HCC biomarkers and the

current role in patients

at risk for hepatocellular carcinoma (HCC)
Robert G. Gish MD
Senior Medical Director Liver Program
St Josephs Medical Center, Phoenix, Az
Clinical Professor of Medicine (Adjunct): University of Nevada, Las Vegas
Medical Director Hepatitis B Foundation, Doylestown, PA

HCC Biomarkers and Surveillance:
Outline
Biomarkers for HCC are not being used primarily to establish a diagnosis of
cancer
HCC biomarkers can be used as risk tests in surveillance protocols to
determine who may develop HCC and to guide changes in imaging method
selection and timing
Alpha beta-protein (AFP) is not FDA cleared as a single test for the diagnosis
or risk of HCC
DCP and AFPL3% are the only biomarkers that are cleared by the FDA with
the intended use as risk markers for HCC
AFP as a single test: for Diagnosis of HCC? (no):
Poor Diagnostic Tool due to
Low sensitivity and/or specificity
Tumor heterogeneity
Patient heterogeneity

Combined use of AFP, AFP-L3 and DCP significantly
Improves the performance characteristics as risk
markers for HCC
Key Challenges and Uses of Biomarkers
in Clinical Practice
Surveillance Guidelines for High-Risk Patients
Approval/Clearance And Reimbursement for
HCC Biomarkers
USA
FDA Clearance
Canada

Health Canada
Europe
CE Mark
Japan
MHLW
AFP Not cleared for HCC
Reimbursed
Yes Yes

Yes
Reimbursed
AFP-L3 Yes
Reimbursed
Yes

Yes

Yes
Reimbursed
DCP Yes
Reimbursed
Yes

Yes

Yes
Reimbursed
The biomarkers have been already approved in almost every region of the globe.
Inclusion of AFP-L3 and DCP Biomarkers Enable
More Comprehensive Surveillance Strategies
1. Kumada T, et al. J Hepatol 1999;30:125-30.
2. Makuuchi M, et al. Hepatol Res. 2008;38:37-51.
3. Koike Y, et al. Cancer 2001;91:561-9.
AFP-L3 DCP
AFP is a glycoprotein with a single asparagine-
linked complex-type carbohydrate chain on each
molecule.

AFP-L3 is an isoform of AFP which has an
additional fucose residue.

Elevation of AFP-L3 is indicative of greater
malignant potential of HCC and is correlated to
shorter doubling time of tumor volume and
increased hepatic arterial supply.
1

DCP is a precursor form of prothrombin, a
coagulation protein.

HCC cells lack the carboxylase protein to change
DCP to prothrombin.

DCP has been reported to be a specific marker
for microinvasion and a high DCP level is
associated with development of portal vein
invasion.
2,3
HCC biomarkers: Intended use
AFP-L3 and DCP are intended for and FDA Cleared for in vitro diagnostic
use as an aid in the risk assessment of patients with chronic liver diseases
for development of HCC in conjunction with other laboratory findings,
imaging studies, and clinical assessment.
The combined use of these biomarkers has been shown to be more
sensitive and effective for the early detection of HCC through
surveillance.
4-6


1. Shimauchi Y, et al. Oncol Rep.2000;7:249-56.
2. Toyoda H, et al. Clin Gastroenterol Hepatol 2006;4:111-7.
3. Volk ML, et al. Cancer Biomarkers 2007;3:79-87.
Inclusion of AFP-L3 & DCP Improves Early Detection
Primary Tool Sensitivity Specificity Conclusions
Colli et al (2006)
1
Ultrasound
48%

(95%
CI
34-62%)
97%

(95%
CI
95-98%)
Ultrasound isinsufficiently sensitive to
detect HCC in many cirrhotics or to
support an effective surveillance
program.
Singal et al (2012)
2
Ultrasound 43.9% 91.5%
Ultrasound is suboptimal when used
alone
Volk et al (2007)
3
AFP, AFP-L3 & DCP 84% 94%
Combined use of AFP, AFP-L3 and DCP
results in enhanced detection of HCC
with minimal false positives
Hann et al (2013)
4
AFP, AFP-L3 & DCP 83% 91%
1. Colli A, et al. Am J Gastroenterol 2006;101:513-23.
2. Singal et al. Cancer Epidemiol Biomarkers Prev. 2012;21:793-9
3. Volk ML, et al. Cancer Biomarkers 2007;3:79-87
4. Hann HW, et al. DDW 2013 Poster Tu 1048
Biomarkers Supplement Ultrasound and Provide
an opportunity for earlier HCC detection
Surveillance strategy Cost per test
1
Advantages Disadvantages
No surveillance $0 No expenditure No chance of diagnosis
Ultrasound
$185

Widely used , low cost Poor sensitivity
Ultrasound + AFP
$216

Widely used
Poor sensitivity, particularly in
early stage HCC
Ultrasound + AFP, AFP-L3,
DCP
$361

Earlier and more sensitive
detection of HCC
Additional costs of AFP-L3 and DCP
CT
$640

High sensitivity High cost, limited access
1. Yu NC, et al. Clin Gastroenterol Hepatol
2011;9:161-7.
Earlier Detection of HCC May Improve Opportunity for
Cost-Effective Interventions
Treatments
(Stage I)
Mean years
survived

Mean cumulative
expenditures
ICERs
(Incremental cost-effectiveness
ratios)
No treatment 1.06 $35,390 N/A
Chemotherapy 1.29 $68,824 Insignificant difference
Radiation 1.47 $65,098 74,404
Liver directed
(Ablation and/or TACE )
2.67 $95,566 25,345
Resection 4.51 $123,807 15,303
Transplant 5.98 $207,473 55,066
Shaya FT, et al. Pharmacoeconomics. 2013 Nov 29.
Resection, classified by the AASLD Guidelines as
curative therapy, is also the most cost effective.
Costs and Performance of HCC Surveillance Tools
Surveillance tests Cost per test
1
References Sensitivity Specificity
Ultrasound $185 Singal, et al. (2012)
2
44% 92%
AFP only $31 Volk, et al. (2007)
3
69% 91%
3 biomarkers
(AFP, AFP-L3, DCP)
$176

$88 (AFP, AFP-L3) + $88
(DCP)
Volk, et al. (2007)
3
88% 91%
Hann, et al. (2013)
4
83% 91%
1. Yu NC, et al. Clin Gastroenterol Hepatol 2011;9:161-7.
2. Singal et al. Cancer Epidemiol Biomarkers Prev. 2012;21:793-9
3. Volk ML, et al. Cancer Biomarkers 2007;3:79-87
4. Hann HW, et al. DDW 2013 Poster Tu 1048
Costs
1
: CT, $640; MRI, $1400
AFP-L3 and DCP Are Reimbursed By Various Insurance
Providers
AFP-L3 and DCP are covered by a number of
public and commercial payers (data on file)

Medicare Clinical Laboratory Fee Schedule
1

AFP-L3 (CPT 82107): $88.54
DCP (83951): $88.54


Some Examples of Health Plan Coverage
Medicare
Medicaid
Tricare
Highmark Blue Cross Blue Shield
EmblemHealth
Capital Blue Cross
Tufts Health Plan
Blue Cross Blue Shield of Delaware
Priority Health
1. Centers for Medicare and Medicaid Services. Clinical Laboratory Fee Schedule. 2013.
Liver Dedicated
Surveillance
Ultrasound (US)
+ HCC biomarkers

in at-risk patients*
Poor/Fair
quality US
Or
abnormal
biomarkers
Good/Excellent
quality US and
normal HCC
biomarkers
#
gadoxetate disodium MRI
(Or dynamic CT)
US surveillance
q6 months with
biomarkers
Negative MRI
Abnormal US
or
increasing
biomarkers

blood tests AFP-L3%/DCP (HCC serum biomarkers); *AASLD Guidelines 2009; # see LI-RADS.
AASLD = American Association for the Study of Liver Diseases; AFP = alpha-fetoprotein; CT = computed tomography;
DCP = des-gamma-carboxy prothrombin; HCC = hepatocellular carcinoma; LI-RADS = Liver Imaging Reporting and Data System;
MRI = magnetic resonance imaging; US = ultrasound.
Proposed Liver Ultrasound Algorithm
Gish, R. ,Gastroenterology & Hepatology, 2014; 10;2:121-123.
HCC Biomarkers Utility Examples
Remember: Not all HCC make all biomarkers
Patient number of AFP-L3, DCP and AFP in those with known HCC (n=685)
Toyoda et al. Clin Gastro and Hep 2006;4:111-117
AFP > 20 ng/mL
DCP > 40mAU/mL
AFP-L3 > 10%
96
110
93
153
45
14
15
159 (23%)
all negative
Combined Biomarker Testing Identifies a Greater Number of
Positive tests in Patients with Known HCC
1
AFP-L3 Positive
15
(20.3%)
DCP Positive
3
(4.1%)
AFP Positive
8
(10.8%)
7
(9.5%)
15
(20.3%)
4
(5.4%)
15
(20.3%)
All Negative
7
(9.5%)
FDA submission data for uTASWako i30
If HCC biomarkers are used prior to
imaging:
In this study of 74 patients, the use of AFP
alone would have detected 45 HCC cases
and missed 29 cases of HCC
Cut-off point Positive cases
AFP 20 ng/mL 45 (60.8%)
AFP-L3 10 % 49 (66.2%)
DCP
7.5 ng/mL

29 (39.2%)
Combination of
AFP, AFP-L3, and DCP
Same as above 67 (90.5%)
Utility when biomarkers are used in combination in patients
without HCC: False Positives, True Negative, True Positives (in the
colored circles)
AFP-L3%
DCP AFP
2
(0.5%)
3
(0.7%)
9
(2.2%)
15
(3.7%)
26
(6.5%)
81(20.2%)
True Negative
258 (64%)
7
(1.7%)
False Positive
AFP: 25%
AFP-L3%: 7%
DCP: 9%
N = 401
FDA submission data for LiBASys
Predictors of HCC Development in Patients with Chronic Hepatitis C
El-Serag, et al. 2014
The development of HCC in
patients with HCV and cirrhosis
can be predicted based on:
high AFP levels
high ALT levels
low platelet levels
older age at AFP test
AFP = -fetal protein; ALT = alanine aminotransferase.
Case 1 Risk assessment of
HCC development by AFP-L3
0.17 ng/mL
AFP-L3
Normal: <10 %
AFP
Normal: <20 ng/mL
DCP
Normal: <7.5 ng/mL
L
e
v
e
l

o
f

B
i
o
m
a
r
k
e
r

0.26 ng/mL 0.23 ng/mL 0.19 ng/mL
Cut-off
Level
3.2 ng/mL
4.3 ng/mL
7.9 ng/mL
13.8%
34.6%
87.7%
24.1 ng/mL
69.7%
Only AFP-L3 increased before HCC
diagnosis by MRI.
-------------------------------------
-----------------
Elevation of AFP-L3 indicates higher risk of
developing HCC and can be a trigger to do
CT/MRI for early detection.
Hann HW, et al. DDW 2013 Poster Tu 1048
Months Before Diagnosis by imaging
MRI dx of HCC
Case 2 Risk Assessment Using
AFP-L3 and DCP Elevation
Months Before Diagnosis by Imaging
L
e
v
e
l

o
f

B
i
o
m
a
r
k
e
r

0.30 ng/mL
0.32 ng/mL
17.65 ng/mL
9.1 ng/mL
6.8 ng/mL
7.4% 7.8%
38.6%
Cut-off
Level
10.8 ng/mL
AFP-L3
Normal: <10 %
AFP
Normal: <20 ng/mL
DCP
Normal: <7.5 ng/mL
AFP-L3 and DCP were increased before
HCC detection in some patients who had
a low (normal) AFP level.
---------------------------------------------------------
Adding AFP-L3 and DCP as risk markers to
conventional surveillance tools such as AFP
and ultrasound can increase chances of
early detection of HCC.
Hann HW, et al. DDW 2013 Poster Tu 1048

MRI dx of HCC
Case 3 DCP Positivity and
Risk Assessment
AFP-L3
Normal: <10 %
AFP
Normal: <20 ng/mL
DCP
Normal: <7.5 ng/mL
L
e
v
e
l

o
f

B
i
o
m
a
r
k
e
r

1.43 ng/mL
6.45 ng/mL
19.86 ng/mL
2.5 ng/mL
2.6 ng/mL
3.5 ng/mL
0.5%
Cut-off
Level
0.5% 0.5% Months Before Diagnosis by Imaging
Some patients have an early AFP-L3
elevation, and some have DCP. DCP
elevation represents a different feature of
HCC development than AFP-L3.
--------------------------------------------------------
Combined use of DCP and AFP-L3 can
increase the sensitivity of HCC
identification.
Hann HW, et al. DDW 2013 Poster Tu 1048

MRI dx of HCC
22
Distribution Pattern of AFP-L3
In patients Who Developed HCC in the future
No HCC
0
2
4
6
8
10
12
14
16
18
20
0-9.9 10-19.9 20-29.9 30-39.9 40-49.9 50-59.9 60-69.9 70-79.9 80-89.9 90-99.9
AFP-L3%
P
a
t
i
e
n
t

n
u
m
b
e
r
0
50
100
150
200
250
300
350
0-9.9 10-19.9 20-29.9 30-39.9 40-49.9 50-59.9 60-69.9 70-79.9 80-89.9 90-99.9
AFP-L3%
P
a
t
i
e
n
t

n
u
m
b
e
r
Sensitivity= 51.3%
Specificity = 92.5%
23
Sensitivities and Specificities: for the
development of future cancer
Cutoff point
AFP-L3
(10%)
AFP
(10ng/mL)
AFP
(100ng/mL)
Sensitivity 51.3% 79.5% 25.6%
Specificity 92.5% 61.6% 94.3%
Relative
Risk
(95% C.I.)
8.2
(4.7-14.3)
5.3
(2.5-11.4)
4.1
(4.6-9.6)
Sherman M, et al. DDW 2005 Poster
24
HCC Occurrence Rate by disease state and if
AFP-L3 10%
0%
10%
20%
30%
40%
50%
60%
0 500 1000 1500 2000
Days
H
C
C

o
c
c
u
r
r
e
n
c
e

r
a
t
e
:

%
All
Start CH
Start LC
Only HBV
Only HCV
0%
10%
20%
30%
40%
50%
60%
0 500 1000 1500 2000
Days
H
C
C

o
c
c
u
r
r
e
n
c
e

r
a
t
e
:

%
All
AFP-L3% < 10%
AFP-L3% 10%
Sherman M, et al. DDW 2005 Poster
Lead Time of AFP-L3 before HCC Dx
Pt.
ID
AFPL3%
elevation period < Dx
IMAGING DATA @ 1st
AFPL3% ELEVATION
IMAGING DATA w/in
3 mos.Dx DATE Pt.
ID
AFPL3%
elevation period
< Dx
IMAGING DATA @
1st
AFPL3% ELEVATION
IMAGING DATA w/in
3 mos.Dx DATE
DAYS # MAX CM # MAX CM DAYS # MAX CM # MAX CM
1 -619 No Tumor 2 1.9 11 -112 No Information 1 1.1
2 -245 4 2.0 1 7.0 12 -9 1 5.0 1 12.0
3 -462 No Information 2 1.2 13 -63 1 2.1 1 2.1
4 -251 1 0.8 1 4.0 14 -593 No Information 2 2.9
5 -105 3 2.0 3 2.6 15 -4 No Information 1 1.2
6 -95 1 1.1 2 1.9 16 -119 No Information 2 0.7
7 -163 No Information 1 2.6 17 -351 No Information 3 1.0
8 -280 1 2.0 2 2.5 18 -140 No Tumor 1 1.0
9 -38 No Information 1 1.0 19 0 No Information 1 4.0
10 -78 No Information 1 1.5 20 -365 No Tumor 2 1.5
Mean -204.6 1.7 2.1 1.6 2.7
Median -129.5
Sherman M, et al. DDW 2005 Poster
No HCC HCC
37-60
Sen
%
Spec
%
PPV
%
NPV
%
AUC P
AFP-L310% 31 5 20 96 14 97 .59 .0005
AFP20
&DCP40
78 10 40 90 11 98 .65 <.0001
AFP20 &
L3>10%
13 6 20 98 28 97 59 <.0001
AFP20&
L310% &
DCP40
10 6 20 99 33 97 .59 <.0001
Performance of elevated AFP-L3 or combination of
biomarkers during months 37 to 48
in predicting HCC during months 37 to 60
*Interpretation: if biomarkers are negative in HCV patients < 3% risk of HCC
False positive rate for biomarkers for future cancer events 1-10%
If all three biomarkers are positive 1/3 chance of HCC in the follow up period
Note: data generated using first generation instrument (LiBASys) and lower sensitivity assays.
*communication from Dr Gish
Additional Utilities of Biomarkers
Biomarkers are useful for risk prediction for HCC recurrence after
therapeutic interventions

Additional uses: Correlation with
Level of dedifferentiation of the cancer
Presence of vascular invasion
Guide evaluation for metastatic disease
Used for liver transplant listing
HCC Biomarkers Literature Review
(Including Off-Label Use)

Use of HCC biomarkers for prognosis
Once an HCC is diagnosed: proposed utility of AFPL3% and DCP

Predicting clinical course
Presence of vascular invasion
Risk of developing metastases
Level of dedifferentiation of HCC tumor
Mortality risk
30
Tumor Doubling Time: higher AFPL3%, more rapid progression in size
AFP-L3 AFP
90 days >90 days
Average 944.3 237.4
SD 1967.0 581.3
P = 0.0600
Tumor doubling time
AFP: ng/mL
P value (90 days vs. >90 days)
90 days >90 days
Average 24.4 10.1
SD 20.8 15.4
P value (90 days vs. >90 days) P = 0.0066
Tumor doubling time
AFP-L3: %
P value: Mann-Whitney U Test
0
10
20
30
40
50
60
70
'
9
0

d
a
y
s
$
'

9
0
>
9
0
> 90
Days
90
Days
0
1000
2000
3000
4000
5000
6000
7000
8000
'
9
0

d
a
y
s
$
'

9
0
>
9
0
90
Days
> 90
Days
AFP-L3 (%)
AFP (ng/mL)
Sherman M, et al. DDW 2005 Poster
Current Biomarkers and Risk of Microvascular
Invasion
Independent predictors of microvascular invasion
Tumor size (< 2, 2-4, > 4 cm)
Odds ratio: 3.4 (95% CI: 1.5-4.1)
Preoperative DCP levels (< 100, 100-500, > 500 mAU/mL)
Odds ratio: 2.2 (95% CI: 1.1-2.4)
Tumor grade (3-grade system)
Odds ratio: 2.2 (95% CI: 1.1-3.7)
Shirabe K, et al. J Surg Oncol. 2007;95:235-240.
Future development of Portal Venous Invasion of HCC
in relation to the Serum DCP level at diagnosis
DCP(-): 183 patients with DCP < 100 mAU/mL
Koike Y, et al. Cancer 2001 Feb 1;91(3):561-9
DCP(+): 37 patients with DCP > 100 mAU/mL
DCP helps predicts clinical course
Current HCC Biomarkers and Risk of Portal Vein
Invasion
AFP-L3 15%
RR: 2.459 (95% CI: 1.005-6.017; P = .0487)
DCP 100 mAU/mL
RR: 3.019 (95% CI: 1.077-8.464; P = .0357)
Number of HCC tumors 2
RR: 4.912 (95% CI: 1.619-14.905; P = .0049)

Hagiwara S, et al. J Gastroenterol. 2006;41:1214-1219.
DCP and pathological variables of HCC
1
well
differentiated
moderately
differenciated
poorly
differenciated
P
I
V
K
A
-

(
m
A
U
/
m
l
)

pV invasion (-)
pV(+)
10
100
1000
10000
100000
P<0.05
P<0.05
1
10
100
1000
10000
100000
P<0.01
400 400
In 100 HCC recipients of LDLT in Kyoto
Note: For DCP 1mAU/mL = 0.092ng/mL
Courtesy of Hiroto Egawa
Survival Rate with Respect to HCC Stage and
AFP-L3 BEFORE Therapies
S
u
r
v
i
v
a
l

R
a
t
e

(
%
)

Stage
Year
Stage
Year
S
u
r
v
i
v
a
l

R
a
t
e

(
%
)

Stage
Year
S
u
r
v
i
v
a
l

R
a
t
e

(
%
)

Stage
Year
S
u
r
v
i
v
a
l

R
a
t
e

(
%
)

AFP-L3 Negative
AFP-L3 Negative
AFP-L3 Negative
AFP-L3 Negative
AFP-L3 Positive
AFP-L3 Positive
AFP-L3 Positive
AFP-L3 Positive
Courtesy of Hiroto Egawa
Survival Rate with Respect to HCC Stage and AFP-L3
AFTER Therapies.
Stage,
Days
Stage,
S
u
r
v
i
v
a
l

R
a
t
e

(
%
)

S
u
r
v
i
v
a
l

R
a
t
e

(
%
)

Days
AFP-L3 Negative
AFP-L3 Positive
AFP-L3 Negative
AFP-L3 Positive
Courtesy of Hiroto Egawa
Results
The 1-, 3-, and 5-year survival rates after HCC diagnosis
progressively decreased with increasing serum AFP
levels, p-value <0.0001.

AFP Levels

Patients (%)
1-Year
survival
rate
3-Year
survival rate
5-Year
survival
rate
<10
196(13)
67% 37% 24%
10-<100
322(22)
56% 25% 15%
100-<1000
238(16)
37% 13% 8%
1000
308(21)
12% 2% 1%
Not tested
416(28)
49% 24% 16%
Tyson et al. Clin Gastroenterol Hepatol. 2011 9(11): 989-994
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 2 4 6 8 10
S
u
r
v
i
v
a
l


Year
<10
10-<100
100-<1000
>=1000
Not tested
Survival of Patients with HCC
Stratified by Serum AFP Levels
Tyson et al. Clin Gastroenterol Hepatol. 2011 9(11): 989-994
*AFP at HCC
diagnosis
Adjusted Hazard
Ratio (95% CI)
P-value
<10 Reference
10-<100 1.50 (1.22-1.83) <0.0001
100-<1000 2.23 (1.80-2.76) <0.0001
1000 4.35 (3.54-5.36) <0.0001
Not tested 1.53 (1.26-1.86) <0.0001
AFP Levels and Mortality Risk in 1480 HCC Patients:
Multivariate Cox Proportional Hazard Model
* Adjusted for age, sex, race/ethnicity, ascites, encephalopathy,
MELD, HCC treatment
Tyson et al. Clin Gastroenterol Hepatol. 2011 9(11): 989-994
0
.2
.4
.6
.8
1
0 1 2 3 4 5 6 7 8 9
R
e
c
u
r
r
e
n
c
e

r
a
t
e

Post-transplant year
0
.2
.4
.6
.8
1
0 1 2 3 4 5 6 7 8 9
R
e
c
u
r
r
e
n
c
e

r
a
t
e

Post-transplant year
400 (n=115) 5-y 13%
>400 (n=29) 5-y 42%
>400(n=29) 5-y 54%
400 (n=107) 5-y 10%
AFP (ng/ml)
P=0.0003
DCP (mAU/ml)

P<0.0001
Courtesy of Hiroto Egawa
Preoperative Tumor Markers and
Risk of Recurrence after LDLT
Variables risk ratio 95% CI p value
Number 11 2.786 1.002-7.752 0.0495
Size >5 cm 7.246 1.639-31.250 0.0083
Beyond MC 1.447 0.376-5.573 0.5909
AFP > 400 1.389 0.352-5.490 0.6389
DCP > 400 6.289 2.320-16.949 0.0003
Preoperative Tumor Markers and
Risk of Recurrence after LDLT
0
.2
.4
.6
.8
1
0 1 2 3 4 5 6
Exceeded (n=44) 5-y 56%
Met (n=93) 5-y 4%
R
e
c
u
r
r
e
n
c
e

r
a
t
e

Post-transplant year
n10 and diameter5cm and DCP 400
7 8
p<0.0001
9
Kyoto Criteria and Recurrence Rate
0
.2
.4
.6
.8
1
0 1 2 3 4 5 6
Exceeded (n=44) 5-y 31%
Met (n=93) 5-y 87%
S
u
r
v
i
v
a
l

r
a
t
e

Post-transplant year
7
p<0.0001
8 9
n10 and diameter5cm and DCP 400
Kyoto Criteria and Patient Survival Rate
Acknowledgments for data, slides and mentorship


Lewis Roberts
Hashem el Serag
Morris Sherman
Hiroto Egawa
Richard Sterling