Identification of carcinoembryonic antigen mRNA in circulating peripheral blood of pancreatic carcinoma and gastric carcinoma patients

Life Scienm, Vol. 59, No.s 25126, pp. 21W2199,1996 +ght 0 1996 Elscvier Scicncc Inc. Printed in the USA. All rights resemd lzo24-3205/% s15.M) t .oo

ELSEVIER

PIISOO24-3205(96)00576-O

IDENTIFICATION OF CARCINOEMBRYONIC ANTIGEN mRNA IN CIRCULATING PERIPHERAL BLOOD OF PANCREATIC CARCINOMA AND GASTRIC CARCINOMA PATIENTS Naomi Omichi Funaki, Junji Tanaka*, Takayuki Kasamatsu, Gakuji Ohshio', Ryo Hosotani, Takashi Okino, and Masayuki Imamura. First

Department

of Surgery, Faculty of Medicine, Kyoto University, 54 Shogoinkawahara-cho,' Sakyo-ku, Kyoto 606, Japan *Department of Surgery, Shiga Medical Center for Adult Diseases (Received infinal formOctober 15,1996) Summary

To

detect

pheral

adenocarcinoma

antigen

(CEA)

patients

mRNA

with

in the

(GC) and also,

gastritis

patients

number the

analysis

was

By this sensitive were

advanced

PC

developed

patient,

metastasis

after

after

analysis.

Positive suggest mRNA

3

of

4

after

a gastric Because

reverse

mRNA. CEA

of

RT-PCR

blood

spreading

reaction.

and 2 of 9 GC for

1 highly

mRNA-positive

the

patients

resection

or

within

control

method

blood,

transcription chain

Except

operation

or

lymphoma

of the small

polymerase

curative

None

from

gastric

from pancreatitis

in the peripheral

that our sensitive

in the peripheral

with

in the peripheral

the

for CBA

palliative

for CEA mRNA

the hematogenous

by

peri-

obtained

or

3 of 9 PC patients

method,

recurrence

the

expected

two-step

positive

blood

carcinomas,

performed

circulating

of carcinoembryonic

(PC)

volunteers.

cells

by an original

patients

peripheral

as controls,

and four healthy

followed

the

carcinoma

without

of carcinoma

in

the presence

pancreatic

carcinoma patient

cells

we analyzed

blood,

9

liver months

patients

blood.

The

for detecting

is practically

useful

of adenocarcinoma

was

results CBA

to find

cells.

Kq, Words:CBA mRNA, RT-PCR,Pancreatic carcinoma, gastric carcinoma hematogenous dissemination

Address First

correspondence

Department

to : Naomi

of Surgery,

54 Shogoinkawahara-cho,

Omichi

Faculty

Sakyo-ku,

Funaki,

M.D.,

of Medicine,

Kyoto

606, Japan

Kyoto

Ph.D. University

CEAmRNAinPeripheralBlood

2188

Despite

many

recurrence

surgical

and

management

metastasis

(1).

diseases,

and

still

Identifying

therefore,

operations

efforts

is

cancer

cells

group.

In

reported of

on the attempts

tumor-associated

polymerase

chain

a

to

system

circulating mRNA

by

the

protein

reaction

of

may

mRNA

adenocarcinoma

cells, has

the

serum mRNA

marrow has

AFP was

(10)

been

using

our we

on

the

line.

protein on

cells the

to

no

amplification

the

present

identify

CEA

excluding

the

application

we

the

from

mRNA

of

All

Patients III.

computed

patients'

Diagnosis

No

trial

Therefore,

the

peripheral (10.11)

CEA-producing patients'

was more

of bone

cell

blood

and

unsatisfactory.

In

sensitive

method

peripheral

genomic

DNA

and

to

blood

also

its

of

analyses by

gastritis

the

profiles

are

and

biopsy.

The

II

necessary, mainly

tumor-forming

diagnosis.

by endoscopic

if made

I ,

echography,

by

and,

some

in

in Tables

made was

pancreatitis

histological

was made

listed

was

radiography

chest

Diagnosis

above-mentioned pancreatitis

and Methods

recurrence

of

tomography,

angiography.

chronic

the

the

to PC and GC patients.

Patients

and

a

CEA like

detection

protocol

circulating

the

amplification

serum

(11).

from

for

marker

blood.

PCR

and the

As

within

nodes

from

line

the

tumor

in

mRNA

new,

indicate

RT-PCR

lymph

described

our

level,

(l-6).

ceils

cells

cell

(6

to

a

the

and

the (AFP)

RT-PCR

measuring as

But

in

protein

blood

cancer

blood

report

cells

serum

thought of

and

developed

(I -fetoprotein

the

identified

was

using

study,

of

intact

peripheral

message

the

isolate

also

three-step

within

have

as a form of mRNA

have

in

cancer

such

investigators

be

the

previously

to constitute

We

secreted

(7-q). on

for

hematogenously

carcinoma

usefulness

clinical secondary

reverse-transcription

well-documented only

the

patients'

But

the

level

with

cells

form

cell

on

technique tested

a

well

the

reported

blood

the

been

performed and

cancer

in

for

decision-making

sevral

the

as

mRNA-producing

level

the

highly-sensitive

protein CEA

blood

problems

be thought

years,

operations,

group

Patients

by

Unlike

of

for

hepatocellular

capture

original,

presence

risk

(RT-PCR)(l-5).

manuscript).

existence

major

to detect

peripheral

an

submitted

pose

may well

recent

curative

high

therapies.

a

risk

for

important

adjuvant

disseminating high

a

Vol. 59, No.s 25/26, 19%

diagnosis

by type of

2189

CEAmRNAinPeripheraiBl~~d

Vol.59,No.s25/26, 19%

TABLE

I

List of PC Patients Serum CEA (ng/ml)

Curative operation

outcome

CEA mRNA

Sex Age (M/F) at test

Timing of blood sampling

l.M

54

pre"

yes

1.6

+

l.M

55

yes

2.6

+

2t4M 3.M 4.M 5.M 6.M 7.M

77 70 42 80 49 50

yes yes no no no no

1.0 1.0 5.0 5.0 5.5 1.0

+ + -

R(-) for 12M R(-) for 8M n-d.+= n-d. n.d. n.d.

8. F

54

yes

1.0

-

R(-)

for 12M

9. F

14

IM after operation RI-) pre pre pre pre pre pre chemo*B 60M after operation 6M after operation

yes

t1.0

-

R(-)

for 6M

after

9M*2Rx3(+)

after

2M R(+)

.--__pre signifies "before operation" M stands for "months" R stands for "recurrence" only this patient had mutinous cyst adenocarcinoma described as n.d. in the case of non-curative operation es chemo signifies "during chemotherapy"

-

*I sZ n3 n4 *=

RNA

extraction

eral

blood

(AGPC)

and

method

heparinized

cDNA

according (12)

whole

synthesis

to

with blood

ethanol].

We

cyanate (6) to

to

for the

citrate

better

solubilized of

and

10

~1 1 of

et to

ordinary

diethyl

al lml

AGPC

5x buffer

(Gibco DTT

from

periph-

In short,

5ml of

Sarcosyl of

of

isothiocyanate/

/ 1OOmM

fl -mercapto-

guanidinium

isothiodescribed according

(13). of

Two

ml

total

method. in

BRL),

5ml

a guanidinium

the one previously in the whole blood

pyrrocarbonate

reverse-transcribed

Industries,LTD,Japan),lOmM

with

quantity

than of RNA

Gillespie

the in

was

the

extracted

D [6M_guanidinium

/ 0.5%

corresponds

by

well

higher

protection of

which

processed (13)

level

report

mixture,

solution

was

guanidinium-phenol-chloroform

modification.

mixed

(pH7.0)

increased

the

RBA

acid

a slight was

isothiocyanate-enriched 25mM-sodium

the

blood,

50~ 1

2mM dNTP

this was

sample further

Extracted

(DEPC)a

of

RNA was treated water

mixture

consisted

(Wako Pure

(Gibco),

Chemical

0.25~ g random hexamer (Pharmacia), 5~ g bovine serum albumin (BSA)(Gibco) and 2OOU M-MLV reverse transcriptase (Gibco,Cat.No. 28025-013). The reverse

transcription

was performed

at 37 "C for one hour.

2190

CEA

Vol. 59,No.s25/26, 1996

mRNAinPeripheralBlood

TABLE List of Pancreatitis

II

or Gastritis

Patients

Diagnosis

Sex Aqe (M/F) at test

Without

Carcinoma CEA mRNA

Serum CEA (ng/ml)

10. M

50

chronic pancreatitis with pseudocyst

1.5

11. M

71

chronic pancreatitis

3.3

12. M

52

chronic pancreatitis

1.2

13. F

70

chronic pancreatitis

1.8

14. M

47

chronic pancreatitis

tl.O

15. M

33

chronic pancreatitis

1.6

16. M

52

acute pancreatitis

t1.0

17. M

31

chronic gastritis

cl.0

18. F

89

chronic gastritis

cl.0 -

of

Preparation human

colon

Type

Cell

eagle's

cancer

medium

purification performed

PCR

1

Co10

with

205

(Pharmacia)

specific

according

and

anti-sense

extends

from exon B2

protocol

which mixture

in

then

CEA

amplify was

First a

265-bp

composed

was one

modified

serum.

mRNA

Prep@

mRNA was

gene

detection (15,16).

sense

within

were Sense

primer

exon

B2,

2 and

(5'-AGGGCTTGGGCAGCTCCGCA-3') C and A) to exon A3.

performed

fragment. of

American

transcription

and

(the 3' two bases, step-PCR

Quick

sequence

both 3

bovine a

reverse

for

are

from

Dulbecco's

to the above method.

published

primer

high-CEA-producing

fetal

using

according

(5'-TCTAACCCACCTGCACAGTA-3')

a

(Purchased

(5'-GACGACCCCACCATTTCCCC-3')

common

PCR

10%

cells

primers

to

205

cultivated

1 ~1 q of mRWA

The

template

Co10

was

supplemented

kit

synthesized primer

line,

(14)

from

using

primers

control

cell

Culture)

extracted

was

positive

The

half

50 of

using ti 1 the

of

primers

1 and

individual

3

PCR

reverse-transcribed

TABLE List of GC Patients Sex

III

and a Gastric Curative operation

Timing of blood sampling

Age

(M/F) at test

2191

CEAmRNAinPeripher~Blood

Vol.59,No.s25/26, 19%

Lymphoma

Patient outcome

Serum CEA mRNA CEA (ng/ml)

19. M

72

post*' chemo#'

no

7.5

+

after

5M LM(+)X2

20. M

87

prex4 early

yes

2.2

-

Rx"(-)

for 15Me6

ca.

21. M

72

2wX7 after operation chemo

yes

1.4

-

R(-)

for 15M

22. M

55

pre early

yes

1.8

-

R(-)

for 12M

ca.

23. M

60

pre

yes

3.5

+

after

24. M

64

pre

no

4.1

-

n.d.*8

7M R(+)

25. M

53

pre chemo

no

1.5

-

n-d.

26. F

64

post chemo

no

t1.0

-

n-d.

27. F

35

post chemo

no

t1.0

-

n-d.

28. M

57

pre (malignant

yes lymphoma)

1.5

-

R(+)

post signifies "after operation" LM stands for "liver metastasis" chemo signifies "during chemotherapy" pre signifies "before operation" R stands for "recurrence" M stands for "months" w stands for "weeks" described as n.d. in the case of non-curative operation

sample

(25~ 1)

of was

BSA,

0.2mM

overlaid

3 minutes Taq

and

50pmole

Norwalk,CT),

and

polymerase

amplification

5~ 1 of of

dNTP

and

with

mineral

then

10

each

DEPC-treated

cooled

oil,

buffer in

(Perkin-Elmer-Cetus,

1~ 1 of

water.

The

for

the

heating

step

at of

Perkin-Elmer-Cetus).

of

from

temperature.

reaction

addition

3-second

denature

the

This

annealing

shuttle-type

"C

2.5 units cycle

94°C ,

was

for of of

followed

fragment

performed

temperature PCR

1 CLg mixture

93

Each

at PCR

the (629: ). As 20-second annealing by the chain extension reaction was small-sized, denaturing

TE(pH8.0),

heat-denatured

to 80 "c

(AmpliTaqQ, consisted

x PCR

primer

was

during the to

effective

to

CBA mRNAin Peripheral Blood

2192

further time

exclude

non-specific

duration

without

preliminary

test.

PCR

product

was

The

second

amplify

set

a

same

as

additional program But

PCR.

primers

24

u 1

the

the

of

unless

noted

test

cDNA

the

was

by

Fugua

half

of

sample

same

as

fragment

"c ),

extension faint, was

by

one-fiftieth

We

were

in

all

able the

controls

of

to

observe fail

The positive

control

with

of

step The with

0.1~ g was

the

negative

CRA

mRNA

calculated

electrophoresis

a

NA.Pharmacia

319-bp

of

except

then

of

(55°C )

35-cycle

PCR

product

mixture

for

the

residual was

difference

/3 -actin

cDNA

93°C ,

Taq denature

heat and

30-second

amplified

band

(corresponding 30 cycles

pair

mixture

at

l-second

annealing

the

PCR

for

detection

fi -actin

primer

was

the

heat-denature

the

amplification

from the patients (data not shown). for CXA mRNA mRNA.

The

detection negative

simultaneously-performed control

one-fiftieth

control

the

first

using

was

1

fi 1)

the

same

the same program.

samples

without

to

template

the

the

60°C

PCR

When

PCR

as

The

constituent

"c , and

of

amplification

50-pmole-each

the

was

2.

extraction,

for

mixture necessity

same

the

CRA

which

50pmole

The

using

30-second

in a new

set under

RNA

3

by

PCR

to

subjected

3-minute

steps.

"c )

the set

and

the

bromide.

80

(72

second

water. was

2

ethidium

The

followed

PCR.

20ng/ml

(17).

of

first

added

for

our

the

(Agarose

for

at

of

gels

protocol

addition

amplified

primer

al

mixture PCR

the

of primer

of

cDNA.

consisted

polymerase (94

the Our

primers.

et

were

the

in

second-step

primers

basically

was

amplified

reported the

was

the

except

shorten

yield

agarose

2.5%

reliability

fragment

of

temperature

containing

one of

PCR

product

on

of

primers

mixture

temperature

PCR

the

for

DEPC-treated

of

annealing

Biotech,Sweden) To

PCR

second

from the annealing 1ofi 1

consisted

constituent

first

to

PCR

One-fiftieth

template

These

and

the

cycles,

the

fragment.

the

for

35

as

Another

individually. the

of

190-bp

amplification

affecting

After used

Vol.59,No.s25126, 1996

for

the

(corresponding

in the first-step

PCR.

PCR

second-step to

1

of

and

fl -actin mRNA samples from the

was the PCR performed control without was

the

ti 1)

of

for

the

first

any

template.

PCR

performed

the

negative

2193

CEAmRNAinPeripheralBlood

Vol.59,No.s25/26, 1996

FIG.1 PCR

amplification

single

of

Co10

P stands

band.

used

the

name

as a marker,

cDNA

forms

for PCR product

and N for that without represent

205

template.

of

the lowest

with

Numbers lOO-bp

primer.

being

a clear template

1.2 and 3 ladder

a lOO-bp

was

band.

Results PCR

amplification

the

first

single

of

band

either

of the

cDNA

serially

was

made

PCR

visualized

second

PCR,

extracted first

an

a

and

band

second

heterodimeric

of

primer

band between

of

CEA

mRNA

CEA

mRNA-negative

1oa,

102,

healthy

10,

gel

pairs

of

10'.

of

0.1 the

visible

sample

small

and

ascertained

5, 2 or 1 Co10 205 blood, which

volunteer's

of

cells

205

(FIG.ZA).

The

After

that

the

weight

visible.

it together

We with

this

is a

PCR product.

Co10

healthy

Co10

TO The

also

amplified

quantity

cDNA

molecular

was

and

normal

of

were

lanes.

template.

(FIG.2B).

intermediate product

clear

bands

205

J.L g PCR

FIG.l,

a

control

Co10

the first and second

Detection

of

in

the

as

sample

PCR

in as

additional

assay,

used

band

with

Iml

our

and

second

from

observed visible

diluted

10-6-diluted

additional

first

this

and/or

a

is

lanes or negative

10-z -diluted

PCR

As were

non-specific

serially

diluted

first

the

in

sensitivity

second between

and

mRNA

cells

products

on positive

CEA

determine

205

PCR

individually

not observed Detection

Co10

second

and

205

cells

volunteer's were

mixed

mixed blood

with

lml

had been pre-checked as system. Total RNA extraction, cDNA synthesis and PCR amplification were performed just as was done on patients' blood. Under our system even five Co10 negative

for

CEA

mRNA

using

our

CEAmRNAin

2194

Vol.59,No.s25/X,1996

Peripheral Blood

FIG.2A PCR

using

FIG.2B

serially

diluted

test the sensitivity. product 2-3P

and

FIG.ZB

represent

indicated

heterodimeric

pair.

derived

represents

second

PCR

positive

the

band

PCR products

FIG.2A the

primer

Colo-205

consisting

to

the first PCR

product.

control

After

cDNA

the

l-3P lane

second

and with

PCR,

a

of the first and second

was also visible.

FIG.3 Even

five

Co10

volunteer's

blood

2-3 represents 205

cell-containing

the blood

sample of

patients,

3

after

second

cells

was

blood

CEA

mRNA

patient

was after

PCR the

sample

in

at

control was

205 cells

patients

the

positive

of

healthy

second

for

PCR.

CEA

was negative

were No-l,

positive

operation

lml

product.

patients'

(33%)(patient

preoperatively

with

detectable

Co10

preoperative

months

mixed

the positive

without

Detection the

205

and

without

samples positive 2

and

was

mR.NA and

(FIG.3). Among 9 PC for CEA mRNA

4)(FIG.4). also

clinically

No.1

positive

7

obvious

2195

CEAmRNAinPeripheralBlood

Vol.59,Nos 25/26, 1996

FIG.4 The

second-step

samples

(lanes

PCR 4-13)

amplification

14). l-3 and 2-3 represent

(lane for

the

first

and

"post"

of

and a healthy

and

second

signifying

patients' sample

the positive

PCR,

before

PC

volunteer's

control

respectively,

and after ZZZ

"pre"

operation.

FIG.5 The

second-step samples

patients' samples

PCR

(lanes

positive

amplification

(lanes

4-10)

11-13).

control

for

l-3

of

pancreatitis

and healthy and

2-3

the

first

and

2

months

he

volunteers'

represent

the

second

PCR,

individually.

recurrence. multiple protein

And

after

liver level

metastases.

was

below

This patients

had been

No.4

patient

was

No.3

preoperative

were date.

negative No.5,

but were

in

1.0

and

an

negative

CEA 7

No.2

patient,

ng/ml,

was

recurrence-free advanced

patient for

6

another

No.8

mRNA

and

preoperative

for CEA mRNA

found

whose

positive

for

during

12 months

with

paraaortic

stage

and

was

and

to

serum CEA

remained

invasion. patients

recurrence-free were

in their peripheral

CEA mRNA.

observed.

9 postoperative

patients

have

advanced blood.

to cases

21%

CEAmRNAinPeripheral Blood

The

second-step

patients (lane 15).

of

positive

As

for

GC

were

patient

operation

to

But

patient

this

after patient months

this

developed

curative months cases CEA-mRNA

of

as

or

No.21 and

CEA

the stomach.

GC

much

advanced mass

in

possible

the

cancer was

been

suffered

the

In

cells

and

and

received

chemotherapy.

ligament 7 22 CEA

No.20

and

remained 2

recurrence-free patients

peripheral from

and PCR.

metastases 5 months CEA mRNA-positive

examined 27

No.19 second

hepatoduodenal

operation.

their

(FIG.5).

carcinoma

liver

was

pancreatitis

after

preoperatively

patient has

as

multiple

No.23

patient

for

(22%)(patient

mRNA

No.24, 25, 26 and observed. their without CEA mRNA in negative

control

acute

blood

patients

locally

curative

patients,

operation

9 for

recurrence

the

layer.

chronic

developed

analysis.

after

mRNA-negative mucosal

2 a

remove

the positive

in the peripheral

positive had

of

PCR, respectively.

with

patients

patients,

23)(FIG.6) No.19

and second

for CEA mRNA

samples

a gastric lymphoma patient gastritis patients (lanes 14-

l-3 and 2-3 represent

the

of

4-12),

13) and chronic

the first

None

FIG.6 amplification

PCR

(lanes

Vol.59,Nos 25/26, 1996

malignant

within weeks

during were blood.

the after

the

15

advanced No.28

lymphoma

of

CEAmRNAiuPeripheraIBlood

Vol.59,No.s 25/X,1996

the

patients

two

Neither

of

positive

(Figure

negative

(Figures

4 and 5).

of

final

Sequencing and

sample that

the

cDNA

6).

the

the

products

without

fail

volunteers

products

randomly-picked

amplified

sequence

healthy

PCR

up

of

the

were

positive

patients'

with

was

totally

control revealed

samples

identical

were

gastritis

chronic

with

Moreover,

the

expected

(data not shown).

Discussion We

have

developed

identify the

CEA

mRNA

analysis

a RT-PCR

an

original,

in

the

of

the

system

has

marrow

been

reported,

cells

(11).

practically

It seems for

carcinoma to

the

cells

reason

carcinoma has

that

multiple

205

the

mixed

cannot

useful

convenient

following

without is set same

exon

needed we

upper The

denaturing

were

hours.

for

This

to

PCR

healthy

only

steps. the

our

than even

In

blood

be

five

its Co10

(FIG.3)

and

(FIG.2).

system of

using

has other

total

RNA

whole

was

blood

(6). The lower PCR primer

only

3' two bases

primer the

in

from

the the

electrophoretic

in the

this

without

annealing

shortened way,

exist

designed

sequences

between

final

may

only

method

In this

100 adenoAnd

extraction

greatly

due

cells.

The

The

adeno-

impossible.

cDNA

the present

-

if a patient

system

detect

donor's

100

CEA mRNA

course

carcinoma

control

targeted PCR

more

simple

so that

shuttle

obtain

of

positive

primers.

is of

blood,

clearly

cell-fractioning

temperature

two

able

of

to

features.

amplified

as

by

low sensitivity

clinically

sensitivity,

two exons

with

is

(11)

mRNA-bearing

expect

feature

able

previous

time-consuming

amplification. and

our

to span

effectively

CEA

disseminating

1 cc

to this high

and

always

diluted

In addition made

of blood.

sampling

were

with

highly

as many

this

to apply

presence

to

Although

node

difficult

attractive We

blood.

lymph

it required

peripheral

blood

most

to detect

the

to identify

hematogenously

sensitivity. cells

also

we

and

system

in the lymphocytes

the

the

RT-PCR

peripheral

(10)

in a few ml of peripheral

massive

contrast, high

in

cells

because

mixed

screening

sensitive

circulating

bone

1000 carcinoma method

very

way

genomic

temperature

time

duration

patients' bands

blood

within

8

2198

CBAmRNAinPeripheralBlood

Using CEA

patients'

mRNA

in

(22%).

samples,

3 of

9 PC

Except

for

one

pancreatic

carcinoma,

recurrence

after

3

operation

Although

residual

the 12

the high

There but

months

are

3

no

carcinoma number From for

and

cancer

this

point

CEA-mRNA being

GC

patients

detected

view,

negative time

clinically

for

cells

and

cannot

possibility

normal

lack

non-cancerous

not

found

among

In

conclusion,

live

our

whole

information

on

gastric

marker

to

carcinomas These

RT-PCR

hematogenous

were

must

well

be

be

anchoring,

Unlike

invasion,

direct

thought

of

a

ruled

be

diagnosed

metastasis-free.

cannot

patients

will

who

the few.

negative

analysis

non-cancerous

without

nested blood

the

carcinoma

belong

blood.

to discriminate

cells,

long

non-cancerous

peripheral

may

patients

this

contamination

cells

a definite

possibly

or

may

of

epithelial

of

after

analysis.

recurrence-free

advanced

are

patients

extended

microdissemination

for such patients.

the

there

on

the

with

of

of

been

peripheral

recurrence,

interval

beneficial

producing

the

patient

had

their

recurrence-free

cases

the

who

in

although

detection

Although

had this

developed

metastasis

after

think

of

far-advanced

(75%)

liver

months

patient we

with

patients or

9

presence

9 GC patients

low CEA-producers, and/or be very may in the circulation may be very cells

some

far-advanced

operation within

the

in 2 of

patient

positive

one

4

of

and

at

4

to detect and

for recurrence.

mRNA

cells

of

repeated as

PC CEA

able (33%)

positive of

observed,

risk group

had

were

curative

mass-reducing during

we

patients

Vol.59, No.s25/26, 1996

the to

few out

be

CEA-

because

between

cancer

epithelial

cells

and

CEA

mRNA

was

in our study.

detection

provide

a

dissemination

of

CEA

mRNA

practically of

from useful

pancreatic

and

C-BRADLEY,

G.

cells. References

K.PITTMAN, J.SOUTHGATE, P.SELBY, Lancet 338 1227-9 (1991)

1.

B.SMITH, E-BLAIR.

2.

M.MATSUMURA,Y.NIWA,N.KATO,Y.KOMATSU,S.SHIINA,T.KAWABE, T.KAWASE, Hepatology

3.

H.TOYOSHIMA,

M.IHORI,

H.D.DATTA,

-20 1418-25 (1994) P.T.ADAMS,

V-H-TERRY,

M.S.ROTH.

Y.SHIRATORI,

W.R.DROBYSKI, J.Clin.Oncol. -12 475-82

M.OMATA.

S.P.ETHIER, (1994)

4. 5.

H.NAITO,

Cancer

-74(9) N.KUZUMAKI,

Y.ISHIKAWAEur.J.Cancer 6.

N.FUNAKI,

J-TANAKA,

Life Sciences 7.

S.HUMPHREYS,

E.R.BANKS,

D.P.Jr.WOOD, RANGNEKAR.

2199

CEAmRNAinPeripheralBlood

Vol.59,No.s25126, 1%

2533-40

J.W.McROBERTS,

J.UCHINO,

R.KOBAYASHI,

T.SHIKANO,

-27(6) 762-5 (1991) S.SETO, T.KASAMATSU, T.KAIDO,

57(17)

1621-1631

J.E.SHIVELY,J.D.BEATTY.

V.M.

(1994)

M.IMAMURA.

(1995) 355-399

Crit.Rev.Oncol.Hematol.~

(1985) 8.

C.J.KELLY,J.M.DALY.

9.

A.H.CHEVINSKY

10.

M.GERHARD,

H.JUHL,

M.NEUMAIER.

J.Clin.Oncol.

11.

M.MORI,

Cancer -70 1398-1408 (1992) Semin.Surg.Oncol. 1 162-166 (1991)

K.MIMORI,

H.KALTHOFF,

H.W.SCHREIBER,

-12(4) 725-729 H.INOUE, G.F.BARNARD,

C.WAGENER,

(1994) K.TSUJI,

Cancer Research -55 3417-3420 N.SACCHI.Anal.Biochem.162 156-9

S.NANBARA,

H.UEO,T.AKIYOSHI.

(1995)

12.

P.CHOMCZYNSKI,

(1987)

13.

D.H.GILLESPIE,K.K.CUDDY,T.KOLBE,D.I.MARKS.Nature

367 390-391

(1994) 14.

T.U.SEMPLE,L.A.QUINN,L.K.WOODS,G.E.MOORE.

15.

-38 1345-1355 H.SCHREWE,

Cancer

Research

(1978) J.THOMPSON,

M-BONA,

M.HASSAUER,J.E.SHIVELY,S.von

L.J.F.HEFTA,

A.MARUYA,

KLEIST,W.ZIMMERMANN.

Mol.Cell.

Biol. 16.

-lO(6) 2738-2748 J.A.THOMPSON, H.PANDE,

R.J.PAKTON,

L.SHIVELY,

R.L.SIMMER,C.W.TODD,A.D.RIGGS,J.E.SHIVELY. Sci.USA 17.

84 2965-2969

(1990)

Proc.Natl.Acad.

(1987)

S.A.FUQU~N.F.FALETTE,W.L.McGUIRE,J.Natl.Cancer 861

A.PADMA,

Inst.82

858-