Clinical Trial Details
— Status: Not yet recruiting
Administrative data
| NCT number |
NCT02448056 |
| Other study ID # |
201504020RINC |
| Secondary ID |
|
| Status |
Not yet recruiting |
| Phase |
N/A
|
| First received |
May 10, 2015 |
| Last updated |
May 14, 2015 |
| Start date |
June 2015 |
| Est. completion date |
May 2025 |
Study information
| Verified date |
May 2015 |
| Source |
National Taiwan University Hospital |
| Contact |
Ja-Der Liang, Master |
| Phone |
886-2-23123456 |
| Email |
jdliangntu[@]ntu.edu.tw |
| Is FDA regulated |
No |
| Health authority |
Taiwan: Ministry of Health and Welfare |
| Study type |
Observational [Patient Registry]
|
Clinical Trial Summary
Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the third leading
cause of cancer-related death worldwide. Treatments of HCC include surgical resection, local
therapies such as radiofrequency ablation and ethanol injection, transarterial
chemoembolization, sorafenib and best supportive care. However, even after successful
treatment such as surgical resection, most patients suffered from recurrence or progression
of the tumor. Because clinical staging systems cannot precisely predict the outcome of
patients with HCC, it's of great interest to search serum biomarkers for HCC. Among them,
alpha-fetoprotein (AFP) is the most well-studied. However, the applicability of AFP for HCC
after surgical resection of tumor or after local therapy is still uncertain.
MicroRNAs (miRNAs), 17- to 25-nucleotide non-coding RNAs, are frequently dysregulated in
cancer and emerging as novel non-invasive biomarker for cancer screening, diagnosis, monitor
therapy efficacy and predict prognosis. MiRNAs are stably expression in serum as their
resistance to endogenous RNase and easily storage with high stability. Several studies have
shown abnormal expression of human serum miRNAs in many cancers such as liver, colorectal,
and pancreatic cancer. The sensitivity of miRNA as a diagnostic biomarker of HCC could be
upto 80%. Using miRNA arrays can generate miRNA signatures and improve the sensitivity and
specificity of biomarker for tumor diagnosis and prognosis prediction.
In this study, the investigators will establish an miRNA platform as biomarkers for
diagnostic or prognostic tools of HCC. The investigators will also compare the miRNA
expression level before and after treatment in the serum and correlate the miRNA expression
between serum and tumor tissue.
Description:
Background:
Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third mortality rate
of cancer in the world (Kamangar et al., 2006). Treatments of hepatocellular carcinoma
include surgical resection, local therapies such as radiofrequency ablation (RFA) and
percutaneous ethanol injection (PEI), transarterial chemoembolization (TACE), and best
supportive care(Bruix and Sherman, 2005). For patients who are not candidates for surgical
intervention or local therapies, sorafenib is recommended (Cheng et al., 2009; Llovet et
al., 2008). However, even after successful treatment such as surgical resection, most
patients suffered from recurrence or progression of the tumor (Bruix and Sherman, 2005;
Llovet et al., 2002; Llovet et al., 2008). Though there are several staging systems of HCC
have been developed, such as the Okuda staging system, AJCC system, BCLC staging system, and
CLIP scoring system, the prognostic value of each staging systems are not consistent(Chen et
al., 2009), and applicability of each staging system depends on the treatment methodology
selected.
Because of the shortcoming of clinical staging systems in predicting the outcome of HCC, it
is of great interest to search serum biomarkers to predict the prognosis HCC (Fujiyama et
al., 2002; Marrero and Lok, 2004). Among them, alpha-fetoprotein (AFP) is the mostly
well-studied. AFP has been used as a marker in diagnosing HCC (Bruix et al., 2001) and as a
prognostic factor for newly diagnosed HCC(1998). AFP is also of value as a predictive factor
for chemotherapy response (Chan et al., 2009). However, the applicability of AFP for HCC
after surgical resection of tumor or after local therapy is still uncertain. For patients
with HCC and normal serum level of AFP, APF could not be used a prognostic factor. Other
biomarkers, such as glipecan-3, are still under development (Marrero and Lok, 2004).
MicroRNAs (miRNAs), 17- to 25-nucleotide non-coding RNAs, are frequently dysregulated in
cancer and emerging as novel non-invasive biomarker for cancer screening, diagnosis, monitor
therapy efficacy and predict prognosis (Wu et al., 2007). MiRNAs are stably expression in
serum as their resistance to endogenous RNase and easily storage with high stability (Aref
et al., 2014). Several studies have shown abnormal expression of human serum miRNAs in many
cancers such as liver, colorectal, and pancreatic cancer (Huang et al., 2010; Liu et al.,
2012; Qi et al., 2013). The sensitivity of miRNA as a diagnostic biomarker of HCC could be
up to 80% (Yin et al., 2014). Using miRNA arrays can generate miRNA signatures and further
improve the sensitivity and specificity of biomarker for tumor diagnosis and prognosis
prediction.
Trial purpose：
1. To identify serum miRNA as a diagnosis or prediction biomarker for HCC.
2. To correlate with the expression level of miRNA between serum, HCC tumor tissue and
adjacent non-tumor tissue.
3. To collect and store deoxyribonucleic acid (DNA) for future exploratory research into
genes/genetic variation that may influence response to treatment in HCC.
4. To collect and store surplus plasma and tumor tissues for future exploratory research
into proteins/genes/genetic variation that may influence response to treatment in HCCs.
Methods： Study design This is a prospective study. All patients need to provide informed
consents. A total of 200 patients aged >20 year-old who are diagnosed HCC in the National
Taiwan University and Far-Eastern Memorial Hospital will be enrolled. The diagnostic
criteria of HCC are according to 2011 AASLD criteria (Bruix et al., 2011). In brief, nodules
more than 1 cm found on ultrasound screening of a cirrhotic liver should be investigated
further with one dynamic studies, either CT scan or contrast MRI. If the appearance is
typical of HCC (i.e., hypervascular with washout in the portal/venous phase) in one
technique, the lesion would be diagnosed as HCC. If the findings are not characteristic or
the vascular profile is not coincidental among techniques the lesion should be biopsied. For
clinical staging, two staging systems will be applied to these patients, including BCLC
staging and clinical TNM system. The clinicopathological data will be collected
prospectively. Clinical outcomes, including treatment toxicity, treatment response as
defined by the revised RECIST criteria, disease-progression, and overall survival will be
recorded.
Specimen and serum collection Blood samples will be collected at 3 time
points—pre-treatment, 7 days after treatment and one month after treatment. Blood samples
will be separated in plasma and mononuclear cells upon collection and will be stored in -20Ç
before further use. The treatment methods include operation, TACE, RFA, PEI, or
anti-angiogenesis agent including sorafenib use.
Tissue collection The HCC tissues (T) and the corresponding non-tumor liver tissues (NT)
will be obtained. The surgical specimens will be frozen immediately after surgery and stored
in -140°C.
Viral marker Hepatitis B surface antigen (HBsAg) and antibody to HCV (anti-HCV) will be
checked using commercially available enzyme linked immunosorbant assay kits for every
patient. For HBsAg positive patient, HBeAg and anti-HBe will be measured by enzyme linked
immunosorbant assay. Serum level of HBV DNA will be measured by in house TaqMan real-time
polymerase chain reaction (PCR). For anti-HCV positive patients, HCV RNA will be measured by
in house TaqMan real-time PCR
RNA isolation, reverse transcription and miRNA array Blood samples drawn into EDTA
containing tubes and centrifuged at 4,000x g for 15 min for plasma separation. Plasma
transferred into a clean micro centrifuge tube and centrifuged again at 12,000x g for 5 min
and 200 ul of plasma will be transferred to a new micro centrifuge tube and stored at -80 _C
until analysis. RNA will isolated using High Pure miRNA Isolation Kit (Roche, Mannheim,
Germany) according to the manufacturer's instructions and then stored at -80 _C until the
experiment.
Reverse transcription reaction For miRNA analyses by quantitative real-time polymerase chain
reaction (qRT-PCR), 100 ng of total RNA will be reverse-transcribed using the TaqMan miRNA
Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA). All reactions will be
performed as specified in the manufacturers protocol.
TaqMan low-density arrays TLDA will be analysed to identify the profile of differentially
expressed miRNAs between the three sets of samples (pre-treatment, 7 days post-treatment,
one month post treatment). In brief, total RNA will reverse-transcribed into cDNA by the
TaqMan MicroRNA Reverse Transcription Kit and Megaplex RT set pool A and B version 3.0
(Applied Biosystems). The RT product will loaded into TaqMan Array Human MicroRNA A+B Cards
Set v3.0 (Applied Biosystems), enabling simultaneous quantitation of 667 human miRNAs.
TaqMan microRNA assays and analysis will be performed on the ABI 7900HT Instrument (Applied
Biosystems). All reactions were performed according to the standard manufacturers'
protocols. Quantitative miRNA expression data were acquired and normalized using ABI 7900HT
SDS software (Applied Biosystems).
Quantitative Real-Time PCR (qRT-PCR) Expression of miRNA detected via TaqMan low-density
arrays will also be studied as well in the HCC tumors and / or their paired normal liver
tissue by qRT-PCR analysis as described previously (Lee et al., 2011). RNU6B snRNA was used
as an endogenous control. Each microRNA assay was performed in triplicate. Expression of
microRNAs was reported as delta Ct value (Ct value of RNU6B - Ct value of target microRNA).
The investigators defined the groups of tumors or cells with high or low expression based on
the median expression value of each microRNA.
Statistical analysis Overall survival and progression-free survival will be evaluated using
Kaplan-Meier's method. Once a specific protein is identified by the proteomic study,
patients will be divided into high expression group and low expression group, according to
the median plasma expression level of the miRNA of all patients; survival difference between
high expression group and low expression group will be analyzed using log-rank test. p-value
less than 0.05 will be regarded as significant.
