AFP-L3% : IS IT AN EARLY MARKER FOR HCC ?

1. AFP-L3% : IS IT AN EARLY
MARKER FOR HCC ?
Prof./ Moustafa Rizk
Clinical pathology department
Faculty of Medicine, Alexandria University

2. AFP is an oncofetal glycoprotein that
frequently reappears in HCC and yolk
sac tumors. However, slightly elevated
serum levels of AFP are also seen in
patients with hepatitis and LC .

3. serum AFP levels of patients with
HCC at the initial detection became
lower than before .

4. This has resulted in a wide overlap
in measured AFP concentrations
among patients with LC and HCC .
(AFP levels between 20-200 ng/ml)

5. However, AFP results are often not
definitive until a tumor has
reached a large size or an
advanced state.
AFP levels between 200-500,000 ng/ml

6. LC is known as an underlying premalignant lesion
of HCC, and hence more specific and sensitive
markers of HCC are desirable in monitoring
patients for evolution of HCC.

7. AFP GLYCOFORMS
HOW ?
1-Isoelectric focusing
2-Affinity electrophoresis
3-Liquid-phase Binding Assay System

8. 1-Isoelectric focusing
Using isoelectric focusing (IEF), AFP isoform Band +II is
relatively specific for HCC. Occasionally, another tumor-
specific isoform, Band +III AFP, is also present in HCC
cases. Furthermore, studies conducted in this lab
suggest that screening for the Band +II isoform allows
early, even preclinical, diagnosis of HCC in high-risk
patients.
Identification and evaluation of the use of alpha feto-protein (AFP) tumour specific variants in HCC diagnosis
Johnson et al Br J Cancer 1997
Liver Cirrhosis Germ cell tumours
Hepatocellular Carcinoma
IEF of Bands I, II and III AFP

9. Band +II AFP - a better diagnostic marker for early diagnosis of HCC
Serum Band+II AFP isoform, but not total serum AFP level, can be used to
differentiate early liver cancer (HCC) from liver cirrhosis.
Identification and evaluation of the use of alpha feto-protein (AFP) tumour specific variants in HCC diagnosis Johnson at al Br J
cancer 1997.

10. During carcinogenesis ( development
of HCC) the sugar chain is modified
by fucosyltransferase.
FucFuc α1
↓
6
FucFuc α1
↓
6
This carbohydrate complex reacts with Lens culinaris (lentil) agglutinin (LCA).
α1,6 fucose residue
N-acetylglucosamine
carbohydrate chain on AFP

11. 2-Affinity electrophoresis
AFP can be fractionated by affinity electrophoresis into 3
glycoforms: L1, L2, and L3 based on the reactivity with
the lectin Lens culinaris agglutinin (LCA).
AFP-L3 binds strongly to LCA via an additional α 1-6
fucose residue attached at the reducing terminus of N-
acetylglucosamine; this is in contrast to the L1
isoform.

13. Glycoforms of Alpha-Fetoprotein (AFP)
Glycoform Affinity for LCA* Comment
AFP-L1 Low Associated with inflamm. Chronic L.
AFP-L2 Intermediate Derived from yolk sac + tumors
AFP-L3 High Expressed by malignant hepatocytes
* LCA - Lens culinaris agglutinin.

14. 3-Liquid-phase Binding Assay System
The L3 form of AFP is separated from other AFP
forms using a special chemical method.
When a specific chemical is added, a fluorescent
reaction is produced and is measured .
The amount of fluorescence produced shows the
level of AFP-L3 and total AFP in the blood.
The more AFP-L3 in the sample, the greater the
chance the patient will develop liver cancer.

15. 3 anti-AFP monoclonal antibodies
LCA binds to the sugar chain
anion-exchange chromatograph
anion 1 conjugated anti-AFP antibody
The peroxidase-labeled anti-AFP monoclonal antibody
quantified fluorometrically
complexes 1 and 2

16. L1L1L3L3
YS8
POD
YS5
L1
L1 + L3
AFP concentrationAFP concentration
L1 + L3
L3
× 100
AFP-L3%AFP-L3%
YS5
L3
LCA
POD
If the AFP-L3% is greater or equal to 10%, the risk is seven-fold.

17.  Taketa et. al. reported in 1993 in a clinical study which tested
AFP-L3% in 424 subjects with acute hepatitis, chronic hepatitis,
liver cirrhosis, HCC, and extrahepatic tumors.
The results were approximately 38% of tumors less than 2 cm in
diameter showed AFP-L3% elevations (≥10%) and these
elevations were observed 4-5 months before detection of HCC
by imaging techniques with a sensitivity of 48% and a specificity
of 81%.
In the same year, Sato et. al. published their data in New
England Journal of Medicine, in which they followed 361 subjects
with L3% elevations within 3 to 18 months, before the HCC was
detected by imaging techniques.
Similar results were observed by Shiraki et. al. and other
investigators at different sites.

18. AFP-L3% is a superior indicator of
relative risk
Leerapun, A. et al.Tthe utility of AFP-L3% in the diagnosis of
hepatocellular carcinoma: evaluation in U .S. referral population.
Gastroenterology, April 2006; 130, S2:774

19. AFP-L3%
Clinical significance – HCC detection
 Additional marker for HCC detection of higher specificity (~90%), medium sensitivity
(~50%) and much higher positive predictive value than AFP (~35% versus ~20%)
demonstrated in many publications.
 Use in addition with AFP can increase the overall sensitivity of detection. -
Volk et al. 2007 Cancer biomarkers
cutoff
cutoff
 Comparison of AFP and AFPL3% levels in cirrhotic patients in contrast
to HCC patients (n=253):

20. This paper lists and reviews serum HCC biomarkers
and categorizes them as: 1. Oncofetal antigens
and glycoprotein antigens,2. enzymes and
isoenzymes, 3. genes, 4. cytokines.
Serum tumor markers for detection of hepatocellular carcinoma. Zhou L, et al., World J Gastroenterol. 2006;
12(8):1175-1181
The authors conclude that “…AFP, AFP-L3 and DCP are the most
useful serum tumor markers for the detection of HCC, and
simultaneous determination of these markers could improve the
accuracy, especially in differentiating HCC from nonmalignant
hepatopathy.”

21. AFP-L3%
Clinical significance – indicator of malignancy
 The interpretation of positive AFP and AFP-L3% results is different, AFP
may be an indicator of tumor mass and inflammatoric processes, AFP-L3%
of high malignant potential.
 AFP-L3% positive HCC has malignant characteristics such as portal vein
invasion and lower tumor classification. Oka et al. 2001 J Gastroenterol
Hepatol
 The AFP-L3% is closely related to HCC differentiation, which can be
considered as useful adjunct in HCC diagnosis. Khien et al. 2001 Int J Biol
Markers

22. Clinical significance – indicator of malignancy
Small HCC tumors, less than 2 cm in diameter, were
more likely to be aggressive cancer in clinical settings, if
AFP-L3% was greater than 10%, the current cutoff for a
positive test.
In non-malignant chronic liver disease, liver cells did not
appear to express AFP-L3%, therefore, a higher level of
AFP-L3% could be an indicator of tumor occurrence and
aggressiveness.

23. Clinical significance – follow-up, prognosis
 AFP-L3% levels before and/or after HCC treatment give indications on
recurrence rate and overall survival.
Aoyagi et al. 1998 Am. Cancer. Soc.
 E. g. study with 112 HCC patients,
PEI and TACE, AFP-L3>18% before
treatment:
Hayashi et al. 1999 Am. J. Gastroent.
 E. g. study with 60 HCC patients (<2 cm
tumors), resection, PEI and TACE, AFP-
L3>10% after treatment:
AFP-L3%+
AFP-L3%-

24. Limitations
1- Not all liver cancers are AFP-secreting.
2- AFP may be elevated due to germ cell
tumors and other carcinomas.
3- Viral infections, cirrhosis, and other liver
diseases may elevate AFP.
4- Samples from patients who are pregnant, less
than one year of age, or with acute or fulminant hepatitis can
show elevated values of AFP-L3% and total AFP .

25. Conclusions
• Both total AFP and AFP-L3% are quantified in a single serum sample.
The AFP-L3% is considered elevated (positive) if it is >10 percent of
the total AFP.
• Elevated AFP-L3% values (>10 percent) are associated with a seven-
fold increase in the risk of developing hepatocellular carcinoma
(HCC) within the next 21 months.
• AFP-L3% becomes elevated three to 21 months before HCC can be
detected by standard imaging techniques.
• While total AFP may be related to clinical stage, the AFP-L3% is an
indicator of biological behavior (aggressiveness) of HCC. AFP-L3%
positive HCC has the potential for rapid growth and early distant
metastasis.
• The high specificity of AFP-L3% makes it a desirable marker for
following patients after therapy. A positive result that subsequently
turns negative for AFP-L3% is usually indicative of successful
treatment of HCC.

26. Thank youProf./ Moustafa Rizk