Tumor Marker Lab

Research in the Tumour Marker Laboratory involves two main areas: screening of novel anticancer drugs and the study and identification of cancer markers for drug targeting, diagnosis and prognosis. Successful application of these markers would be very useful in: 1) screening for the presence of cancer; 2) diagnosing a specific type of cancer; 3) determining prognosis and 4) monitoring the course of remission following treatment. Our focus is on cancers commonly found in Southeast Asia including Hong Kong and China such as lung cancer, hepatocellular carcinoma (HCC) and nasopharyngeal carcinoma (NPC).

Screening for the anticancer properties of novel drugs is performed using both in-vitro cell culture systems and in-vivo animal models in our lab. Currently, we are actively involved in the screening of novel drugs for the treatment of lung cancer and NPC. The pre-clinical evaluation of such drugs has laid down the foundation for their use in the clinic for patient treatment. Elucidation of protein targets of different drugs also allows us to find better markers for a particular cancer type.

With the use of state-of-the-art equipment, analyses of global differences in protein / peptide expression patterns are performed in our lab using 2D-PAGE, MALDI-TOF, SELDI, HPLC and ICAT. Specific proteins of interest are also studied using immunohistochemical technique. Differentially expressed proteins / peptides, in combination with clinical data, are then evaluated for their potential as tumour markers which are then further characterized by molecular technique such as quantitative RT-PCR.

Current and previous research topics from our lab include:
1.Identification of differentially expressed proteins using proteomic approaches
2.Evaluation of novel drugs for their anticancer properties
3.Identification and evaluation of the use of alpha feto-protein (AFP) tumour specific variants in HCC diagnosis
4.Development of computer algorithms (Neural Network, Classification Tree) integrating tumor marker information and patient variables for improving the diagnosis of cancers such as HCC

Publications

Identification of differentially expressed proteins using proteomic approaches

2D-PAGE
Metabolic enzymes involved in glycolysis and gluconeogenesis including pyruvate kinase, phosphopyruvate hydratase, phosphoglycerate mutase A, and fructose-bisphosphate aldolase were found to be present at higher levels in HCC cell lines as compared to a hepatoblastoma cell line, HepG2. On the other hand, enzymes involved in energy metabolism, including nicotinate-nucleotide pyrophosphorylase and adenylate kinase 3, and heat shock protein isoforms hsp60 and hsp70, were found to be higher in HepG2 cells. These suggest that the energy metabolism and regulation of intracellular homeostasis may be different between HepG2 and HCC cells.

Figure 1. 2D-PAGE of HepG2 cell lysate

ICAT
Using Isotope-Coded Affinity Tags (ICAT), we have compared the proteomes of two NPC cell lines, NP69 which is a normal NP cell line and C666-1, a nasopharyngeal carcinoma cell line. Several differentially expressed proteins have been identified including NF-kB p65, Syntaphilin, Kringle-containing transmembrane protein of kremen 2 gene and seven transmembrane helix receptor. In addition, two novel proteins were found including proteins for MCG:43116 and MCG:39573.

Evaluation of novel anticancer drugs

In-vitro drug testing
We have previously shown that over 85% of nasopharyngeal carcinomas (NPC) in Hong Kong demonstrate moderate to strong expression of the epidermal growth factor receptor (EGFR), overexpression of which was associated with poor prognosis. Results from our lab indicate that cetuximab (C225), a humanized chimeric monoclonal anti-EGFR antibody, was capable of enhancing the effects of an anti-cancer drug, paclitaxel, in an additive manner (figure 2). This suggests the possibility of using cetuximab in combination with paclitaxel to improve therapeutic outcomes of NPC patients.

Figure 3. Immunohistochemical staining of the epidermal growth factor receptor (EGFR) in an NPC tissue sample

Models
Apart from in-vitro models for drug testing, our lab also develops in-vivo mouse models to further investigate the effectiveness of novel anticancer drugs. This allows us to evaluate a certain drug in a more similar context as seen in human cancer patients.

Identification and evaluation of the use of alpha feto-protein (AFP) tumour specific variants in HCC diagnosis

Serum -fetoprotein (AFP) is commonly increased in patients with hepatocellular carcinoma, with concentrations ranging from 10 to >500 μg/L. Concentrations greater than 500 μg/L are usually considered diagnostic of hepatocellular carcinoma (HCC). However, moderately increased serum AFP (10–500 μg/L) is also common in nonmalignant chronic liver diseases, leading to low specificity of the AFP test for HCC. This represents a serious drawback as most cases of HCC arise in patients with concurrent chronic liver disease

Figure 1 IEF of Bands I, II and III AFP. LC = Liver Cirrhosis;
HCC = Hepatocellular Carcinoma; GCT = Non-seminomatous germ cell tumours

Using isoelectric focusing (IEF), we have shown that AFP isoform Band +II is relatively specific for HCC. Occasionally, another tumor-specific isoform, Band +III AFP, is also present in HCC cases. We have successfully determined that while Band +I AFP is disialylated AFP, Band +II AFPs are composed of monosialylated AFP. 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.