This document summarizes a study examining circulating free tumor DNA (cfDNA) as a diagnostic biomarker for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) in Nigerian patients. The study found that cfDNA levels were higher in patients with HCC compared to non-HCC patients, and that cfDNA concentration alone had moderate accuracy for diagnosing HCC. The diagnostic accuracy was improved when cfDNA concentration was combined with alpha-fetoprotein levels. While the common TP53 gene mutation was found in HCC patients, detection of this mutation had lower diagnostic utility than cfDNA concentration alone or combined with AFP. The study concludes that cfDNA quantification alone or combined with AFP may be useful for

1. Table 1.Clinical characteristics of study participants
CIRCULATING FREE TUMOUR DNA CONCENTRATION IS A USEFUL DIAGNOSTIC
BIOMARKER IN HBV-RELATED HEPATOCELLULAR CARCINOMA IN AFRICANS
BACKGROUND
Sub-Saharan Africa (SSA) has one of the highest incidence rates
of hepatocellular carcinoma (HCC) in the world1
. In West Africa,
HCC is the most common cancer among men and second most
common among women2
.
Hepatitis B (HBV) and aflatoxin exposure through contaminated
groundnut consumption are the most common aetiologies**. The
TP53 gene 249Ser mutation induced by aflatoxin is common in
HCC in West African patients.
Circulating free tumour DNA (cfDNA) is released by necrotic HCC
cells into blood and can be detected using quantitative PCR,
providing a potential “liquid biopsy” of cancer3
. In West Africa, the
common TP53 249Ser gene mutation has been shown to be
detectable in cfDNA4
. cfDNA detection is therefore an attractive
biomarker for cancer diagnosis in resource limited settings, where
access to radiology and histopathology services is limited.
OBJECTIVES
1. To measure and compare cfDNA levels in patients with and
without HBV-related HCC
2. To measure and compare TP53 mutation frequency in cfDNA
in patients with and without HBV-related HCC
3. To determine the diagnostic accuracy of cfDNA quantification
and TP53 mutation detection in cfDNA for HBV-related HCC
diagnosis
RESULTS
 All HCC were advanced stage IV tumours
 All patients with HBV infection had genotype E virus
 None were co-infected with hepatitis delta virus
CONCLUSIONS
REFERENCES
MATERIALS & METHODS
Sixty-six Nigerian patients underwent liver disease evaluation with
clinical examination, liver function tests, alpha-fetoprotein and
ultrasound.
Access to CT scans and MRI is severely limited in Nigeria, therefore a
modified criteria for HCC diagnosis appropriate for resource-limited
settings was used. HCC was diagnosed by characteristic findings on
ultrasound or histopathology and an elevated alpha-fetoprotein
>400IU/mL.
cfDNA was isolated from 250μL of plasma using QiaAmp circulating
DNA kit (Qiagen). Plasma cfDNA was quantified with real time PCR
using TaqMan TP53 249Ser Mutation detection assay
(Invitrogen***lower limit of detection) and TP53 Reference assay,
performed on a StepOnePlus instrument (Invitrogen). Targetted
sequencing of the TP53 249 codon was performed in a subgroup of 16
patients to confirm the findings of the Taqman TP53 assay.
 cfDNA levels are elevated in advanced HCC in West Africans
and have moderate accuracy for HCC diagnosis.
 cfDNA concentration combined with AFP had excellent
accuracy for HCC diagnosis.
 Though TP53 mutation is common in HCC, TP53 mutation
detection appears dependent on cfDNA concentration and
has lower utility for HCC diagnosis.
 Our data suggest cfDNA quantification alone or in
combination with AFP may prove useful for HCC diagnosis in
resource-poor settings.
1. Parkin DM et al; Cancer J Clin 2005; 55(2). 2.Parkin DM et al; Lancet Oncol 2008; 9(7)
3. Alix=Panabieres C et al; Annu Rev Med 2012; 63. 4. Kirk GD et al; J Nat Cancer Ins 2000; 92(2)
Contact Information
Dr S Knapp: s.knapp@imperial.ac.uk; Dr J Howell: j.howell@imperial.ac.uk
S Knapp1
, J Howell1
, Y Sherwani1
, M Shariff1
, L Karanfilian1
, N Ladep1,2
, E Okeke2
, S Taylor-Robinson1
, M Thursz1
.
Department of Hepatology, Imperial College, London, UK1
; Deparment of Medicine, Jos University Teaching Hospital; Jos, Nigeria2
.
Healthy Controls
(n=16)
HBV CLD, no
cirrhosis
(n=11)
HBV cirrhosis
(n=11)
HBV HCC
(n=26)
Mean Age
(+/-sd)
44.19
+/-16.75
42.09
+/-11.23
45.00
+/- 11.39
47.50
+/-16.50
Gender (%) 7 M (43.8%)
9 F (56.2%))
6 M (54.5%)
5 F (45.5%)
9 M (81.8%)
2 F (18.2%))
17 M (63.4%)
9 F (36.6%)
Mean α-FP
(+/- sd)
2.14
+/- 1.70
3.72
+/- 3.04
175.21
+/- 547.27
15434.38
+/- 13730.97
cfDNA concentration in plasma is higher in HCC
compared with non-HCC patients
cfDNA concentration has moderate HCC diagnostic accuracy alone
and good accuracy when combined with AFP
P=0.0003 P=0.004
AUC=0.762 AUC=0.958
ROC cfDNA concentration alone
for HCC diagnosis
ROC cfDNA concentration and AFP
for HCC diagnosis
TP53 Ser249 concentration has poor HCC diagnostic accuracy alone and
does not improve accuracy of cfDNA concentration for HCC diagnosis
AUC=0.661 AUC=0.778
TP53 mutation mutation is more frequent in HCC than non-HCC patients
ROC TP53 mutation concentration alone
for HCC diagnosis
ROC TP53 mutation concentration with cfDNA
concentration for HCC diagnosis
TP53 mutation concentration in cfDNA
by disease stage
Table 2.Number with TP53 mutation (p=0.006)
HCC Cirrhosis CLD Healthy controls
0
1
2
3
ConcentrationTP53SerDNA(ng/μL)
P=0.026
cfDNA concentration in HCC and no HCC patients cfDNA concentration by liver disease stage
TP53 249Ser
positive
(n=17)
TP53 249Ser
negative
(n=47)
Healthy
controls
(n=16)
2 (12.5%) 14 (87.5%)
HBV CLD,
no cirrhosis
(n=11)
3 (27.3%) 8 (72.7%)
HBV cirrhosis
(n=11)
1 (9.1%) 10.(91.9%)
HBV HCC
(n=26)
11 (42.3%) 15 (57.7%)