Dynamics

of

amino

acid

metabolism

of

primary

human

liver

cells

in

bioreactors

The

kinetics

of

amino

acids

ammonia

-LRB-

-RRB-

and

urea

-LRB-

UREA

-RRB-

in

liver

cell

bioreactor

runs

were

analyzed

and

simulated

by

a

two-compartment

model

consisting

of

a

system

of

differential

equations

The

model

parameters

most

of

them

representing

enzymatic

activities

were

identified

and

their

values

discussed

with

respect

to

the

different

liver

cell

bioreactor

performance

levels

The

nitrogen

balance

based

model

was

used

as

a

tool

to

quantify

the

variability

of

runs

and

to

describe

different

kinetic

patterns

of

the

amino

acid

metabolism

in

particular

with

respect

to

glutamate

-LRB-

GLU

-RRB-

and

aspartate

-LRB-

ASP

-RRB-

Introduction

Bioreactor

technology

for

extracorporeal

liver

support

using

primary

human

liver

cells

has

been

developed

within

the

last

decade

-LSB-

-RSB-

The

design

of

a

bioreactor

for

maintaining

the

hepatocyte

's

full

functionality

is

of

great

importance

The

used

multi-compartment

bioreactor

consists

of

three

interwoven

independent

capillary

membrane

systems

Two

of

them

-LRB-

compartments

and

in

the

following

aggregated

to

the

perfusion

compartment

-RRB-

provide

decentralized

plasma

flow

and

the

third

one

-LRB-

compartment

-RRB-

provides

oxygen

supply

to

the

cells

which

are

localized

in

the

extracapillary

space

-LRB-

compartment

liver

cell

compartment

-RRB-

This

three-dimensional

spatial

structure

represents

an

artificial

equivalent

of

the

hepatic

vasculature

at

the

lobular

level

The

bioreactor

is

integrated

into

a

perfusion

system

that

enables

monitoring

and

control

of

system

conditions

-LRB-

see

Fig.

-RRB-

Previous

studies

have

shown

that

primary

liver

cells

reconstitute

to

tissue-like

structures

after

inoculation

into

the

bioreactor

and

that

they

maintain

metabolic

activities

over

several

weeks

-LSB-

-RSB-

This

bioreactor

therefore

provides

a

valuable

tool

to

analyze

the

dynamics

and

network

structures

of

the

physiological

and

molecular

interactions

of

liver

cells

under

standardized

conditions

that

closely

reflect

the

situation

in

the

natural

organ

Fig.

of

the

liver

cell

bioreactor

with

the

perfusion

circuit

the

two

inflow

streams

and

the

outflow

stream

Measured

data

were

acquired

from

the

waste

Recently

data

from

this

bioreactor

system

has

been

analyzed

statistically

as

well

as

by

fuzzy

cluster

and

rule

based

data

mining

and

pattern

recognition

methods

in

order

to

identify

early

performance

predictors

for

the

bioreactor

's

long-term

performance

-LSB-

-RSB-

With

respect

to

the

prominent

role

of

hepatocytes

in

the

amino

acid

metabolism

the

analysis

and

modeling

of

metabolic

pathways

of

amino

acids

could

provide

important

information

on

the

functional

state

of

hepatocytes

cultured

in

vitro

In

initial

studies

the

role

of

amino

acid

regulation

in

extracorporeal

liver

support

systems

was

analyzed

-LSB-

-RSB-

More

recently

three

different

modeling

paradigms

i.e.

correlation

networks

Bayesian

networks

and

systems

of

differential

equations

were

applied

to

characterize

the

concentration

profiles

of

six

amino

acids

and

related

nitrogen-containing

compounds

in

bioreactor

cultures

of

primary

human

liver

cells

-LSB-

-RSB-

The

present

work

reports

on

the

model

based

analysis

of

the

measured

kinetics

of

amino

acids

as

well

as

ammonia

-LRB-

-RRB-

and

urea

-LRB-

UREA

-RRB-

of

liver

cell

bioreactor

runs

Materials

and

methods

Cells

and

bioreactors

Primary

human

liver

cells

were

isolated

according

to

a

method

described

elsewhere

-LSB-

-RSB-

from

human

donor

livers

-LRB-

n

-RRB-

that

were

not

suitable

for

transplantation

due

to

organ

injury

After

isolation

cells

were

cultured

within

bioreactors

over

days

-LRB-

This

broad

variability

in

duration

is

caused

by

their

use

for

liver

support

from

the

seventh

day

onwards

and

by

the

performance

Low

performance

runs

were

finished

earlier

High

performance

runs

were

continued

for

a

longer

period

of

time

However

only

the

first

days

of

the

runs

were

analyzed

in

this

study

-RRB-

Concentrations

of

free

amino

acids

in

the

culture

perfusates

were

determined

by

an

automated

reversed

phase

high

performance

liquid

chromatography

system

-LRB-

RP-HPLC

-RRB-

with

precolumn

derivatization

using

the

ophthaldialdehyde

method

-LSB-

-RSB-

and

UREA

concentrations

were

determined

using

routine

clinical

analyzers

-LRB-

Roche

Diagnostics

Heidelberg

Germany

-RRB-

With

respect

to

the

model

based

analysis

two

compartments

of

the

multi-compartment

capillary

membrane

bioreactor

system

were

considered

The

liver

cell

compartment

with

the

volume

mL

contains

the

liver

cells

in

the

inter-capillary

space

The

perfusion

compartment

with

the

volume

mL

supplies

a

stream

through

the

inside

of

the

capillaries

This

perfusion

stream

carries

the

concentrations

of

the

compounds

that

are

supplied

to

or

removed

from

the

bioreactor

Decentralized

mass

exchange

at

low

gradients

is

achieved

by

independent

perfusion

of

two

medium

capillary

systems

enabling

different

perfusion

modes

-LSB-

-RSB-

In

this

study

counter-directional

medium

flow

was

performed

to

facilitate

rapid

matter

distribution

Due

to

the

high

flow

rate

of

the

perfusion

stream

-LRB-

mL

min

-RRB-

an

almost

ideal

mixing

within

the

perfusion

compartment

can

be

assumed

-LRB-

Fig.

-RRB-

To

this

perfusion

compartment

two

time-variant

inflow

streams

are

added

with

the

flow

rates

FA

-LRB-

t

-RRB-

and

FB

-LRB-

t

-RRB-

as

defined

by

Eq

-LRB-

-RRB-

FA

-LRB-

t

-RRB-

follows

a

step

function

from

mL

h

down

to

mL

h

switching

at

tA

-LRB-

day

-RRB-

FB

-LRB-

t

-RRB-

switches

from

up

to

mL

h

at

the

time

tB

-LRB-

see

Table

-RRB-

The

outflow

rate

-LRB-

t

-RRB-

to

the

waste

equals

the

sum

of

both

inflow

rates

-LRB-

This

is

realized

by

overflow

keeping

a

constant

pressure

in

the

perfusion

compartment

-RRB-

The

inflow

rate

FA

-LRB-

t

-RRB-

carries

the

amino

acids

and

at

the

concentrations

cAi

-LRB-

see

Table

-RRB-

The

inflow

rate

FB

-LRB-

t

-RRB-

carries

only

the

amino

acid

aspartate

-LRB-

ASP

-RRB-

at

the

concentration

μmol

L

i.e.

cBi

for

all

i

The

time

courses

of

the

concentrations

-LRB-

t

-RRB-

in

the

outflow

stream

may

be

considered

to

describe

the

response

of

the

medium

to

the

inoculation

of

the

bioreactor

with

cells

These

concentrations

are

in

steady-state

at

-LRB-

-RRB-

cAi

prior

to

inoculation

Due

to

the

almost

ideal

mixing

in

the

perfusion

compartment

the

concentrations

-LRB-

t

-RRB-

in

the

perfusion

stream

are

the

same

as

in

the

outflow

stream

Table

variables

ci

and

i

for

the

liver

cell

compartment

and

the

perfusion

compartment

respectively

their

initial

values

ci

-LRB-

-RRB-

and

i

-LRB-

-RRB-

before

inoculation

of

the

bioreactor

with

liver

cells

the

concentration

cAi

in

the

inflow

stream

fed

with

the

flow

rate

FA

-LRB-

t

-RRB-

according

to

Eq

-LRB-

-RRB-

and

the

stoichiometric

coefficients

si

for

nitrogenNameModel

variables

ci

and

Ici

-LRB-

-RRB-

i

-LRB-

-RRB-

cAi

-LSB-

μmol

L

-RSB-

Stoichiometric

coefficient

Data

A

data

set

with

the

elements

ci

j

k

-LRB-

i

j

k

K

-RRB-

for

kinetic

variables

and

bioreactor

runs

at

K

time

points

tk

was

analyzed

The

kinetics

over

the

first

days

of

the

bioreactor

runs

were

analyzed

here

-LRB-

tk

days

-RRB-

Concentrations

of

and

UREA

were

measured

daily

-LRB-

tk

days

-RRB-

Amino

acid

concentrations

were

determined

up

to

the

third

day

daily

and

every

third

day

afterwards

-LRB-

tk

days

-RRB-

The

samples

for

the

measurement

of

the

ci

j

k

were

taken

from

the

waste

of

the

liver

cell

bioreactor

system

-LRB-

see

Fig.

-RRB-

which

was

emptied

daily

after

accumulation

of

the

outflow

stream

over

the

period

of

h

The

model

based

analysis

described

focuses

on

the

amino

acid

metabolism

as

quantified

by

the

measured

time

series

of

the

concentrations

of

amino

acids

-LRB-

i

see

Table

-RRB-

as

well

as

of

-LRB-

i

-RRB-

and

UREA

-LRB-

i

-RRB-

Each

run

was

labeled

by

Lj

-LCB-

low

medium

high

-RCB-

describing

the

performance

with

respect

to

the

long-term

maintenance

of

the

functionality

of

the

liver

cells

within

the

bioreactor

Seven

runs

were

labeled

high

-LRB-

j

-RRB-

seven

runs

medium

-LRB-

j

-RRB-

and

four

runs

low

-LRB-

j

-RRB-

This

performance

labeling

was

provided

by

an

expert

based

on

his

assessment

of

altogether

variables

that

were

measured

to

quantitatively

characterize

the

system

Differential

equation

system

The

differential

equation

system

-LRB-

-RRB-

was

developed

to

describe

the

measured

kinetics

of

the

amino

acids

as

well

as

of

and

UREA

-LRB-

Table

-RRB-

This

model

takes

into

account

two

compartments

as

described

in

Cells

and

bioreactors

the

perfusion

compartment

and

the

liver

cell

compartment

The

Eq

-LRB-

-RRB-

describe

the

dynamics

of

the

components

i

in

the

perfusion

compartment

by

four

terms

The

first

and

second

term

represent

the

fresh

medium

inflow

with

the

volumetric

rates

FA

-LRB-

t

-RRB-

and

FB

-LRB-

t

-RRB-

respectively

from

the

reservoir

into

the

perfusion

compartment

with

the

volume

The

reservoir

concentrations

cAi

of

the

components

i

are

specified

in

Table

The

volumetric

rates

FA

-LRB-

t

-RRB-

and

FB

-LRB-

t

-RRB-

are

specified

by

Eq

-LRB-

-RRB-

The

third

term

in

Eq

-LRB-

-RRB-

denotes

the

outflow

from

the

perfusion

compartment

into

the

waste

with

the

volumetric

rate

-LRB-

t

-RRB-

The

last

term

describes

the

diffusion

between

the

perfusion

and

the

liver

cell

compartment

with

its

rate

being

proportional

to

the

difference

of

the

concentration

i

in

the

perfusion

compartment

and

the

concentration

ci

in

the

liver

cell

compartment

The

parameter

is

proportional

to

the

diffusion

coefficient

and

assumed

to

be

the

same

for

all

compounds

to

simplify

parameter

identification

The

Eqs

-LRB-

-RRB-

formulate

the

changes

of

the

concentrations

ci

in

the

liver

cell

compartment

with

the

volume

due

to

the

exchange

with

the

perfusion

compartment

-LRB-

the

first

term

on

the

right

side

of

each

differential

equation

-RRB-

and

the

metabolic

reactions

-LRB-

the

following

term

-LRB-

s

-RRB-

on

the

right

side

of

each

differential

equation

-RRB-

as

drawn

schematically

in

Fig.

The

model

drawn

in

Fig.

and

its

Eqs

-LRB-

-RRB-

were

constructed

based

on

the

mean

kinetics

of

measured

variables

averaged

over

the

seven

high-performance

runs

as

shown

in

Fig.

The

kinetics

of

these

seven

individual

runs

are

highly

correlated

with

their

correlation

coefficients

being

greater

than

for

at

least

six

of

the

seven

high-performance

runs

and

the

amino

acids

MET

SER

THR

ARG

HIS

GLY

TRP

LEU

VAL

PHE

and

ILE

-LSB-

-RSB-

The

dynamics

of

these

amino

acids

are

characterized

by

monotonously

almost

exponentially

decreasing

kinetics

-LSB-

-RSB-

Their

dynamics

were

therefore

described

by

first

order

reactions

as

formulated

by

the

second

term

in

Eq

-LRB-

-RRB-

The

observed

decreasing

kinetics

of

amino

acid

concentrations

is

the

net

result

of

anabolic

and

catabolic

reactions

where

the

catabolism

of

amino

acids

-LRB-

which

was

considered

here

only

-RRB-

surpasses

the

amino

acid

synthesis

and

release

by

proteolytic

activities

-LRB-

which

were

neglected

here

-RRB-

The

second

term

with

the

parameters

in

Eq

-LRB-

-RRB-

can

be

interpreted

mainly

as

amino

acid

uptake

by

transamination

and

oxidative

deamination

-LRB-

see

Table

-RRB-

In

addition

to

the

amino

acids

with

highly

correlated

and

monotonously

decreasing

kinetics

the

time

courses

of

the

concentrations

of

the

amino

acids

ALA

LYS

and

TYR

were

for

simplification

also

modeled

using

Eq

-LRB-

-RRB-

The

kinetics

of

the

amino

acids

ASP

ASN

GLU

and

GLN

were

modeled

in

greater

detail

by

Eqs

-LRB-

-RRB-

Glutamate

-LRB-

GLU

-RRB-

is

known

to

be

the

central

compound

of

amino

acid

metabolism

Its

kinetics

were

found

in

-LSB-

-RSB-

to

be

highly

correlated

with

ASP

ASP

was

fed

by

the

inflow

rate

FB

-LRB-

t

-RRB-

-LRB-

see

Cells

and

bioreactors

-RRB-

to

avoid

or

compensate

the

exhaustion

of

ASP

which

is

essential

for

elimination

via

the

urea

cycle

The

degradation

of

amino

acids

is

interpreted

in

a

simplified

form

by

transamination

to

GLU

for

the

amino

acids

LEU

HIS

ARG

VAL

TRP

PHE

ILE

ALA

TYR

and

LYS

-LRB-

denoted

by

AAg

in

Fig.

-RRB-

The

amino

acids

MET

SER

GLY

and

THR

-LRB-

denoted

by

AAn

in

Fig.

-RRB-

however

are

not

transaminated

forming

GLU

but

deaminated

via

different

pathways

forming

The

parameters

with

the

indices

as

shown

in

Fig.

and

Table

describe

the

metabolic

fluxes

between

ASP

ASN

GLU

GLN

and

They

can

be

interpreted

in

a

simplified

way

by

reactions

catalyzed

e.g.

by

the

enzymes

asparaginase

aspartate

aminotransferase

-LRB-

AST

-RRB-

glutamate

oxalacetate

transaminase

-LRB-

GOT

-RRB-

glutamine

synthetase

-LRB-

GS

-RRB-

and

glutamate

dehydrogenase

-LRB-

GLDH

-RRB-

In

general

the

biochemical

reactions

were

assumed

to

be

either

of

linear

or

bilinear

nature

-LRB-

first

or

second

order

-RRB-

In

two

cases

however

additional

assumptions

were

required

to

obtain

sufficient

model

fits

-LRB-

-RRB-

For

the

aggregated

modeling

of

the

elimination

via

the

urea

cycle

in

the

presence

of

ASP

a

non-linear

Michaelis-Menten-type

kinetics

was

used

-LRB-

see

Eq

-RRB-

This

reflects

the

observation

that

the

UREA

formation

rate

does

not

significantly

increase

any

more

at

strongly

elevated

ASP

concentrations

-LRB-

see

ASP

and

UREA

in

Fig.

-RRB-

-LRB-

-RRB-

Protein

synthesis

i.e.

anabolism

seems

to

be

switched

on

or

is

strongly

increased

after

the

third

day

This

was

modeled

using

Eq

-LRB-

-RRB-

The

total

protein

-LRB-

PROT

-RRB-

was

not

measured

but

a

corresponding

variable

was

hypothetically

introduced

by

Eq

-LRB-

-RRB-

to

improve

the

model

fit

of

the

other

variables

to

the

measured

data

for

t

days

Fig.

of

the

model

Eqs

-LRB-

-RRB-

The

numbers

within

the

circles

denote

the

indices

i

of

the

corresponding

concentrations

ci

and

the

numbers

at

the

arrows

denote

the

indices

m

of

the

corresponding

model

parameters

pm

-LRB-

The

diffusion

process

modeled

by

the

parameter

is

not

shown

here

-RRB-

AAg

denotes

the

amino

acids

LEU

HIS

ARG

VAL

TRP

PHE

ILE

ALA

TYR

and

LYS

AAn

denotes

the

amino

acids

MET

SER

GLY

and

THRFig

kinetics

-LRB-

SD

-RRB-

of

measured

variables

-LRB-

amino

acids

UREA

-RRB-

averaged

over

the

kinetics

of

the

n

high

performance

runs

-LRB-

n

-RRB-

Table

pm

of

the

model

-LRB-

-RRB-

with

interpretation

estimated

values

and

confidence

intervals

-LSB-

pmlow

pmhigh

-RSB-

enzyme

activities

AST

aspartate

aminotransferase

GOT

glutamate

oxalacetate

transaminase

GS

glutamine

synthetase

GLDH

glutamate

dehydrogenase

values

and

units

of

the

parameters

pm

of

the

model

-LRB-

-RRB-

as

identified

by

the

model

fit

to

the

mean

kinetics

averaged

over

the

seven

high

performance

runs

-LRB-

Fig.

-RRB-

first

and

second

principal

componentmInterpretation/enzymespm

-LSB-

pmlow

pmhigh

-RSB-

-LSB-

-RSB-

mL

h

Aminotransferases

-LRB-

Transamination

-RRB-

-LSB-

-RSB-

day

-LSB-

-RSB-

day

-LSB-

-RSB-

day

-LSB-

-RSB-

day

-LSB-

-RSB-

day

-LSB-

-RSB-

day

-LSB-

-RSB-

day

-LSB-

-RSB-

day

-LSB-

-RSB-

day

-LSB-

-RSB-

day

Other

specific

-LSB-

-RSB-

day

-LSB-

-RSB-

day

-LSB-

-RSB-

day

-LSB-

-RSB-

day

-LSB-

-RSB-

day

μmol

-LSB-

-RSB-

day

-LSB-

-RSB-

day

-LSB-

-RSB-

day

Urea

Cycle

Km

-LRB-

ASP

-RRB-

-LSB-

-RSB-

day

μmol

-LSB-

-RSB-

μmol

L

-LSB-

-RSB-

day

μmol

-LSB-

-RSB-

day

GLDH

Protein

-LSB-

-RSB-

day

-LSB-

-RSB-

The

model

parameters

were

identified

by

fitting

the

model

to

the

measured

data

ci

j

k

minimizing

the

scaled

mean

square

error

-LRB-

mse

-RRB-

as

defined

by

Eq

-LRB-

-RRB-

for

run

j

The

model

fitting

error

mse

was

scaled

by

the

square

of

the

maximum

of

the

respective

measured

variable

According

to

Eq

-LRB-

-RRB-

the

kinetics

i

-LRB-

t

-RRB-

obtained

from

the

simulation

of

Eqs

-LRB-

-RRB-

with

the

initial

values

listed

in

Table

were

averaged

over

h

-LRB-

i.e.

over

the

time

interval

of

the

accumulation

of

the

bioreactor

outflow

in

the

waste

where

the

samples

for

the

measurements

were

taken

from

daily

-RRB-

and

then

compared

with

the

measured

data

ci

j

k

The

differential

equations

were

solved

using

a

Runge-Kutta

fourth

order

algorithm

The

parameter

identification

by

model

fitting

minimizing

the

mse

was

performed

using

a

simplex

search

method

MATLAB

tools

-LRB-

The

MathWorks

Inc.

Natick

MA

USA

-RRB-

were

used

for

all

calculations

Results

and

discussions

The

mean

time

profiles

of

the

measured

variables

each

averaged

over

the

high

performance

runs

are

shown

in

Fig.

The

results

of

the

model

fitting

to

these

averaged

data

are

shown

in

Fig.

and

the

identified

model

parameters

are

listed

in

Table

-LRB-

third

column

-RRB-

The

confidence

intervals

for

the

parameter

values

-LRB-

fourth

and

fifth

column

in

Table

-RRB-

were

identified

by

repeated

model

fitting

to

the

randomly

disturbed

time

series

data

with

means

and

standard

deviations

as

shown

in

Fig.

The

fit

of

the

modeled

kinetics

to

the

measured

data

is

acceptable

for

of

the

variables

with

the

exception

of

lysine

-LRB-

LYS

-RRB-

and

alanine

-LRB-

ALA

-RRB-

The

initial

increase

of

the

LYS

kinetics

could

be

hypothetically

explained

by

proteolytic

activities

The

final

increase

of

ALA

can

be

explained

by

the

ALA

aspartate

transferase

activity

The

decreasing

kinetics

of

out

of

the

amino

acids

is

explained

by

transamination

and

oxidative

deamination

forming

GLU

and

The

feeding

of

ASP

after

the

third

day

results

not

only

in

an

increase

of

the

concentration

of

ASP

but

also

of

asparagine

-LRB-

ASN

-RRB-

GLU

and

glutamine

-LRB-

GLN

-RRB-

due

to

the

activities

of

the

enzymes

AST

GOT

GS

and

asparaginase

-LRB-

alternatively

or

additionally

the

activity

of

asparagine

synthetase

could

be

included

in

the

model

-RRB-

Fig.

and

simulated

kinetics

of

the

concentrations

of

amino

acids

and

UREA

The

measured

kinetics

-LRB-

dots

-RRB-

are

those

of

the

mean

profiles

shown

in

Fig.

-LRB-

n

-RRB-

The

simulated

kinetics

-LRB-

t

-RRB-

-LRB-

thin

lines

-RRB-

are

those

obtained

from

the

model

-LRB-

-RRB-

The

simulated

kinetics

-LRB-

t

-RRB-

were

averaged

according

to

Eq

-LRB-

-RRB-

over

the

past

h

-LRB-

thick

lines

-RRB-

in

order

to

use

them

for

model

fitting

to

the

measured

data

that

were

acquired

from

the

waste

-LRB-

outflow

accumulated

over

h

-RRB-

Table

lists

the

model

parameter

values

pm

with

the

respective

model

fitting

error

mse

as

identified

by

individual

model

fitting

to

the

seven

high

performance

runs

Figs.

show

the

results

of

the

model

fitting

to

the

data

for

the

individual

high

performance

runs

and

respectively

The

kinetics

of

run

-LRB-

not

shown

here

-RRB-

is

very

similar

to

the

mean

kinetics

of

the

seven

high

performance

runs

shown

in

Fig.

The

quasi-stationary

concentrations

of

several

amino

acids

such

as

MET

and

LEU

are

lower

in

run

-LRB-

see

Fig.

-RRB-

than

in

run

-LRB-

compare

Fig.

-RRB-

This

results

in

a

value

which

is

about

higher

for

run

than

for

run

-LRB-

see

Table

-RRB-

While

the

variability

of

the

parameter

values

can

be

explained

by

differences

in

the

liver

cell

material

obtained

from

different

individual

donors

-LRB-

having

different

age

weight

liver

damage

etc.

-RRB-

it

is

not

entirely

clear

at

this

stage

how

to

explain

such

different

values

of

the

parameter

that

represents

diffusion

and

potentially

other

phenomena

taking

place

across

and

beyond

the

membrane

between

the

perfusion

and

the

liver

cell

compartment

These

issues

are

addressed

in

greater

detail

in

-LSB-

-RSB-

as

well

as

in

studies

described

in

-LSB-

-RSB-

to

elucidate

the

dynamics

of

electrolyte

distribution

in

the

bioreactor

without

liver

cells

using

tracer

experiments

and

input/output

systems

analysis.Table

of

the

parameters

pm

of

the

model

-LRB-

-RRB-

with

the

values

of

the

scaled

mean

square

error

-LRB-

mse

-RRB-

as

identified

by

individual

model

fits

to

the

seven

high

performance

runs

.01620.01430.02020.01150.01350.01450.0321

Fig.

data

of

the

variables

of

bioreactor

run

with

the

simulated

kinetics

of

the

model

fitted

to

the

data

of

and

simulated

kinetics

as

in

Fig.

but

for

run

The

model

which

was

developed

using

the

kinetic

patterns

of

high

performance

runs

was

also

fitted

to

the

data

of

the

runs

with

medium

and

low

performance

The

aim

of

these

model

studies

is

to

elucidate

causes

of

low

liver

cell

bioreactor

performance

The

fit

of

the

model

with

parameters

to

bioreactor

runs

results

in

an

array

of

parameters

Three

selected

parameters

with

their

values

as

identified

by

fitting

the

model

to

the

individual

runs

are

listed

in

Table

Considering

a

tolerable

model

fitting

error

mse

of

about

or

less

the

model

fit

proved

satisfactory

for

six

of

the

seven

high

performance

runs

-LRB-

with

the

exception

of

run

-RRB-

and

for

two

of

the

seven

medium

performance

runs

-LRB-

and

-RRB-

However

the

model

fit

was

not

acceptable

for

the

high

performance

run

the

medium

performance

runs

and

as

well

as

all

four

low

performance

runs

The

main

reason

for

the

insufficient

model

fits

is

that

the

measured

kinetics

of

leucine

-LRB-

LEU

-RRB-

isoleucine

-LRB-

ILE

-RRB-

valine

-LRB-

VAL

-RRB-

ALA

and

other

amino

acids

are

not

decreasing

in

these

runs

This

behavior

is

shown

in

Fig.

for

run

In

these

cases

some

parameter

values

e.g.

for

were

found

to

be

zero

-LRB-

negative

parameter

values

were

set

to

zero

see

Table

-RRB-

These

zero

parameter

values

cause

that

the

medium

and

low

performance

runs

displayed

in

Fig.

are

artificially

close

to

each

other

Run

labeled

as

a

high

performance

one

appears

to

be

a

medium

performance

run

according

to

the

model

simulation

results

-LRB-

see

Figs.

and

-RRB-

Table

for

the

liver

cell

bioreactor

runs

tB

as

used

in

Eq

-LRB-

-RRB-

the

star

-LRB-

-RRB-

for

run

denotes

that

the

feed

FB

-LRB-

t

-RRB-

was

switched

on

during

the

period

from

the

second

to

the

fourth

day

the

scaled

mean

square

error

-LRB-

mse

-RRB-

and

the

parameters

and

as

used

in

Eq

-LRB-

-RRB-

were

identified

by

model

fitting

to

the

data

of

the

high

performance

runs

-LRB-

averaged

-RRB-

and

of

the

individual

high

performance

runs

medium

performance

runs

and

low

performance

runs

No.tB

-LSB-

d

-RSB-

mse

-LSB-

-RSB-

-LSB-

d

-RSB-

-LSB-

d

-RSB-

-LSB-

d

-RSB-

-LRB-

averaged

-RRB-

Fig.

and

simulated

kinetics

as

in

Fig.

but

for

run

of

the

first

and

second

principal

components

of

the

model

parameter

matrix

-LCB-

-RCB-

as

identified

for

the

liver

cell

bioreactor

runs

-LRB-

high

performance

runs

medium

performance

runs

low

performance

runs

-RRB-

Figure

displays

the

identified

model

parameters

of

the

liver

cell

bioreactor

runs

after

principal

component

analysis

-LRB-

PCA

-RRB-

of

the

parameter

array

for

the

first

and

second

principal

components

in

a

biplot

-LSB-

-RSB-

The

first

principal

component

represents

of

the

total

parameter

variance

and

the

first

and

second

principal

components

together

represent

almost

of

this

variance

Most

of

the

parameters

-LRB-

with

the

exception

of

-RRB-

representing

transamination

and

oxidative

deamination

activities

mainly

constitute

the

first

principal

component

of

the

parameter

array

-LRB-

see

Table

for

these

parameters

-RRB-

These

parameters

are

correlated

The

correlation

coefficients

calculated

between

the

parameters

estimated

over

the

runs

have

values

between

and

with

the

median

of

and

the

mean

of

The

values

of

these

parameters

are

high

for

six

of

the

seven

high

performance

runs

and

low

for

run

as

well

as

for

the

medium

and

low

performance

runs

as

shown

in

Table

for

parameter

The

value

of

the

parameter

is

strongly

correlated

with

the

bioreactor

performance

-LRB-

p-value

by

the

two-sided

t-test

of

pm

values

of

high

versus

medium

or

low

performance

runs

-RRB-

The

mean

value

of

the

parameter

averaged

over

the

high

performance

runs

equals

-LRB-

-RRB-

The

parameter

is

zero

for

all

low

performance

runs

In

Fig.

the

medium

and

low

performance

runs

are

displayed

in

the

left

part

describing

low

transamination

and

oxidative

deamination

activities

whereas

six

of

the

seven

high

performance

runs

-LRB-

with

the

exception

of

run

-RRB-

are

displayed

in

the

right

part

representing

high

transamination

and

oxidative

deamination

activities

Some

parameters

are

highly

correlated

over

the

runs

with

a

correlation

coefficient

r

of

more

than

e.g.

the

parameters

and

-LRB-

representing

the

catabolism

of

methionine

and

glycine

-RRB-

as

well

as

and

Also

the

parameter

is

highly

correlated

-LRB-

r

-RRB-

with

the

parameters

and

The

high

correlation

of

the

parameters

and

representing

the

transamination

of

LEU

ILE

and

VAL

can

be

hypothetically

explained

by

the

fact

that

LEU

ILE

and

VAL

are

branched-chain

amino

acids

which

can

not

be

transaminated

in

hepatocytes

To

transaminate

these

amino

acids

the

activity

of

non-parenchymal

liver

cells

is

required

Among

the

high

performance

runs

there

are

further

individual

differences

that

are

displayed

in

Fig.

by

a

low

second

principal

component

-LRB-

e.g.

high

values

of

the

parameters

and

for

run

-RRB-

and

a

high

-LRB-

e.g.

low

values

of

the

parameters

and

for

runs

and

-RRB-

The

parameters

and

represent

the

uptake

of

histidine

and

glutamine

-LRB-

see

Table

-RRB-

As

shown

in

Fig.

for

run

the

high

parameter

value

results

in

a

low

stationary

concentration

of

HIS

and

the

high

parameter

value

results

in

high

and

low

GLU

levels

These

effects

could

be

related

to

the

amino

acid

transport

system

N

-LRB-

-RRB-

that

mediates

specifically

the

uptake

of

histidine

and

glutamine

-LSB-

-RSB-

The

parameter

that

represents

the

activity

of

GS

was

found

to

be

very

low

for

the

high

performance

runs

and

-LRB-

see

Table

-RRB-

GS

plays

an

important

role

in

the

spatial

organization

-LRB-

zonation

-RRB-

of

the

liver

and

is

exclusively

expressed

in

pericentrally

located

hepatocytes

-LSB-

-RSB-

The

low

GS

activity

in

the

high

performance

runs

and

could

therefore

be

caused

by

a

high

proportion

of

periportal

versus

pericentral

hepatocytes

in

the

cell

preparations

for

these

two

runs

Conclusion

The

kinetics

of

amino

acids

and

the

related

nitrogen-containing

compounds

and

UREA

in

a

primary

human

liver

cell

bioreactor

were

analyzed

and

modeled

using

a

differential

equation

system

The

model

focuses

on

the

kinetics

of

GLU

and

ASP

as

well

as

on

the

formation

and

elimination

of

and

the

synthesis

of

UREA

It

describes

the

degradation

of

amino

acids

by

transamination

oxidative

deamination

and

other

specific

reactions

In

addition

the

activities

of

selected

enzymes

such

as

AST

GOT

GS

and

GLDH

as

well

as

in

a

more

aggregated

form

the

activities

of

urea

cycle

enzymes

are

included

The

differential

equation

system

does

not

represent

a

fully

mechanistic

but

rather

a

phenomenological

model

since

essential

metabolic

activities

had

to

be

neglected

because

they

can

not

be

identified

based

on

the

measured

data

The

differential

equation

system

allows

the

analysis

of

a

number

of

representative

liver

cell

functions

in

terms

of

their

kinetic

behavior

The

identification

of

the

model

parameters

by

fitting

the

model

responses

to

the

measured

data

was

used

to

generate

hypotheses

about

the

causes

of

specific

differences

between

the

bioreactor

runs

The

model

fits

were

found

to

be

very

satisfactory

for

eight

high

and

medium

performance

runs

The

model

is

however

inadequate

for

low

performance

runs

and

with

respect

to

the

LYS

kinetics

also

for

high

performance

runs

This

is

probably

caused

by

the

neglection

of

proteases

activities

in

the

model

that

appear

to

be

relevant

for

low

and

medium

performance

runs

Both

protein

synthesis

and

degradation

could

not

be

modeled

in

detail

due

to

the

lack

of

representative

protein

measurements

The

applied

model

based

analysis

of

data

obtained

from

the

bioreactor

system

can

be

used

to

quantitatively

evaluate

the

functional

state

of

liver

cell

cultures

under

high

performance

conditions

intended

for

clinical

application

in

extracorporeal

liver

support

systems

The

approach

can

also

be

used

to

study

the

effect

of

several

exogenous

factors

e.g.

of

hormones

or

drugs

on

hepatocyte

metabolism

in

vitro

The

model

based

analysis

methods

applied

here

therefore

provide

suitable

tools

for

in

silico

studies

supplementing

in

vitro

studies

of

hepatocyte

functions

in

a

systems

biological

way

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


Metinel

Node

The

First

Lymph

Node

Draining

a

Metastasis

Contains

Tumor-Reactive

Lymphocytes

Background

We

previously

identified

tumor-reactive

lymphocytes

in

the

first

lymph

nodes

that

drain

the

primary

tumor

In

this

study

we

performed

lymphatic

mapping

to

investigate

the

possibility

of

finding

the

first

lymph

nodes

that

drain

metastases

and

of

learning

whether

these

lymph

nodes

contained

tumor-reactive

lymphocytes

suitable

for

adoptive

immunotherapy

The

global

cancer

registry

Globocan

shows

million

new

incident

cases

and

million

deaths

worldwide

due

to

cancer

in

A

total

of

million

persons

are

estimated

to

be

alive

with

cancer

-LRB-

within

years

of

diagnosis

-RRB-

Leading

causes

of

death

from

cancer

in

order

are

those

of

the

lung

-LRB-

million

-RRB-

stomach

-LRB-

-RRB-

and

liver

-LRB-

-RRB-

Most

of

these

deaths

were

caused

by

metastatic

disease

and

they

verify

the

fact

that

surgery

and

oncological

therapy

do

not

cure

most

patients

with

disseminated

cancer

The

incidence

of

cancer

will

continue

to

increase

especially

in

developing

countries

largely

due

to

smoking

infections

and

lifestyles

resembling

those

of

people

living

in

industrialized

countries

Thus

intensified

efforts

are

warranted

both

to

prevent

cancer

and

to

find

new

modalities

of

treating

patients

with

metastatic

cancer

Tumor

cells

spread

by

lymphogenous

and

hematogenous

routes

into

the

systemic

circulation

to

disseminate

the

disease

The

importance

of

tumor

lymphangiogenesis

has

lately

been

proven

in

studies

where

high

levels

in

colorectal

cancer

specimens

of

lymphangiogenic

peptides

and

vascular

endothelial

growth

factor

-LRB-

VEGF

-RRB-

C

and/or

VEGF-D

promote

tumor

lymphangiogenesis

and

metastasis

The

sentinel

node

concept

implies

that

the

lymphatic

drainage

from

a

primary

tumor

first

drains

to

a

certain

locoregional

lymph

node

specific

for

each

individual

and

that

its

tumor

status

is

representative

for

the

entire

lymphatic

field

The

concept

is

established

in

staging

for

breast

and

malignant

and

has

also

been

evaluated

in

several

other

types

of

malignancies

such

as

colon

cancer

ovarian

cancer

pancreatic

cancer

and

urinary

bladder

cancer

The

technique

has

been

used

as

a

diagnostic

tool

to

improve

staging

and

to

tailor

the

extent

of

regional

lymph

node

dissections

Probably

most

solid

tumors

disseminate

first

through

the

lymphatics

and

thereafter

enter

the

systemic

circulation

via

lymphovenous

shunts

present

in

the

first

draining

lymph

nodes

Approximately

half

of

the

lymphatic

fluid

entering

a

lymph

node

continues

directly

into

the

systemic

circulation

therefore

lymphogenic

spreading

of

the

tumor

cells

may

be

mandatory

for

hematogenic

dissemination

However

to

our

knowledge

only

one

study

has

previously

evaluated

lymphatic

mapping

in

metastases

where

liver

metastases

were

investigated

Because

it

has

been

proven

that

both

metastases

and

primary

tumors

have

the

capacity

to

and

induce

lymphangiogenesis

we

assume

that

lymphatic

drainage

from

metastases

may

have

an

equal

importance

in

metastases

as

the

sentinel

node

concept

in

many

solid

primary

tumors

for

correct

staging

We

have

previously

experienced

surprises

in

lymphatic

mapping

within

patients

with

colon

and

urinary

bladder

regarding

primary

tumor

lymphatic

drainage

We

believe

that

a

similar

technique

may

be

of

use

in

surgery

for

liver

metastases

and

other

solid

metastatic

tumors

for

correct

staging

and

thereby

for

giving

the

patients

the

most

appropriate

therapy

We

previously

showed

that

a

sentinel

node

derived

from

a

primary

tumor

contains

specific

tumor-reactive

lymphocytes

that

have

immunological

reactivity

toward

the

tumor

in

patients

with

colon

and

in

patients

with

urinary

bladder

cancer

Through

the

injection

of

Patent

blue

dye

or

radioactive

tracer

subserosally

during

surgery

around

the

colonic

tumor

or

transurethrally

around

the

urinary

bladder

tumor

the

sentinel

nodes

were

found

A

biopsy

was

performed

and

the

nodes

immunological

role

and

function

were

studied

After

preparation

the

lymphocytes

were

identified

isolated

and

proliferated

in

vitro

by

stimulation

of

interleukins

-LRB-

ILs

-RRB-

and

tumor

antigen

Lymphocytes

from

sentinel

nodes

proliferated

dose

dependently

and

secreted

interferon

-LRB-

IFN

-RRB-

γ

on

stimulation

with

tumor

homogenate

The

aim

of

the

present

study

was

to

investigate

lymphatic

drainage

from

metastases

and

identify

first

draining

lymph

nodes

We

also

analyzed

tumor-reactive

immunological

properties

in

lymphocytes

derived

from

these

lymph

nodes

MATERIALS

AND

METHODS

Patients

Nineteen

patients

-LRB-

men

and

women

average

age

years

-RRB-

with

metastases

from

solid

tumors

were

included

in

the

study

Patient

characteristics

are

shown

in

Table

The

operations

were

performed

during

the

period

of

November

to

January

The

study

was

approved

by

the

local

ethical

committee

and

each

patient

provided

informed

consent

Table

Patient

characteristicsaPatientAge

-LRB-

y

-RRB-

SexPrimary

tumorOrigin

metinel

nodeTracerNumber

of

metinel

nodesMetinel

node

pos/neg

for

metastasesSuccessful

expansionAliveFollow-up

time

-LRB-

mo

-RRB-

local

melanomaSubcutaneous

posNoNo

negNoNo

posYesNo

cancerLiver

posYesbNo

cancerGroin

lymph

posNoNo

cancerIntra-abdominal

local

melanomaGroin

lymph

melanomaTruncal

posNoNo

cancerCervical

lymph

cancerTruncal

lymph

local

cancerLiver

melanomaCervical

lymph

negYesNo

positive

neg

negative

Mal

malignant

PB

Patent

blue

dye

MI

missing

information

RT

radioactive

tracer

-LRB-

MBq

Tc-nanokolloid

-RRB-

a

Data

regarding

the

expansions

for

patients

and

also

exist

in

unpublished

data.b

Patient

died

on

the

projected

day

of

transfusion

The

lymph

nodes

that

drained

metastases

were

identified

by

injecting

approximately

mL

Patent

blue

dye

-LRB-

PB

-RRB-

-LRB-

Guerbet

Paris

-RRB-

subserosally

or

subcutaneously

in

four

places

around

one

of

the

metastasis

or

in

the

tissues

close

to

the

local

recurrences

with

a

needle

After

the

injection

of

PB

the

first

draining

lymph

nodes

turned

blue

within

to

minutes

and

were

regarded

as

metastases-draining

lymph

nodes

In

our

previous

work

a

cutoff

was

sufficient

to

identify

sentinel

nodes

to

primary

tumors

We

have

experienced

that

during

this

period

of

time

the

first

draining

lymph

nodes

turn

blue

Waiting

longer

means

that

a

second

echelon

of

draining

lymph

nodes

may

be

colored

blue

which

does

not

represent

the

first

draining

lymph

nodes

We

arbitrarily

applied

the

same

limit

when

detecting

lymph

nodes

that

drain

metastases

These

lymph

nodes

were

either

immediately

removed

or

marked

with

sutures

The

sources

of

the

liver

metastases

in

our

study

were

from

colorectal

cancer

in

five

patients

and

in

one

patient

each

from

ovarian

cancer

breast

cancer

cholangiocarcinoma

and

leiomyosarcoma

Four

patients

had

subcutaneous

lymph

node

metastases

from

disseminated

malignant

melanoma

and

one

patient

each

had

metastases

from

ovarian

cancer

breast

cancer

and

squamous

cell

carcinoma

of

the

tongue

Two

patients

had

intra-abdominal

local

recurrences

after

surgery

for

colon

cancer

and

one

patient

had

an

intra-abdominal

local

recurrence

after

surgery

for

pancreatic

cancer

Because

of

these

liver

metastases

five

patients

underwent

partial

right-sided

liver

resections

-LRB-

patients

and

-RRB-

and

three

patients

underwent

partial

left-sided

liver

resection

-LRB-

patients

and

-RRB-

The

patient

with

metastases

resulting

from

ovarian

cancer

-LRB-

patient

-RRB-

had

several

large

bulky

cystic

tumors

and

surgeons

performed

an

almost

complete

right-sided

liver

resection

In

four

cases

surgery

was

performed

for

solitary

colorectal

liver

metastases

-LRB-

patients

and

-RRB-

and

in

the

remaining

five

cases

tumor-reducing

surgery

was

performed

In

six

cases

a

preoperative

lymphoscintigraphy

was

performed

by

a

subcutaneous

injection

of

to

MBq

Tc-nanokolloid

radioactive

tracer

in

quadrants

around

the

metastasis

The

lymphoscintigraphies

were

performed

to

plan

the

surgical

procedure

by

localizing

the

lymph

node

or

nodes

draining

the

metastasis

The

position

of

the

metastasis-draining

lymph

node

or

nodes

was

marked

on

the

skin

with

a

felt-tip

pen

When

the

lymphoscintigraphy

was

not

performed

on

the

day

of

surgery

a

new

peritumoral

injection

of

radioactive

tracer

was

made

at

start

of

surgery

The

draining

nodes

were

intraoperatively

identified

by

the

use

of

a

handheld

gamma

detection

tube

In

four

of

six

cases

a

simultaneous

intraoperative

injection

of

PB

was

performed

Preparation

of

Specimens

Lymph

nodes

draining

metastases

and

nondraining

lymph

nodes

-LRB-

for

analytical

purposes

-RRB-

were

surgically

removed

and

immediately

taken

to

the

laboratory

on

ice

In

addition

one

slice

of

the

whole

metastasis

-LRB-

including

the

invasive

margin

-RRB-

was

also

dissected

and

sent

for

analysis

Frequently

most

of

the

lymph

node

or

nodes

were

used

for

the

expansion

procedure

The

remaining

specimens

underwent

routine

histopathological

examinations

with

hematoxylin

and

eosin

staining

At

the

laboratory

slices

mm

were

cut

from

the

central

and

peripheral

part

of

the

lymph

nodes

for

analysis

by

flow

cytometry

-LRB-

FACS

-RRB-

and

proliferation

analyses

The

metastasis

was

also

analyzed

by

FACS

and

a

preparation

of

the

whole

tumor

-LRB-

homogenate

-RRB-

was

used

Immunological

Evaluation

Single

cell

suspensions

were

made

from

the

lymph

nodes

and

metastases

by

gentle

pressure

with

a

loose-fitting

glass

homogenizer

as

previously

described

Briefly

cells

were

washed

in

phosphate-buffered

saline

containing

fetal

calf

serum

and

then

stained

with

fluorophore

conjugated

antibodies

toward

the

cell

surface

markers

and

-LRB-

Becton

Dickinson

-RRB-

Thereafter

the

cells

were

investigated

with

a

FACSCalibur

-LRB-

Becton

Dickinson

-RRB-

For

cell

cultures

single

cell

suspensions

of

metastasis-draining

lymph

node

derived

cells

were

resuspended

in

RPMI

proliferation

media

containing

human

AB

serum

-LRB-

Sigma

-RRB-

penicillin-streptomycin

-LRB-

Sigma

-RRB-

and

glutamine

-LRB-

Sigma

-RRB-

For

preparation

of

the

antigen

source

the

metastasis

was

homogenized

by

an

Ultra-turrax

homogenizer

in

volumes

-LRB-

w/v

-RRB-

of

phosphate-buffered

saline

followed

by

minutes

denaturation

at

°C

This

tumor

homogenate

was

then

used

for

in

vitro

activation

and

clonal

expansion

of

lymphocytes

The

metastasis-draining

lymph

node

derived

lymphocytes

were

finally

dispersed

into

cell

culture

flasks

at

cells/mL

of

proliferation

media

supplemented

with

IU

of

-LRB-

Proleukin

Chiron

-RRB-

per

milliliter

of

culture

every

to

days

Tumor

homogenate

was

diluted

to

and

and

added

to

the

cell

cultures

after

to

days

After

to

weeks

the

cell

cultures

were

restimulated

by

irradiated

peripheral

blood

mononuclear

cells

from

the

patient

These

cells

were

collected

by

Ficoll-Hypaque

and

used

as

antigen-presenting

cells

for

restimulation

together

with

further

addition

of

tumor

homogenate

To

verify

clonal

expansion

of

T

lymphocytes

Vβ

repertoire

analyses

were

performed

in

a

few

cases

To

test

for

functionality

single

cell

suspensions

were

investigated

for

and

cytokine

production

IFN-γ

and

After

an

average

period

of

weeks

expanded

cells

were

counted

evaluated

by

FACS

and

investigated

for

the

exclusion

of

malignant

cells

and

bacteria

before

they

were

considered

ready

for

transfusion

Cells

were

washed

three

times

in

saline

solution

-LRB-

Natriumklorid

Baxter

Viaflo

mg/mL

Baxter

-RRB-

containing

human

serum

albumin

-LRB-

Baxter

-RRB-

and

prepared

in

a

sterile

erythrocyte

transfusion

bag

at

cells/mL

The

transfusions

took

place

at

the

surgical

ward

as

an

intravenous

transfusion

over

the

course

of

hour

Our

criterion

for

adoptive

transfer

is

a

transfusion

of

lymphocytes

to

a

patient

regardless

of

the

number

of

transferred

cells

In

our

previous

studies

-LRB-

M.K.

unpublished

data

-RRB-

of

adoptive

transfer

originating

from

sentinel

nodes

on

average

million

clonally

expanded

autologous

tumor-reactive

lymphocytes

were

transfused

to

each

patient

and

in

that

pilot

study

the

disease

of

all

patients

responded

to

therapy

We

do

not

know

the

optimal

number

of

cells

to

be

transfused

for

the

best

or

maximum

immune

response

but

according

to

our

experience

million

transfused

cells

-LRB-

the

average

number

of

transfused

cells

in

the

metinel

node

study

-RRB-

may

be

sufficient

The

explanation

may

be

that

the

cells

proliferate

and

promote

division

of

effector

T

cells

and

memory

T

cells

when

they

are

stimulated

by

their

antigen

-LRB-

which

is

derived

from

the

tumor

-RRB-

The

cell

expansion

continues

in

the

patient

RESULTS

Metinel

Nodes

Lymph

nodes

draining

liver

metastases

intra-abdominal

recurrences

and

subcutaneous

metastases

were

identified

in

all

cases

with

an

average

number

of

nodes

found

per

patient

-LRB-

range

-RRB-

-LRB-

Table

-RRB-

We

denoted

these

metastasis-draining

lymph

nodes

metinel

nodes

Seventeen

-LRB-

-RRB-

of

the

analyzed

metinel

nodes

were

positive

for

metastatic

disease

In

-LRB-

-RRB-

of

patients

where

data

were

available

all

metinel

nodes

were

positive

for

metastatic

disease

-LRB-

range

-RRB-

and

in

-LRB-

-RRB-

of

patients

all

metinel

nodes

-LRB-

range

-RRB-

were

negative

for

metastatic

disease

Tracer

was

injected

around

liver

metastases

-LRB-

n

-RRB-

intra-abdominal

recurrences

-LRB-

n

-RRB-

or

close

to

subcutaneous

metastases

-LRB-

n

-RRB-

Draining

metinel

nodes

appeared

visually

blue

-LRB-

Fig.

-RRB-

within

to

minutes

after

the

injection

of

PB

or

were

found

to

be

disease

positive

by

lymphoscintigraphy

-LRB-

Fig.

-RRB-

No

complications

due

to

the

lymph

node

mapping

procedure

were

observed

The

method

seems

valid

for

detecting

the

draining

metinel

node

regardless

of

tumor

type

because

the

following

were

identified

by

this

method

colorectal

cancer

-LRB-

n

-RRB-

malignant

melanoma

-LRB-

n

-RRB-

ovarian

cancer

-LRB-

n

-RRB-

breast

cancer

-LRB-

n

-RRB-

pancreatic

cancer

-LRB-

n

-RRB-

squamous

cell

cancer

-LRB-

n

-RRB-

cholangiocarcinoma

-LRB-

n

-RRB-

and

leiomyosarcoma

-LRB-

n

-RRB-

Thus

we

conclude

that

lymph

nodes

draining

liver

metastases

or

local

recurrences

can

readily

be

identified

by

this

simple

safe

and

quick

procedure

Fig.

-LRB-

A

-RRB-

After

intraoperative

injection

of

Patent

blue

dye

around

liver

metastasis

in

patient

the

metinel

node

in

the

hepatoduodenal

ligament

is

blue

-LRB-

B

-RRB-

Preoperative

percutaneous

injection

of

MBq

Tc-nanokolloid

around

ovarian

cancer

groin

local

recurrence

-LRB-

patient

-RRB-

demonstrates

a

medially

and

distally

situated

metinel

node

after

minutes

In

all

cases

of

surgery

for

liver

metastases

lymph

nodes

draining

the

area

of

the

metastases

were

found

within

the

liver

hilum

or

hepatoduodenal

ligament

The

average

number

of

metinel

nodes

from

liver

metastases

were

and

-LRB-

-RRB-

of

of

the

analyzed

metinel

nodes

were

positive

for

metastatic

disease

Two

patients

underwent

surgery

to

treat

intra-abdominal

local

recurrences

from

colon

cancer

-LRB-

patients

and

-RRB-

resections

of

the

recurrence

and

reresection

of

the

bowel

en

bloc

were

performed

One

patient

with

pancreatic

cancer

-LRB-

patient

-RRB-

underwent

tumor-reducing

surgery

to

treat

a

local

intra-abdominal

recurrence

after

a

previous

Whipple

operation

Four

patients

underwent

surgery

with

lymph

node

biopsies

performed

to

treat

subcutaneous

metastases

from

disseminated

malignant

melanoma

-LRB-

patients

and

-RRB-

and

one

patient

each

underwent

surgery

to

treat

subcutaneous

metastases

from

disseminated

breast

cancer

-LRB-

patient

-RRB-

groin

lymph

node

metastasis

due

to

ovarian

cancer

-LRB-

patient

-RRB-

and

subcutaneous

metastases

related

to

squamous

cellular

carcinoma

of

the

tongue

-LRB-

patient

-RRB-

-LRB-

Table

-RRB-

Immunology

We

previously

demonstrated

the

presence

of

tumor-reactive

T

lymphocytes

in

primary

tumor-draining

sentinel

nodes

from

patients

with

and

urinary

bladder

cancers

Here

we

investigated

whether

signs

of

immune

recognition

also

occurred

in

metinel

nodes

draining

metastases

Single

cell

suspensions

were

collected

from

identified

metinel

nodes

and

investigated

by

flow

cytometry

for

the

presence

of

T

lymphocytes

B

lymphocytes

and

natural

killer

-LRB-

NK

-RRB-

cells

-LRB-

Table

-RRB-

Most

of

the

metinel

nodes

were

predominated

by

T

helper

cells

with

an

average

ratio

of

-LRB-

range

-RRB-

However

five

patients

-LRB-

patients

and

-RRB-

displayed

an

increase

in

the

fraction

of

cytotoxic

T

cells

Two

of

these

patients

had

malignant

melanoma

The

metinel

nodes

contained

an

average

of

-LRB-

range

-RRB-

B

lymphocytes

and

an

average

of

-LRB-

range

-RRB-

NK

cells

Table

Immunology

of

single

cell

suspensions

collected

from

metinel

nodesPatientTime

in

culture

-LRB-

d

-RRB-

No

of

cells

at

start

-LRB-

millions

-RRB-

No

of

cells

at

end

-LRB-

millions

-RRB-

Ratio

of

cells

start/endStart

-LRB-

gated

-RRB-

ratio

and

ratio

and

startRatio

and

and

and

and

and

and

and

and

and

and

and

and

and

and

and

and

and

and

and

and

and

cells

are

crucial

for

initializing

both

the

cellular

and

humoral

immune

response

toward

for

example

tumors

cells

recognize

antigens

expressed

on

major

histocompatibility

complex

II

and

have

the

capacity

to

catalyze

the

immune

response

in

B

cells

Antigen

presenting

cells

-LRB-

APCs

-RRB-

and

cells

cells

have

compared

with

cells

a

long-term

capacity

of

acting

both

as

effector

T

cells

and

as

memory

cells

cells

are

mainly

effector

cells

and

will

act

during

a

few

weeks

but

further

recruitment

of

CD

cells

is

maintained

by

cells

The

expansion

procedure

aims

to

increase

the

number

of

cells

and

the

ratio

can

be

regarded

as

a

measurement

of

how

well

this

can

be

achieved

The

average

ratio

of

cells

at

the

start

of

the

procedure

was

for

patients

who

underwent

successful

expansions

and

for

patients

who

underwent

unsuccessful

expansions

Consequently

the

ratios

were

higher

for

patients

with

successful

expansions

To

test

for

recognition

of

tumor

antigen

the

number

of

metinel

node

acquired

lymphocytes

from

nine

patients

-LRB-

patients

and

-RRB-

were

increased

successfully

by

the

addition

of

low

dose

and

tumor

homogenate

An

average

of

cells

-LRB-

range

-RRB-

were

collected

and

the

cells

were

expanded

on

average

for

days

before

transfusion

At

the

time

of

transfusion

a

mean

of

tumor-reactive

T

cells

-LRB-

range

-RRB-

were

returned

-LRB-

Table

-RRB-

In

patient

cells

were

expanded

and

prepared

for

transfusion

but

the

patient

suddenly

died

without

receiving

any

cells

No

side

effects

from

transfusion

of

expanded

T

lymphocytes

were

seen

and

the

patients

were

discharged

from

the

hospital

on

the

same

day

of

the

transfusion

The

expansions

were

successful

in

five

of

seven

patients

who

had

colorectal

primary

tumor

one

of

two

in

patients

with

ovarian

cancer

and

breast

cancer

and

one

patient

each

with

cholangiocarcinoma

and

leiomyosarcoma

The

following

expansions

failed

in

all

patients

malignant

melanoma

-LRB-

n

-RRB-

pancreatic

cancer

-LRB-

n

-RRB-

and

squamous

cellular

cancer

-LRB-

n

-RRB-

An

analysis

of

the

Vβ

repertoire

from

patient

was

investigated

by

flow

cytometry

of

metinel

node

acquired

lymphocytes

-LRB-

Fig.

-RRB-

and

revealed

an

increase

and

clonal

expansion

of

Vβ

families

and

In

addition

analysis

of

the

Vβ

repertoire

in

two

independently

cultured

metinel

nodes

derived

from

one

breast

cancer

metastasis

-LRB-

patient

-RRB-

-LRB-

data

not

shown

-RRB-

demonstrated

clonal

expansion

after

days

short-term

culture

of

the

same

T

cell

receptor

families

in

both

nodes

-LRB-

Vβ

Vβ

Vβ

and

Vβ

-RRB-

We

conclude

that

cultures

of

metinel

node

acquired

lymphocytes

expand

in

the

presence

of

autologous

tumor

extract

and

Fig.

In

patient

the

T

cell

receptor

Vβ

repertoire

was

investigated

by

flow

cytometry

in

two

sentinel

-LRB-

nodes

at

the

day

of

operation

and

after

in

vitro

cell

culture

-LRB-

days

-RRB-

Clonal

expansion

of

Vβ

families

and

were

detected

in

the

T

cell

population

Cells

from

patient

stimulated

at

the

end

of

the

expansion

demonstrated

a

high

production

of

IFN-γ

of

pg/mL

-LRB-

Fig.

-RRB-

and

no

detectable

-LRB-

data

not

shown

-RRB-

indicating

a

response

In

an

additional

patient

-LRB-

patient

-RRB-

the

stimulated

IFN-γ

secretion

was

pg/mL

while

only

a

low

production

of

pg/mL

was

found

again

demonstrating

a

response

pattern

Fig.

Amount

of

interferon

gamma

secreted

into

the

supernatant

measured

by

enzyme-linked

immunosorbent

assay

Data

presented

are

for

patient

after

days

of

in

vitro

cell

culture

According

to

more

detailed

analyses

of

some

cases

we

found

that

some

T

cells

expressed

Fox

indicating

that

these

cells

in

fact

were

T

regulatory

cells

DISCUSSION

We

show

that

it

is

possible

to

find

the

first

lymph

nodes

draining

various

types

of

metastases

by

lymphatic

mapping

by

means

of

dye

or

radioactive

tracers

We

have

named

these

metastasis-draining

lymph

nodes

metinel

nodes

to

show

both

the

analogy

and

difference

to

the

well-known

term

sentinel

node

which

means

the

first

node

to

receive

lymphatic

drainage

from

a

primary

tumor

In

studied

cases

we

demonstrated

three

different

ways

of

identifying

these

nodes

by

injection

of

tracers

subserosally

around

liver

or

intra-abdominal

metastases

close

to

subcutaneous

metastases

or

into

local

lymph

node

metastases

The

fact

that

it

is

possible

to

expand

the

metinel

node

derived

lymphocytes

via

in

vitro

expansions

during

several

weeks

of

time

together

with

our

analysis

that

they

contain

lymphocytes

that

show

clonal

expansion

toward

tumor

antigens

and

produce

high

levels

of

IFN-γ

in

the

expansions

are

evidence

that

they

contain

tumor-reactive

lymphocytes

These

cells

are

mainly

T-helper

cells

that

have

developed

an

immunological

response

toward

the

metastatic

cells

We

suggest

that

these

T

cells

may

be

used

in

future

trials

of

adoptive

cellular

therapy

of

disseminated

solid

cancer

Studies

have

demonstrated

that

tumor

growth

beyond

a

certain

size

required

and

that

the

amount

of

neovascularization

peritumorally

was

a

predictor

of

metastatic

disease

Far

less

is

known

about

the

specific

factors

regulating

the

lymphangiogenesis

around

a

tumor

and

its

possible

importance

much

as

a

result

of

the

difficulty

of

discriminating

between

lymphatics

and

blood

vessels

However

during

recent

years

the

molecular

pathways

signaling

for

lymphangiogenesis

have

been

described

and

these

studies

have

revealed

that

lymphangiogenesis

is

a

major

component

of

the

metastatic

process

The

lymphatic

capillaries

consist

of

thin-walled

low-pressure

vessels

that

drain

the

interstitium

When

the

surrounding

interstitial

fluid

pressure

increases

the

walls

of

the

lymphatics

are

stretched

and

open

to

passage

of

cells

or

fluids

The

lymphatic

capillaries

are

joined

into

major

lymphatic

collecting

vessels

and

filtered

through

at

least

one

lymph

node

before

entering

the

venous

circulation

After

entering

the

node

approximately

half

of

the

lymphatic

fluid

continues

directly

into

the

blood

before

the

rest

drains

into

an

efferent

lymphatic

vessel

A

malignant

tumor

causes

a

peritumoral

increase

of

lymphatic

vessels

mediated

through

VEGF

Studies

have

shown

that

high

levels

of

the

cytokine

cascade

of

VEGF-C

and

VEGF-D

promote

the

tumoral

lymphangiogenesis

and

that

inhibition

of

their

joint

receptor

suppresses

the

effects

Kawakami

et

al.

showed

that

high

levels

of

VEGF-C

and

its

receptor

correlated

with

increased

lymph

node

metastases

and

lymphatic

invasion

in

human

colorectal

cancer

White

et

al.

concluded

that

patients

operated

on

for

colon

cancer

had

an

overexpression

of

VEGF-D

but

not

of

its

receptor

which

correlated

with

a

decrease

in

disease-free

interval

and

overall

survival

A

study

based

on

patients

with

breast

cancer

showed

that

lymph

vessels

were

present

more

often

in

metastatic

axillary

lymph

nodes

-LRB-

-RRB-

than

in

nonmetastatic

lymph

nodes

-LRB-

-RRB-

and

that

intranodal

and

perinodal

lymphatic

endothelial

cell

proliferation

fractions

were

higher

in

metastatically

involved

lymph

nodes

Taken

together

all

these

findings

support

lymphangiogenesis

in

metastases

Thus

a

tumor

causes

an

upregulation

of

lymphangiogenic

factors

that

leads

to

an

increase

in

peritumoral

lymphatic

vessels

in

both

tumors

and

metastases

It

is

likely

that

these

newly

developed

lymph

vessels

connect

to

the

preexisting

lymphatic

vessels

and

lymph

nodes

Shed

tumor

cells

may

invade

these

newly

formed

vessels

and

the

dissemination

of

tumor

cells

is

facilitated

The

present

study

shows

that

this

process

can

be

visualized

by

lymphatic

mapping

and

the

first

draining

lymph

node

from

the

metastasis

the

metinel

node

may

be

identified

Animal

studies

showed

that

metastases

had

the

capacity

to

metastasize

Mice

inoculated

with

pulmonary

venous

blood

from

mice

having

pulmonary

metastases

developed

disseminated

cancer

disease

after

injection

into

the

tail

vein

showing

that

pulmonary

metastases

shed

viable

circulating

cells

Similarly

Hoover

and

demonstrated

that

mice

who

had

undergone

amputation

of

their

primary

tumors

and

now

only

had

pulmonary

metastases

experienced

spread

of

the

tumor

cells

specifically

into

the

lungs

after

parabiosis

In

surgery

for

colorectal

liver

metastases

it

is

not

standard

to

perform

a

biopsy

of

perihepatic

lymph

nodes

Nakamura

et

al.

reported

a

survival

of

in

node-positive

patients

but

the

study

cohort

was

small

Most

studies

identify

few

survivors

after

liver

resection

for

colorectal

liver

metastases

involving

positive

perihepatic

lymph

nodes

These

lymph

node

metastases

are

unlikely

to

be

of

lymphatic

origin

from

the

primary

colonic

tumor

instead

they

may

be

explained

by

hematogenous

spread

to

the

liver

followed

by

lymphogenic

spreading

from

the

liver

metastases

to

the

perihepatic

lymph

nodes

Correct

perihepatic

lymphatic

staging

is

beyond

dispute

not

only

because

the

increased

morbidity

in

patients

having

perihepatic

lymph

node

metastases

but

also

because

of

the

improvements

in

systemic

therapies

In

the

nine

cases

of

resected

liver

metastases

in

our

study

all

metinel

nodes

were

found

among

perihepatic

nodes

within

the

hepatoduodenal

ligament

or

liver

hilum

Most

of

the

liver

is

drained

by

deep

hepatic

lymphatics

the

upper

inner

part

of

the

liver

drains

to

nodes

around

the

end

of

inferior

caval

vein

and

the

lower

inner

part

drains

to

hepatic

nodes

through

the

liver

hilum

The

superficial

part

of

the

liver

lymphatic

drainage

is

divided

into

two

main

directions

the

posterior

surface

drains

into

pericaval

nodes

and

the

inferior

anterior

and

superior

surface

drains

into

hepatic

nodes

through

the

liver

hilum

In

all

cases

of

resected

liver

metastases

the

injections

of

PB

were

performed

subserosally

on

the

inferior

anterior

or

superior

surface

of

the

liver

Our

identified

perihepatic

metinel

nodes

were

located

corresponding

to

the

superficial

lymphatic

drainage

of

the

liver

Despite

the

prognostic

importance

of

these

perihepatic

lymph

nodes

we

have

only

found

one

article

in

the

literature

describing

lymphatic

mapping

originating

from

liver

metastases

Kane

et

al.

demonstrated

in

a

study

of

patients

with

colorectal

liver

metastases

that

it

was

possible

to

perform

lymphatic

mapping

by

the

intraoperative

injection

of

isosulfan

blue

dye

in

a

peritumoral

and

subcapsular

manner

The

basic

premise

was

that

the

occurrence

of

extrahepatic

metastases

within

the

normal

lymphatic

drainage

of

the

liver

was

considered

to

be

remetastases

from

the

colorectal

metastases

They

found

blue-colored

nodes

in

seven

patients

-LRB-

-RRB-

all

within

the

liver

hilum

falciform

ligament

or

gallbladder

fossa

In

three

of

these

patients

the

nodes

were

not

detected

by

the

surgeon

before

the

mapping

procedure

Like

our

results

they

concluded

that

hepatic

lymphatic

mapping

was

safe

rapid

and

simple

technique

to

perform

in

humans

The

importance

of

lymphatic

mapping

in

liver

surgery

for

metastases

was

underlined

by

Grobmyer

et

al.

who

concluded

that

routine

lymph

node

sampling

had

a

low

yield

in

patients

without

suspected

perihepatic

disease

by

computed

tomographic

scan

or

positron

emission

tomography

or

at

surgical

exploration

Further

another

study

demonstrated

that

microscopic

perihepatic

positive

lymph

node

disease

is

associated

with

the

same

poor

prognosis

as

in

macroscopic

positive

disease

and

suggests

therefore

that

lymph

node

dissection

should

be

systematic

Lymphoscintigraphy

has

been

used

to

find

sentinel

nodes

from

primary

tumors

but

to

our

knowledge

it

has

not

been

reported

as

a

tool

to

locate

lymph

nodes

draining

subcutaneous

metastases

The

radioactive

tracer

facilitated

the

intraoperative

identification

of

the

metinel

nodes

by

a

gamma

probe

used

as

a

complement

to

PB

In

those

cases

the

lymphoscintigraphy

was

made

days

before

surgery

and

new

injections

of

radioactive

tracers

were

provided

on

the

day

of

surgery

One

to

three

metinel

nodes

were

detected

in

our

six

patients

In

the

late

the

immunosurveillance

hypothesis

was

supporting

the

fact

that

tumor

cells

possessed

new

antigenic

potentials

and

provoked

an

immunological

reaction

that

could

eradicate

a

tumor

completely

In

the

last

decade

the

hypothesis

has

been

proven

in

mice

and

humans

The

role

of

the

immune

system

in

cancer

diseases

is

demonstrated

by

immunodeficient

patients

having

both

an

overrepresentation

of

virus-induced

malignancies

and

an

increased

probability

of

developing

cancer

of

nonviral

cause

Cardiac

transplant

recipients

have

for

example

been

shown

to

have

a

higher

prevalence

of

lung

tumors

than

the

general

population

Much

data

also

supports

the

fact

that

both

the

cellular

and

the

humoral

immune

systems

develop

immune

responses

against

tumors

Bevacizumab

a

monoclonal

antibody

against

VEGF

functions

as

an

inhibitor

of

angiogenesis

inhibits

growth

of

human

tumor

cells

in

mice

and

prolongs

the

overall

median

survival

in

human

metastatic

colorectal

cancer

The

presence

of

tumor-reactive

lymphocytes

in

sentinel

is

an

example

of

the

function

of

the

cellular

immune

system

and

a

positive

prognostic

factor

for

survival

in

colorectal

and

ovarian

cancer

In

our

earlier

study

we

made

a

single

cell

suspension

of

the

lymphocytes

from

sentinel

nodes

They

were

expanded

in

vitro

stimulated

by

tumor

homogenate

and

antigen-presenting

cells

In

the

present

expansions

we

proved

it

was

possible

to

find

tumor-reactive

lymphocytes

in

metinel

nodes

and

to

perform

adoptive

immunotherapy

based

on

them

The

preliminary

results

seems

promising

with

total

or

partial

regression

of

some

liver

and

lung

metastases

-LRB-

M.K.

unpublished

data

-RRB-

In

this

study

the

expansions

were

performed

in

exactly

the

same

way

as

in

our

previous

studies

except

that

the

source

of

the

lymphocytes

was

derived

from

lymph

nodes

draining

metastases

that

is

metinel

nodes

The

fact

that

we

succeeded

in

nine

cases

in

proliferating

the

lymphocytes

via

in

vitro

expansions

over

more

than

a

month

is

evidence

for

the

presence

of

tumor-reactive

lymphocytes

Because

the

expansions

were

continuously

stimulated

with

autologous

tumor

homogenate

and

antigen-presenting

cells

together

with

IL

only

lymphocytes

that

were

a

priori

stimulated

by

antigens

derived

from

the

tumor

could

proliferate

and

survive

for

such

a

long

time

Figure

demonstrates

the

Vβ

repertoire

in

cells

in

two

metinel

nodes

in

patient

thus

showing

which

types

of

T-helper

cell

receptors

were

found

at

the

end

of

expansion

The

figure

shows

clonal

expansion

of

Vβ

and

demonstrating

the

presence

of

tumor-reactive

lymphocytes

toward

three

different

types

of

tumor

antigens

These

collective

facts

point

out

the

presence

of

tumor-reactive

type

T-helper

lymphocytes

in

metinel

nodes

T

helper

lymphocytes

play

a

central

role

in

initiating

the

adoptive

immune

defense

In

of

patients

the

cells

survived

until

the

time

for

transfusion

and

patients

underwent

intravenous

adoptive

autologous

T

cell

transfusion

Approximately

half

of

all

patients

who

underwent

the

expansion

procedure

died

as

a

result

of

a

combination

of

too

few

cells

at

the

start

and

the

initial

presence

of

immunosuppressive

factors

that

not

could

be

overcome

Six

of

eight

patients

who

received

transfusions

are

still

alive

although

the

follow-up

time

varies

-LRB-

Table

-RRB-

Our

results

are

encouraging

because

the

prognosis

in

these

type

of

cases

is

usually

poor

In

this

study

we

have

shown

that

it

is

possible

to

find

the

draining

lymph

nodes

from

different

types

of

metastases

We

named

these

nodes

metinel

nodes

and

they

contain

tumor-reactive

lymphocytes

preferentially

T-helper

cells

with

response

We

have

also

demonstrated

that

it

is

possible

to

proliferate

these

cells

in

vitro

and

if

the

expansion

procedure

is

successful

to

use

the

cells

for

adoptive

immunotherapy

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

solid tumors
metastases
sentinel node
lymphatic mapping
immunotherapy