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.

MALDI-TOF Mass Spectrometer

SELDI		Multidimensional HPLC

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:

1. Hui EP. Poon TC. Teo PM. Mo F. Zee B. Leung SF. Ho S. Mok TS. Kwan WH. Johnson PJ. Chan AT. A prospective study of pre-treatment cell kinetics and clinical outcome in nasopharyngeal carcinoma. Radiotherapy & Oncology. 69(1):53-62, 2003 Oct.
2. Ma BB. Poon TC. To KF. Zee B. Mo FK. Chan CM. Ho S. Teo PM. Johnson PJ. Chan AT. Prognostic significance of tumor angiogenesis, Ki 67, p53 oncoprotein, epidermal growth factor receptor and HER2 receptor protein expression in undifferentiated nasopharyngeal carcinoma--a prospective study. Head & Neck. 25(10):864-72, 2003 Oct.
3. Poon TC. Yip TT. Chan AT. Yip C. Yip V. Mok TS. Lee CC. Leung TW. Ho SK. Johnson PJ. Comprehensive proteomic profiling identifies serum proteomic signatures for detection of hepatocellular carcinoma and its subtypes. Clinical Chemistry. 49(5):752-60, 2003 May.
4. Hui EP. Chan AT. Pezzella F. Turley H. To KF. Poon TC. Zee B. Mo F. Teo PM. Huang DP. Gatter KC. Johnson PJ. Harris AL. Coexpression of hypoxia-inducible factors 1alpha and 2alpha, carbonic anhydrase IX, and vascular endothelial growth factor in nasopharyngeal carcinoma and relationship to survival. Clinical Cancer Research. 8(8):2595-604, 2002 Aug.
5. Poon TC. Mok TS. Chan AT. Chan CM. Leong V. Tsui SH. Leung TW. Wong HT. Ho SK. Johnson PJ. Quantification and utility of monosialylated alpha-fetoprotein in the diagnosis of hepatocellular carcinoma with nondiagnostic serum total alpha-fetoprotein. Clinical Chemistry. 48(7):1021-7, 2002 Jul.
6. Poon TC. Chan AT. Zee B. Ho SK. Mok TS. Leung TW. Johnson PJ. Application of classification tree and neural network algorithms to the identification of serological liver marker profiles for the diagnosis of hepatocellular carcinoma. Oncology. 61(4):275-83, 2001.
7. Poon TC. Johnson PJ. Proteome analysis and its impact on the discovery of serological tumor markers. Clinica Chimica Acta. 313(1-2):231-9, 2001 Nov.
8. Johnson PJ. Poon TC. Hjelm NM. Ho CS. Blake C. Ho SK. Structures of disease-specific serum alpha-fetoprotein isoforms. British Journal of Cancer. 83(10):1330-7, 2000 Nov.
9. Chan MH. Shing MM. Poon TC. Johnson PJ. Lam CW. Alpha-fetoprotein variants in a case of pancreatoblastoma. Annals of Clinical Biochemistry. 37 (Pt 5):681-5, 2000 Sep.
10. Chan AT. Ho S. Teo PM. Tjong J. Choi J. Lee WY. Chang AR. Kwan WH. Leung WT. Johnson PJ. Assessment of proliferating cell nuclear antigen in nasopharyngeal carcinoma tissue and its relation to clinical findings. Oral Oncology. 33(1):13-8, 1997 Jan.
11. Johnson PJ. Leung N. Cheng P. Welby C. Leung WT. Lau WY. Yu S. Ho S. 'Hepatoma-specific' alphafetoprotein may permit preclinical diagnosis of malignant change in patients with chronic liver disease. British Journal of Cancer. 75(2):236-40, 1997.
12. Chan AT. Ho S. Teo PM. Law V. Tjong J. Yu P. Chang AR. Kwan WH. Leung WT. Johnson PJ. In vitro uptake of bromodeoxyuridine by human nasopharyngeal carcinoma (NPC) and its relation to clinical findings. European Journal of Cancer. Part B, Oral Oncology. 32B(1):50-4, 1996 Jan.
13. Chan AT. Ho S. Yim AP. Chang AR. Cheng P. Yuen J. Leung TW. Johnson PJ. Primary mediastinal malignant germ cell tumour. Single institution experience in Chinese patients and correlation with specific alpha-fetoprotein bands. Acta Oncologica. 35(2):221-7, 1996.
14. Ho S. Cheng P. Yuen J. Chan A. Leung N. Yeo W. Leung T. Lau WY. Li AK. Johnson PJ. Isoelectric focusing of alphafetoprotein in patients with hepatocellular carcinoma--frequency of specific banding patterns at non-diagnostic serum levels. British Journal of Cancer. 73(8):985-8, 1996 Apr.

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.

Figure 2. Typical mass spectra of ICAT-labelled peptides

Figure 3. PSD spectrum of a D8-labelled peptide (m/z 1427.8). The sequence was identified as EASLVVTCR

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 1. NPC cell lines, HK1, Hone1 and C666-1 are used for in-vitro drug testing.

Figure 2. The percentage of cell death (mean ± SEM) of HK1 cells after combined treatment with paclitaxel and cetuximab at different concentrations.

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.

Figure 4. Nude mouse with cell line derived xenograft

Figure 5. Histological section of a xenograft derived from C666-1 cells

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.

Figure 2. Band +II AFP - a better diagnostic marker for early diagnosis of HCC

Figure 3. Serum Band+II AFP isoform, but not total serum AFP level, can be used to differentiate early liver cancer (HCC) from liver cirrhosis (LC)