from

μg

of

total

RNA

using

Applied

Biosystems

Chemiluminescent

RT-IVT

Labelling

Kit

v

using

manufacturer

's

protocol

μg

of

labelled

cRNA

were

hybridized

to

each

pre-hybed

microarray

in

a

volume

at

°C

for

h.

Array

hybridization

and

chemiluminescence

detection

were

performed

using

Applied

Biosystems

Chemiluminescence

Detection

Kit

following

manufacturer

's

protocol

Images

were

collected

for

each

microarray

using

the

analyser

Images

were

auto-gridded

and

the

chemiluminescent

signals

were

quantified

corrected

for

background

and

spot

and

spatially

normalized

TaqMan

PCR

validation

Sufficient

RNA

remained

from

of

the

initial

samples

for

TaqMan

polymerase

chain

reaction

-LRB-

PCR

-RRB-

validation

in

a

series

of

targets

RNA

was

reverse

transcribed

using

a

High

Capacity

cDNA

Archive

Kit

-LRB-

Applied

Biosystems

CA

USA

-RRB-

Primers

and

probes

for

TaqMan

PCR

were

obtained

by

using

Applied

Biosystems

pre-designed

TaqMan

Gene

Expression

Assays

PCR

was

carried

out

using

an

ABI

PRISM

Sequence

Detection

System

-LRB-

Applied

Biosystems

-RRB-

Analysis

of

relative

gene

expression

data

was

performed

using

the

ΔΔCT

method

-LSB-

-RSB-

with

dehydrogenase

-LRB-

GAPDH

-RRB-

as

an

endogenous

control/reference

assay

Statistical

analysis

Microarrays

were

analysed

using

Spotfire

DecisionSite

for

Functional

Genomics

-LRB-

Spotfire

AB

Goteborg

Sweden

-RRB-

and

R

version

a

free

language

and

environment

for

statistical

computing

and

graphics

-LRB-

R

Development

Core

Team

-RRB-

Arrays

were

initially

normalized

and

genes

were

deemed

undetectable

and

therefore

excluded

from

final

gene

lists

if

they

had

a

signal-to-noise

ratio

of

less

than

three

-LRB-

S/N

-RRB-

in

greater

than

of

the

arrays

An

ANOVA

test

was

used

to

generate

p

values

for

statistical

differences

between

groups

Their

p

values

were

then

adjusted

for

multiple

comparisons

using

the

technique

described

by

Benjamini

and

Yekutieli

-LSB-

-RSB-

Genes

were

deemed

statistically

different

between

groups

if

they

had

an

adjusted

p

and

an

average

fold-change

difference

of

greater

than

Hierarchical

clustering

was

performed

based

on

the

statistically

different

genes

to

determine

whether

samples

grouped

appropriately

Gene

ontology

analysis

was

performed

using

an

online

database

known

as

the

Panther

classification

system

-LRB-

-RRB-

Correlations

between

microarray

and

TaqMan

expression

data

were

measured

using

the

Pearson

coefficient

Results

Unsupervised

clustering

of

all

samples

demonstrated

clustering

into

two

major

groups

-LRB-

data

not

shown

-RRB-

comprising

the

N

group

and

the

T

group

-LRB-

see

Table

-RRB-

There

was

no

tendency

for

FVPTC

to

cluster

independently

with

classic

morphology

PTC

confirming

the

close

relationship

of

these

variants

of

PTC

To

identify

potential

markers

of

malignancy

ANOVA

with

false

discovery

rate

correction

was

used

to

compare

the

benign

and

malignant

thyroid

cohorts

A

p

value

cut-off

of

and

fold-change

difference

of

yielded

statistically

significant

probes

Of

these

corresponded

to

fully

annotated

probes

and

are

listed

in

Table

Table

differentially

expressed

in

malignant

vs

benign

thyroid

tissueGene

NameGene

SymbolAdjusted

p

probe

IDGenes

up-regulated

in

malignant

vs

benign

Active

BCR-related

Adaptor-related

protein

complex

alpha

Apoptosis

caspase

activation

X

interacting

domain

death

Brain

abundant

membrane

attached

signal

protein

Brain

acyl-CoA

Bromodomain

adjacent

to

zinc

finger

domain

Calcium/calmodulin-dependent

protein

kinase

Cathepsin

antigen

-LRB-

homing

function

and

Indian

blood

group

system

-RRB-

Chemokine

-LRB-

C-X-C

motif

-RRB-

ligand

Chromosome

open

reading

frame

Docking

protein

kDa

-LRB-

downstream

of

tyrosine

kinase

-RRB-

Epidermodysplasia

verruciformis

FXYD

domain

containing

ion

transport

regulator

FXYD

domain

containing

ion

transport

regulator

Genethonin

Hypothetical

gene

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Intercellular

adhesion

molecule

-LRB-

-RRB-

human

rhinovirus

Jun

dimerization

protein

Lectin

galactoside-binding

soluble

-LRB-

galectin

-RRB-

Major

vault

Matrix

metalloproteinase

-LRB-

membrane-inserted

-RRB-

Milk

fat

globule-EGF

factor

and

neuronal

cell

adhesion

Phospholipase

Promyelocytic

Protein

inhibitor

of

activated

STAT

protein

Protein

tyrosine

phosphatase

receptor

type

Rho

GDP

dissociation

inhibitor

-LRB-

GDI

-RRB-

calcium

binding

protein

-LRB-

calgizzarin

-RRB-

Similar

to

rat

tricarboxylate

carrier-like

nuclear

body

Stimulated

by

retinoic

acid

gene

Syndecan

-LRB-

N-syndecan

-RRB-

Tax

interaction

protein

domain

family

member

Tenascin

C

-LRB-

hexabrachion

-RRB-

Thymosin

beta

Y

Tissue

inhibitor

of

metalloproteinase

-LRB-

erythroid

potentiating

activity

collagenase

inhibitor

-RRB-

Topoisomerase

-LRB-

DNA

-RRB-

II

alpha

Transforming

growth

factor

beta

Transgelin

Tyrosine

activation

protein

eta

Yamaguchi

sarcoma

viral

related

oncogene

Genes

down-regulated

in

malignant

vs

benign

Aldehyde

oxidase

Ankyrin

Aspartate

Aspartate

ATPase

Cu

transporting

beta

Brain-specific

protein

Casein

Cellular

retinoic

acid

binding

protein

Centromere

protein

Ceroid-lipofuscinosis

neuronal

Chloride

channel

Chondroitin

Chromosome

open

reading

frame

Chromosome

open

reading

frame

Chromosome

open

reading

frame

Clusterin-like

-LRB-

retinal

-RRB-

Component

of

oligomeric

golgi

complex

Coxsackie

virus

and

adenovirus

Crystallin

alpha

Cytosolic

sialic

acid

Dicarbonyl/l-xylulose

DnaJ

-LRB-

-RRB-

homolog

subfamily

B

member

beta

-LRB-

S.

cerevisiae

-RRB-

Extracellular

link

domain

containing

Family

with

sequence

similarity

member

Fatty

acid

binding

protein

Fc

fragment

of

IgG

binding

Fibroblast

growth

factor

receptor

Friedreich

ataxia

region

gene

Glutamate-ammonia

ligase

-LRB-

glutamine

synthase

-RRB-

Glycine

amidinotransferase

-LRB-

l-arginine/glycine

amidinotransferase

-RRB-

Glycoprotein

Growth

hormone

HLA

complex

group

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Hypothetical

protein

Ionized

calcium

binding

adapter

molecule

gene

gene

Lectin

mannose-binding

Leiomodin

-LRB-

smooth

muscle

-RRB-

Likely

ortholog

of

rat

protein

LIM

domain

kinase

Low

density

lipoprotein-related

protein

-LRB-

deleted

in

tumors

-RRB-

Low

density

lipoprotein-related

protein

Matrilin

Metallothionein

-LRB-

functional

-RRB-

Metallothionein

-LRB-

functional

-RRB-

metallothionein

-LRB-

functional

-RRB-

metallothionein

metallothionein

Metallothionein

-LRB-

functional

-RRB-

metallothionein

-LRB-

functional

-RRB-

metallothionein

metallothionein

Metallothionein

-LRB-

functional

-RRB-

metallothionein

metallothionein

metallothionein

-LRB-

functional

-RRB-

Metallothionein

-LRB-

functional

-RRB-

Metallothionein

-LRB-

functional

-RRB-

Metallothionein

Metallothionein

Metallothionein

Metallothionein

Metallothionein

Metallothionein

Methionine

adenosyltransferase

II

Mitogen-activated

protein

kinase

Myc-induced

nuclear

antigen

NIMA

-LRB-

never

in

mitosis

gene

a

-RRB-

related

kinase

PDZ/coiled-coil

domain

binding

partner

for

the

rho-family

GTPase

Phospholipase

receptor

Phospholipase

C-like

Phosphotidylinositol

transfer

protein

Polycystic

kidney

and

hepatic

disease

-LRB-

autosomal

recessive

-RRB-

like

Polymerase

-LRB-

DNA

directed

-RRB-

Potassium

channel

subfamily

K

member

Potassium

channel-interacting

protein

Potassium

inwardly-rectifying

channel

subfamily

J

member

Pre-B

cell

leukemia

transcription

factor

Protein

kinase

cAMP-dependent

catalytic

Protein

phosphatase

regulatory

subunit

hypothetical

protein

member

RAS

oncogene

Ras

association

-LRB-

RalGDS/AF

-RRB-

domain

family

Sarcoglycan

delta

-LRB-

kDa

dystrophin-associated

glycoprotein

-RRB-

Serum

deprivation

response

-LRB-

phosphatidylserine

binding

protein

-RRB-

and

multiple

ankyrin

repeat

domains

Solute

carrier

family

member

Solute

carrier

family

member

Solute

carrier

family

-LRB-

iodide

transporter

-RRB-

member

SPARC

related

modular

calcium

binding

Syndecan

-LRB-

heparan

sulfate

proteoglycan

cell

surface-associated

fibroglycan

-RRB-

Syntaxin

T-box

Thioredoxin-like

Thyroid

stimulating

hormone

Tissue

inhibitor

of

metalloproteinase

Trefoil

factor

-LRB-

intestinal

-RRB-

Trefoil

factor

-LRB-

intestinal

-RRB-

UDP-N-acetyl-alpha-d-galactosamine

polypeptide

N-acetylgalactosaminyltransferase

-LRB-

-RRB-

homolog

-LRB-

S.

pombe

-RRB-

WW

domain

containing

WW

domain

containing

Zinc

finger

protein

Zinc

finger

protein

type-like

ANOVA

with

p

value

correction

yielded

probes

-LRB-

up

down-regulated

in

PTC

-RRB-

significantly

different

-LRB-

p

-RRB-

between

the

malignant

and

benign

thyroid

tissues

Supervised

hierarchical

clustering

was

performed

on

the

arrays

based

on

the

statistically

significant

probes

to

determine

whether

the

samples

would

segregate

appropriately

The

resulting

heat

map

can

be

observed

in

Fig.

Benign

lesions

and

tumours

clustered

together

with

the

exception

of

one

tumour

sample

that

clustered

with

the

benign

group

-LRB-

-RRB-

Fig.

clustering

of

samples

This

heat

map

shows

the

clustering

of

the

samples

based

on

the

probes

found

to

be

differentially

regulated

in

benign

vs

malignant

thyroid

tissue

Clustering

was

performed

using

the

unweighted

pair

group

method

with

arithmetic

mean

with

Euclidian

distance

as

the

similarity

measure

Average

value

was

used

as

the

ordering

function

A

binomial

statistics

tool

was

used

to

compare

classifications

of

multiple

clusters

of

lists

to

a

reference

list

-LRB-

i.e.

the

complete

human

genome

-RRB-

to

statistically

determine

over

or

under-representation

of

Panther

classification

categories

Biological

processes

over-represented

in

the

up-regulated

PTC

cohort

included

tumour

suppressor

oncogenesis

DNA

replication

cell

cycle

and

cell

adhesion

-LRB-

p

-RRB-

Genes

involved

in

homeostasis

and

other

homeostasis

activities

were

highly

over-represented

in

the

down-regulated

cohort

-LRB-

p

-RRB-

ANOVA

tests

were

used

to

determine

which

genes

were

differentially

regulated

in

the

FVPTC

cohort

only

Fifteen

genes

were

identified

including

cluster

of

differentiation

-LRB-

-RRB-

CTSC

CTSH

CTSS

human

leucocyte

antigen

A

-LRB-

HLA-A

-RRB-

HLA-DMA

HLA-DRA

osteoclast

stimulating

factor

-LRB-

-RRB-

and

a

previously

uncharacterized

gene

-LRB-

noname

-RRB-

Microarray

results

were

validated

using

a

reverse

transcription

reaction

followed

by

TaqMan

PCR

for

gene

targets

The

ΔΔCT

method

-LSB-

-RSB-

was

used

to

analyse

relative

gene

expression

data

GAPDH

was

used

as

an

endogenous

control

and

was

chosen

as

an

arbitrary

calibrator

sample

Gene

expression

profiles

for

TaqMan

PCR

were

plotted

in

conjunction

with

those

for

microarray

results

in

Fig.

Pearson

co-efficient

was

used

to

directly

compare

data

from

microarray

analysis

and

TaqMan

RT-PCR

Table

depicts

genes

differentially

expressed

in

both

benign

vs

malignant

and

FVPTC

vs

classic

morphology

PTC

Fig.

PCR

validation

of

microarray

experiments

Profile

charts

of

gene

expression

levels

comparing

results

obtained

by

microarray

analysis

-LRB-

n

-RRB-

to

TaqMan

PCR

analysis

-LRB-

n

-RRB-

for

six

genes

Plots

for

those

genes

with

multiple

probes

are

also

displayed

where

appropriateTable

between

TaqMan

and

microarray

dataGenePearson

's

r

coefficienttwo-tailed

pGenes

differentially

expressed

in

FVPTC

vs

classic

0.00010.740.0002CTSC0.530.0170CTSH0.710.0005CTSS0.620.0037DPP60.760.0001ETHE10.300.2008HLA-A0.77

NONAME

Genes

differentially

expressed

in

benign

vs

Gene

expression

profiles

for

TaqMan

PCR

and

microarray

results

were

compared

using

the

Pearson

coefficient.aGenes

have

more

than

one

probe

ID

on

microarray

Discussion

The

primary

aim

of

this

study

was

to

generate

an

overview

of

molecular

markers

of

malignancy

in

PTC

with

a

view

to

identifying

discriminators

between

common

sub-types

-LRB-

classic

PTC

and

FVPTC

-RRB-

using

genome-wide

expression

microarray

technology

validated

by

TaqMan

RT-PCR

To

this

end

lesions

that

were

well

characterized

histologically

were

selected

The

application

of

microarray

analysis

designed

to

identify

transcripts

strongly

associated

with

each

group

of

interest

yielded

a

gene

list

of

genes

that

were

differentially

expressed

between

cohorts

Significant

down-regulation

of

Coxsackie

virus

receptor

was

recorded

in

the

malignant

cohort

of

thyroid

lesions

The

Coxsackievirus

B

and

adenovirus

receptor

-LRB-

CAR

-RRB-

plays

a

dual

role

as

a

homotypic

junctional

adhesion

protein

and

as

a

viral

receptor

It

is

biologically

plausible

that

altered

expression

may

impact

on

the

morphology

peculiar

to

PTC

given

its

association

with

cellular

adhesion

CAR

has

been

shown

to

be

differentially

expressed

in

various

human

adenocarcinomas

and

differential

expression

may

represent

a

new

factor

in

thyroid

tumourigenesisigenesis

-LSB-

-RSB-

Rab

expression

was

also

down

regulated

in

the

malignant

cohort

The

Rab

small

G

protein

family

is

composed

of

approximately

members

Many

of

them

are

ubiquitous

and

are

expressed

and

participate

in

transport

processes

such

as

endocytosis

and

exocytosis

-LSB-

-RSB-

Other

gene

targets

demonstrating

significant

down-regulation

in

the

malignant

group

were

syndecans

and

Syndecans

are

transmembrane

proteoglycans

expressed

on

adherent

cells

Changes

in

syndecan

expression

have

been

postulated

to

influence

cell

adhesion

migration

and

the

structure

of

focal

contacts

and

the

cytoskeleton

-LSB-

-RSB-

The

abundance

of

metallothionein

genes

in

the

cohort

of

genes

down-regulated

in

PTC

is

interesting

partly

because

of

the

sheer

number

of

isoforms

detected

-LRB-

Table

-RRB-

There

have

been

many

studies

showing

increased

metallothionein

expression

in

a

plethora

of

cancer

types

but

few

showing

decreased

expression

-LSB-

-RSB-

Although

previous

microarray

experiments

showed

metallothionein

genes

to

be

down-regulated

in

thyroid

tumours

to

a

certain

extent

-LSB-

-RSB-

have

detected

so

many

as

the

current

study

Apart

from

microarray

experiments

there

has

been

little

reported

in

the

literature

regarding

metallothioneins

in

thyroid

cancer

An

early

report

by

Nartey

et

al.

-LSB-

-RSB-

showed

metallothioneins

to

be

expressed

actively

in

certain

human

thyroid

neoplastic

tissues

but

not

in

normal

thyroid

tissue

which

would

seem

to

contradict

the

current

authors

findings

In

contrast

to

this

a

later

immunohistochemical

study

showed

an

absence

of

metallothionein

expression

in

of

PTCs

-LSB-

-RSB-

Interestingly

in

three

of

the

seven

positive

PTCs

metallothionein

positivity

was

restricted

to

areas

of

follicular

differentiation

In

one

of

the

only

recent

studies

Huang

et

al.

-LSB-

-RSB-

followed

up

their

initial

microarray

experiment

by

showing

that

is

down

regulated

in

PTC

via

hypermethylation

The

biological

significance

of

low

metallothionein

expression

in

thyroid

tumours

is

therefore

still

poorly

understood

however

it

is

interesting

to

speculate

that

metallothioneins

may

have

roles

as

tumour

suppressors

in

thyroid

carcinoma

Many

genes

identified

such

as

-LSB-

-RSB-

LYN

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

BAX

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

and

-LSB-

-RSB-

have

been

previously

reported

in

both

microarray

and

conventional

experiments

in

thyroid

cancer

The

data

generated

in

this

study

corroborates

the

importance

of

several

biological

processes

in

the

progression

of

thyroid

neoplasia

For

example

expression

was

up

regulated

in

the

PTC

cohort

compared

with

benign

lesions

paralleling

the

increased

protein

expression

of

this

gene

target

identified

at

the

protein

level

using

immunohistochemistry

-LSB-

-RSB-

has

also

been

suggested

as

a

biomarker

of

malignancy

in

the

context

of

colorectal

carcinoma

as

long

ago

as

-LSB-

-RSB-

Correlation

of

highlighted

features

with

the

current

state

of

knowledge

of

the

molecular

pathology

of

thyroid

neoplasia

goes

some

way

towards

providing

an

external

validation

of

the

data

obtained

from

the

system

However

additional

validation

using

TaqMan

RT-PCR

was

performed

In

general

microarray

and

TaqMan

data

correlated

well

with

approx.

of

comparisons

having

p

Candidate

genes

were

selected

contingent

on

results

identified

as

over-represented

biological

processes

-LRB-

oncogenesis

cell

cycling

DNA

replication

and

homeostasis

-RRB-

using

the

Panther

binomial

statistics

tool

as

opposed

to

the

more

traditional

method

of

selecting

the

most

highly

disregulated

genes

This

may

account

for

the

poor

correlation

observed

with

certain

genes

Some

genes

such

as

and

had

excellent

correlation

between

microarray

and

TaqMan

results

whereas

others

such

as

BAX

and

TSHR

showed

poor

correlation

despite

previous

studies

implicating

them

in

thyroid

cancer

-LSB-

-RSB-

Where

there

was

discordance

in

the

data

it

could

be

accounted

for

by

differences

in

the

sequences

targeted

by

the

TaqMan

target

sequences

and

the

microarray

probe

In

those

cases

the

array

and

pre-designed

TaqMan

assays

interrogated

different

exons

or

alternative

splice

variants

This

finding

highlights

the

importance

of

matching

the

targets

to

be

validated

from

microarray

data

sets

Analysis

of

differentially

expressed

transcripts

in

the

FVPTC

revealed

many

transcripts

showing

similar

expression

level

patterns

in

both

FVPTC

and

classic

morphology

PTC

Examples

of

these

transcripts

are

included

in

Table

In

addition

somewhat

surprisingly

unsupervised

clustering

of

all

samples

showed

no

tendency

for

FVPTC

to

cluster

independently

of

classic

morphology

PTC

emphasizing

the

very

close

relationship

of

these

PTC

variants

-LRB-

data

not

shown

-RRB-

However

analysis

of

differentially

expressed

genes

in

FVPTC

exclusive

of

those

identified

in

classic

morphology

PTC

revealed

genes

exhibiting

differential

expression

in

FVPTC

compared

with

benign

lesions

and

conditions

outwith

of

classic

morphology

PTC

These

genes

displayed

a

narrow

gamut

of

function

represented

by

the

transcripts

involved

-LRB-

Table

-RRB-

TaqMan

RT-PCR

was

performed

for

all

of

these

targets

to

confirm

the

array

findings

and

correlation

with

the

array

data

was

strong

-LRB-

see

Table

-RRB-

Aberrant

expression

of

two

major

groups

of

transcripts

was

noted

in

FVPTC

Relatively

increased

expression

of

class

major

histocompatibility

complex

-LRB-

MHC

-RRB-

genes

-LRB-

HLA-A

-RRB-

and

aberrant

expression

of

class

MHC

genes

-LRB-

HLA-DMA

HLA

DRA

-RRB-

and

associated

genes

-LRB-

e.g.

represents

the

invariant

membrane

bound

moiety

of

class

II

HLA

molecules

and

regulates

the

biology

and

functions

of

MHC

class

II

molecules

and

is

a

surface

marker

of

monocytes/macrophages

-RRB-

were

the

most

significant

findings

Additionally

relatively

up-regulated

expression

of

members

of

the

cathepsin

family

-LRB-

cathepsin

C

cathepsin

H

cathepsin

S

and

-RRB-

was

striking

in

the

FVPTC

group

HLA

expression

is

generally

associated

with

immune

functions

such

as

T

cell

interaction

and

antigen

presentation

The

presence

of

prominent

HLA

transcript

expression

especially

among

class

in

FVPTC

is

intriguing

One

potential

cause

of

this

was

that

tumour-infiltrating

leucocytes

were

responsible

for

this

finding

However

haematoxylin

eosin

-LRB-

H&E

-RRB-

stained

slides

of

each

case

were

reviewed

to

specifically

identify

the

degree

of

tumour

infiltration

by

leucocytes

Although

a

mild

lymphocytic

infiltrate

was

noted

in

some

cases

there

was

no

apparent

over

representation

of

lymphocytes

in

the

follicular

variant

compared

to

benign

lesions

and

classic

morphology

PTC

This

raises

the

clear

possibility

that

the

findings

represent

aberrant

increased

expression

of

class

HLA

transcripts

by

the

epithelium

of

FVPTC

This

is

an

unexpected

finding

as

over-expression

of

MHC

class

molecules

would

be

expected

to

increase

tumour

immunogenicity

A

similar

aberrant

expression

of

HLA

transcripts

has

been

recently

described

in

ovarian

neoplasms

-LSB-

-RSB-

Rangel

et

al.

-LSB-

-RSB-

concluded

over-expression

of

HLA-DRA

might

represent

a

novel

biomarker

for

malignancy

and

this

also

seems

biologically

plausible

in

the

FVPTC

setting

A

recent

paper

has

also

described

HLA-DRA

expression

in

ret/PTC-activated

papillary

thyroid

carcinoma

but

not

in

surrounding

normal

thyroid

follicles

-LSB-

-RSB-

Yu

et

al.

-LSB-

-RSB-

showed

discordant

expression

of

-LRB-

-RRB-

and

HLA-DR

in

Hashimoto

thyroiditis

an

autoimmune

condition

associated

with

increased

incidence

of

PTC

and

sharing

molecular

features

such

as

ret/PTC

expression

-LSB-

-RSB-

Hwang

et

al.

-LSB-

-RSB-

have

drawn

attention

to

aberrant

expression

of

HLA-DRA

in

ret/PTC-activated

PTC

Expression

of

HLA-DRA

may

in

some

way

explain

the

propensity

for

PTC

to

metastasize

to

lymph

nodes

and

often

apparently

reside

there

without

markedly

worsening

prognosis

Cathepsins

C

-LRB-

dipeptidyl-peptidase

I

-RRB-

H

and

S

showed

up-regulation

in

FVPTC

compared

to

benign

lesions

Cathepsins

are

a

family

of

proteases

that

play

an

important

role

in

protein

degradation

They

are

key

players

in

the

proliferative

invasive

and

metastatic

potential

of

malignant

tumour

cells

Their

expression

in

the

relatively

biologically

indolent

FVPTC

is

intriguing

and

it

remains

possible

that

cathepsins

have

cellular

roles

outside

of

those

involved

in

invasion

and

dissemination

of

tumour

cells

as

indeed

has

been

suggested

by

others

-LSB-

-RSB-

For

example

cathepsin

L

has

recently

been

shown

to

play

a

role

in

nuclear

transcriptional

activation

and

cathepsins

are

now

recognized

to

play

a

role

in

MHC

class

II

antigen

presentation

-LSB-

-RSB-

has

no

defined

role

in

carcinogenesis

although

outside

of

its

role

in

ossification

it

is

also

known

to

have

roles

in

signal

transduction

and

protein

binding

which

may

be

relevant

to

carcinogenesis

and

particularly

FVPTC

A

recent

paper

highlights

the

role

of

bone

mineralization

proteins

including

osteopontin

and

osteoclast

stimulating

factors

as

potential

biomarkers

of

malignant

tumours

in

general

-LSB-

-RSB-

Indeed

in

addition

to

elevated

expression

increased

expression

of

osteopontin

was

seen

in

PTC

-LRB-

data

not

shown

-RRB-

however

in

our

data

osteopontin

does

not

appear

to

be

specifically

up-regulated

in

FVPTC

and

this

finding

has

also

been

noted

by

other

researchers

-LSB-

-RSB-

In

any

case

osteopontin

is

known

to

be

a

downstream

effector

of

ret/PTC

-LSB-

-RSB-

and

mutated

BRAF

-LSB-

-RSB-

where

it

acts

in

association

with

another

transcript

showing

increased

expression

in

both

classic

morphology

PTC

and

FVPTC

A

particular

focus

of

this

study

was

to

compare

transcriptome

profiles

for

PTCs

with

classic

morphology

and

FVPTC

given

the

propensity

for

FVPTC

lesions

to

prove

problematic

from

a

diagnostic

perspective

Although

the

study

confirms

the

close

relationship

between

the

two

most

common

variants

of

PTC

a

narrow

portfolio

of

genes

and

in

particular

gene

functions

was

elucidated

in

the

FVPTC

cohort

The

targets

identified

are

easily

amenable

to

analysis

by

more

established

techniques

such

as

TaqMan

RT-PCR

with

associated

potential

as

additional

markers

for

application

in

the

FNAC

setting

Clearly

the

potential

biomarkers

identified

in

this

study

will

require

prospective

evaluation

in

the

context

of

real

clinical

diagnostic

situations

in

the

future

to

consolidate

their

merit

as

adjunctive

tests

in

the

diagnostic

setting

and

to

validate

their

altered

expression

states

in

the

pathobiology

of

PTC

development

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

papillary thyroid carcinoma
biomarker
follicular variant
microarray


Virtual reality training for endoscopic surgery : voluntary or obligatory ? Introduction Virtual reality -LRB- VR -RRB- simulators have been developed to train basic endoscopic surgical skills outside of the operating room . An important issue is how to create optimal conditions for integration of these types of simulators into the surgical training curriculum . The willingness of surgical residents to train these skills on a voluntary basis was surveyed . Endoscopic surgery requires dedicated skills such as three-dimensional orientation in a two-dimensional representation of the operating field and complex instrument handling -LSB- 8 , 7 , 5 -RSB- . Training of these skills in the operating room -LRB- OR -RRB- is under pressure due to planning issues and ethical considerations . Virtual reality -LRB- VR -RRB- simulators have been developed to train basic endoscopic surgical skills outside of the OR . Several simulators have been validated and found adequate for the transfer of skills from the simulator to the OR -LSB- 2 , 6 , 9 , 13 , 14 -RSB- . However , discussion arises on how to integrate these simulation-based training modalities in the surgical training curriculum . A questionnaire was distributed to 245 Dutch surgical residents to explore the perspective of the trainee on this issue . Approximately 75 % of residents felt that endoscopic skills training outside the OR is useful -LSB- 12 -RSB- . In another study , sixty Dutch gynaecology residents responded positively -LRB- 3.9 on a five-point Likert scale -RRB- with regard to training in laparoscopic skills before real surgery -LSB- 10 -RSB- . Fifty-five percent of these 60 residents did not have the opportunity to train laparoscopic skills . However , those that did appeared to train only once or twice a year and 33 % did not use available skills trainers voluntarily at all . We hypothesized that insufficient simulator access might be the reason for this contradiction . Therefore , we investigated the willingness of surgical residents to train in endoscopic skills on a voluntary basis when VR simulators were indeed readily available . We also evaluated the effect of competitive incentives on the frequency and duration of simulator training . Materials and methods Equipment , tasks and scoring system This study is performed with the LapSim virtual reality simulator , which uses the Virtual Laparoscopic Interface -LRB- VLI -RRB- hardware , -LRB- Immersion Inc. , San Jose , CA , USA -RRB- The VLI has an interface with a 2600 MHz hyperthreading processor Pentium IV computer running Windows XP and is equipped with 256 random-access memory -LRB- RAM -RRB- , a GeForce graphics card and an 18-inch thin-film transistor -LRB- TFT -RRB- monitor . The systems feature LapSim Basic Skills 3.0 software -LRB- Surgical Science Ltd , Göteburg , Sweden -RRB- , from the LapSim Basic Skills package , consisting of nine tasks . A training program was designed that included all nine tasks : camera navigation , instrument navigation , coordination , grasping , lifting and grasping , cutting , clipping and cutting , suturing and fine dissection -LSB- 4 -RSB- . The computer stores and displays between seven and eleven parameters of performance per task . These parameters are related to time , errors or efficiency of handling . Tasks can be adjusted to different levels of difficulty . The training program for this study was set at an advanced level with thresholds that are based on the performance of 30 experienced endoscopic surgeons -LRB- more than 100 endoscopic procedures -RRB- . The scoring system is two-tiered . First , for any given parameter the system determines whether or not the participant passes or fails the test . Secondly , if a participant passes , a score of between 0 % and 100 % is attached to his or her performance on that particular parameter . The overall score per task is determined by the sum score of the parameters , divided by their number . Hence , an overall score of 100 % can only be obtained by scoring 100 % on each of the individual parameters measured during performance of the particular task . An outcome score of 100 points is given to those participants who score a 100 % on the task performed . Logically , a score of 85 % thus translates into 85 points . A maximum overall score of 900 could be obtained -LRB- i.e. , 100 points on each of the nine tasks measured -RRB- . Participants Twenty-one surgical residents , ranging from postgraduate year -LRB- PGY -RRB- - one level to PGY 6 level , with different endoscopic surgical experience , were given unlimited access to the simulator . Seven residents were at the beginning of their surgical educational program -LRB- PGY 1 and PGY 2 -RRB- and therefore inexperienced in endoscopic surgery . Seven residents were in the middle -LRB- PGY 3 and PGY 4 -RRB- and eight residents were at the end -LRB- PGY 5 and PGY 6 -RRB- of their surgical educational program . Setting and incentives In the period May 2005 to January 2006 a simulator was placed in the general room for surgical residents at the surgical ward of the University Medical Centre in Utrecht . Before the study , residents were instructed on how to operate the simulator , and allocated a personal login number for the simulator . By placing the simulator in the general residents ' room , it was readily and easily accessible 24 hours a day . The room is secured by a code-locked door and accessible by residents only . During the first four months , there were no additional incentives other than the permanent -LRB- 24-hour -RRB- accessibility to the residents for training on the simulator . After these four months , a competitive element was introduced in which the frequency of training was also rewarded -LRB- bi-weekly -RRB- . The overall end-score was calculated every other week by adding this frequency bonus to the highest scores for each task . These overall end-scores for each resident were publicly announced to the complete department of surgery and the winner -LRB- the resident with the highest score -RRB- was awarded a prize . Questionnaire After eight months all residents were requested to fill out a questionnaire . Ten questions were presented on a five-point Likert scale , concerning their perception of their own experience level in endoscopic procedures , their opinion of the possibility to develop and train endoscopic skills within the current surgical curriculum , and their opinion about the application of virtual reality as a means to training endoscopic skills . Value 1 was assigned to `` totally agree '' , value 5 to `` totally disagree '' . In addition , the residents were asked about their frequency of usage of the simulator . If a participant indicated little usage , he or she was questioned why , and what could motivate increased usage . Results In the first four months only two of the 21 residents -LRB- 10 % -RRB- trained on the simulator , for a total of 163 minutes . One resident was a PGY 2 , the other one a PGY 5 . In the second period of four months the number of trainees increased to seven residents -LRB- 33 % , two PGY 2 , two PGY 3 , one PGY 5 and two PGY 6 -RRB- . The duration of training increased to 738 minutes , thereby constituting an average increase of 23.9 minutes per subject . Fifty-eight percent of training was performed during night shifts . All 22 residents -LRB- 100 % -RRB- replied to the questionnaire . The total training time , as an accumulation of estimation on individual training time , was 4140 minutes . The actual cumulated training time for all residents was 901 minutes -LRB- 22 % -RRB- . Thirteen out of 15 residents who did not train at all -LRB- 86 % -RRB- stated that this was due to a lack of time during the day . One resident -LRB- 7 % -RRB- stated he had been not interested enough to train and indicated that he had alternative priorities . Another resident -LRB- 7 % -RRB- stated that she was fully occupied due to an intensive-care traineeship and maternity leave . Residents suggested that the use of the VR trainers could be enhanced by incorporating a mandatory VR training into the surgical curriculum -LRB- 9x = people agreeing -RRB- , to oblige certain skills level on VR simulator before starting endoscopic surgery in the OR -LRB- 3x -RRB- , to implement competitive training with coaching -LRB- 2x -RRB- , to diminish working pressure -LRB- 2x -RRB- , to have more-advanced exercises available on the simulator -LRB- 3x -RRB- , to place the VR simulator in a location other than in the residents ' room -LRB- 1x -RRB- . Only two stated that more initiative of residents was required to improve outcome . Figure 1 refers to the perception of residents ' own experience level in endoscopic procedures , their opinion of the possibility to develop and train endoscopic skills within the current surgical curriculum and their opinion about the application of virtual reality as a means to training endoscopic skills . Fig. 1 . Results of questionnaire In general , the opinion of the residents on their own experience level , on the possibility to develop and train endoscopic skills during their training and on the role of virtual reality varies considerably -LSB- standard deviation -LRB- SD -RRB- 0.44 to 1.39 -RSB- . Their opinion on obligation of VR training to improve endoscopic skills and having VR training as a mandatory part of the basic skills training is most uniform . Residents do not have a marked positive , nor a marked negative opinion on the presented statements on receiving enough training for acquiring basic skills -LRB- mean 2.63 , SD 1.19 -RRB- , on receiving sufficient training time in the OR to train in endoscopic skills -LRB- mean 3.47 SD 1.17 -RRB- and on acquiring a satisfactory level of basic psychomotor skills -LRB- mean 2.42 , SD 1.16 -RRB- . The same applies to their opinion on the representation of their training results on the simulator -LRB- mean 2.98 , SD 0.83 -RRB- ; as well as on the statement that thresholds should be reached before training in the OR is allowed -LRB- mean 2.47 , SD 1.39 -RRB- . There is one statement they do not agree with ; I will not train unless it is obligatory -LRB- mean 4.26 , SD 1.10 -RRB- . Discussion Virtual reality training has the potential to improve and professionalize the training in endoscopic basic psychomotor skills -LSB- 2 , 6 , 9 , 13 , 14 -RSB- . Training results can be shown instantly to demonstrate objective performance and progress of performance assessment . However , one of the main concerns in acquisition of expensive equipment for educational purposes is its effectiveness . While initial enthusiasm about new innovative equipment is usually high among the surgical community , actual usage tends to be disappointing -LSB- 3 -RSB- . This study was undertaken to evaluate the aptitude for training on a voluntary basis when a VR simulator was readily available . Free unlimited access to a VR simulator without obligation or assessment in our setting did not seem to motivate surgical residents to use the simulator for improvement of their psychomotor endoscopic skills level . The addition of a competitive element and a desirable prize had only a marginal effect on the frequency and duration of training . We believe that the effort required to provide this incentive is disproportionate to its marginal effect . The majority of residents -LRB- 86 % -RRB- stated that ` lack of time due to high working pressure ' is the most important reason for not using the simulator . Following a recent European guideline , as set by the European Commission , a working week for a resident in training is being reduced from 70 to 48 hours -LSB- 1 , 11 -RSB- . This may have led to an increase in pressure during working hours , with little time available for voluntary training . However , spare time has increased vastly compared to the former curriculum . Residents did not use personal free time for VR simulator training to improve their skills . The perception of their own experience level in endoscopic procedures and the possibility of developing and training in endoscopic skills within the current surgical curriculum was in general neutral . Therefore no conclusions can be drawn from this . There is favorable , uniform opinion on the desirability of integration of skills training into the curriculum . In addition , residents believe skills training ought to be mandatory for marked improvement of their psychomotor skills . Interestingly , the disagreement on the statement of not training unless it is obligatory -LRB- mean 4.26 , SD 1.10 -RRB- appears to have no bearing in reality , because our study shows very limited use of the simulator . This incongruence might be caused by political correctness or by a discrepancy between intentions and actions . It must be said that our result reflects the quantity of training on a voluntary basis of 22 residents in a single institute only , and might therefore not represent the attitude of national or international surgical residents . In conclusion , the acquisition of expensive devices to train basic psychomotor skills for endoscopic surgery is probably only effective when it is a mandatory part of the curriculum .

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

virtual reality simulation
curriculum design
resident participation