Wai-Wai Cheng

Identification of serum amyloid A protein as a potentially useful biomarker to monitor relapse of nasopharyngeal cancer by serum proteomic Profiling

Purpose: Nasopharyngeal cancer (NPC) is a common cancer in Hong Kong, and relapse can occur frequently. Using protein chip profiling analysis, we aimed to identify serum biomarkers that were useful in the diagnosis of relapse in NPC. Experimental Design: Profiling analysis was performed on 704 sera collected from 42 NPC patients, 39 lung cancer patients, 30 patients with the benign metabolic disorder thyrotoxicosis (TX), and 35 normal individuals (NM). Protein profile in each NPC patient during clinical follow up was correlated with the relapse status. Results: Profiling analysis identified two biomarkers with molecular masses of 11.6 and 11.8 kDa, which were significantly elevated in 22 of 31 (71%) and 21 of 31 (68%) NPC patients, respectively, at the time of relapse (RP) as compared with 11 patients in complete remission (CR; RP versus CR, P = 0.009), 30 TX (RP versus TX, P < 0.001), or 35 NM (RP versus NM, P < 0.001). The markers were also elevated in 16 of 39 (41%) lung cancer patients at initial diagnosis. By tryptic digestion, followed by tandem mass spectrometry fragmentation, the markers were identified as two isoforms of serum amyloid A (SAA) protein. Monitoring the patients longitudinally for SAA level both by protein chip and immunoassay showed a dramatic SAA increase, which correlated with relapse and a drastic fall correlated with response to salvage chemotherapy. Serum SAA findings were compared with those of serum Epstein-Barr virus DNA in three relapsed patients showing a similar correlation with relapse and chemo-response. Conclusions: SAA could be a useful biomarker to monitor relapse of NPC.

Remarkable Application of Serum EBV EBER-1 in Monitoring Response of Nasopharyngeal Cancer Patients to Salvage Chemotherapy

Abstract: Nineteen consecutive patients with metastatic or recurrent nasopharyngeal cancer (NPC) receiving combination chemotherapy were monitored for EBV DNA in their serum. EBV DNA (EBER‐1) concentration in serum was measured before, during, and after chemotherapy. Thirteen patients had additional multiple prechemotherapy readings. There was a significant lead time from first detection of serum EBER‐1 to clinical recurrence in 62% of patients by a mean of 17.4 weeks (range: 8–74.5 weeks; mean = 28.2 weeks if confined to the 8 patients with significant lead time). The median EBER‐1 concentration was significantly higher in those with distant metastasis as compared to those with loco‐regional recurrence only (17,468 vs. 684 pg/mL serum; p= 0.046, Mann‐Whitney U test). Among the 13 patients who responded to chemotherapy, 4 exhibited clinical complete remission (CR) who were only found in the group with EBER‐1 DNA drop to background level, while the magnitude of EBER‐1 drop did not discriminate partial remission (PR) and stable disease (SD) patients clearly. Subsequent profile of EBER‐1 DNA showed concordance with clinical course of either continuous remission or later progression. EBER‐1 DNA in serum can become a useful adjunctive surrogate marker to monitor chemotherapeutic response in NPC patients with distant metastasis or advanced local recurrence.

Role of MIF/CXCL8/CXCR2 signaling in the growth of nasopharyngeal carcinoma tumor spheres

Macrophage migration inhibitory factor (MIF) and CXCL8 (also named IL-8) are strongly expressed in the tissues of nasopharyngeal carcinoma (NPC). However, their role in the growth of NPC has not been fully examined. This study aims to evaluate the functions of MIF and CXCL8 on the growth of NPC tumor spheres. The elevated expression of CXCL8 in tumor over normal tissues was confirmed in 37 pairs of biopsies from NPC patients. In the in vitro study, all the poorly differentiated NPC cell lines, including the EBV-positive C666-1, and the EBV-negative CNE-1, CNE-2, SUNE-1, HNE-1 and HONE-1 cells, were found to express CXCL8 and MIF. Therefore, the EBV-positive C666-1 cell was selected to examine for the role of MIF and CXCL8 in the growth of the NPC tumor spheres. Functional study showed that the growth of C666-1 tumor spheres, under the nutrient poor or growth factor supplemented culture conditions, could be inhibited by the CXCL8 specific peptide inhibitor. The growth of the tumor spheres could also be reduced by the CXCR2 specific inhibitor SB225002 or the PI3K/AKT inhibitor LY294002, indicating that the endogenously produced CXCL8 plays an autocrine role in the growth of the tumor spheres. Further mechanistic studies revealed that the gene expression of CXCL8 could be reduced by the MIF specific small interfering RNA (siRNA) or NF-κB inhibitor parthenolide, and the growth of tumor spheres was also reduced after MIF siRNA transfection. Taken together, the present study highlights the role of MIF/CXCL8/CXCR2 axis in the growth of NPC tumor spheres. Chemotherapeutic interference of this signaling pathway may help to control the growth of the NPC tumor.

Abstract 3153: A new circulating plasma/serum Epstein-Barr virus (EBV) DNA test by locked nucleic acid (LNA) PCR technique with enhanced sensitivity in diagnosis & monitoring of patients suffering from nasopharyngeal carcinoma (NPC) and other EBV associated malignancies

Circulating plasma/serum Epstein-Barr virus (EBV) test has been established as routine first line diagnostic test for Nasopharyngeal Carcinoma (NPC). This real time quantitative PCR test is useful in primary diagnosis of NPC as well as monitoring distant metastases of NPC to lung, liver and distant lymph nodes. However, it is still lacking sensitivity in diagnosis of early stage I/II NPC and local relapse in nasopharynx. Detection sensitivity of this PCR test relies on detecting small fragments of circulating EBV DNA shed into the blood stream from NPC tumor lesions which underwent apoptosis/necrosis. Previous literature already showed that the smaller the molecular size of the circulating EBV DNA targets detected, the higher the detection sensitivity. Using Locked Nucleic Acid (LNA) PCR technique, we have designed a new set of LNA primers for PCR amplification of a much smaller EBV gene target (BamH1-W DNA at 46 bp) than the conventional EBV gene target (BamH1-W at 76 bp). Using this LNA marker, we successfully achieved detection sensitivity of 95.7% in 46 NPC patients (44/46 positive) and detection specificity of 98.3% in 59 normal blood screening subjects (58/59 negative). 89.1% (i.e. 41/46) of the NPC patients had higher EBV DNA gene copies per mL in the LNA marker than the conventional marker. Folds of gene copies per mL of increase of LNA marker over those of conventional marker varied from 6 folds up to 3800 folds in 27/46 NPC patients. The results were confirmed in a validation set of 141 NPC patients (113/141) in a multicenter study under the umbrella of NPC Area of Excellence (AoE) program in Hong Kong with 79.6% (113/142) of NPC patients having higher gene copies per mL of LNA marker than the conventional marker initially tested. On longitudinal monitoring of the new LNA marker in 9 NPC patients for a period of 4.9 months to 5.6 months, higher sensitivity of detection was also achieved at disease onset, during the clinical course of disease and at distant metastases (2/9) than the conventional marker. Three patients with Lymphoepithelioma Like Carcinoma of Lung (LELC) and 3 patients with extra nodal nasal NK/T cell lymphoma were monitored from 3 months to 19.3 months. Detection sensitivity was also higher in the new LNA marker in 3/3 LELC and 1/2 NK/T lymphoma with disease progression whereas 1 NK/T lymphoma with remission had LNA marker remained negative. With such enlightening findings, we are expanding the scope of this new test to many more NPC patients as well as other EBV associated malignancies. We are also recruiting patients in embarking on a large scale prospective study. Citation Format: Timothy T.C. Yip, Hazel Y.Y. Kwok, Sharon S.Y. You, Dora L.W. Kwong, Alvin H.W. Fong, W.W. Cheng, Christopher Lai, Anthony C.C. Shek, Victor W.S. Ma, Maria LiLung, Roger K.C. Ngan. A new circulating plasma/serum Epstein-Barr virus (EBV) DNA test by locked nucleic acid (LNA) PCR technique with enhanced sensitivity in diagnosis & monitoring of patients suffering from nasopharyngeal carcinoma (NPC) and other EBV associated malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3153.

Abstract 1734: Preclinical study of HSP-90 inhibitor drug, AUY922 showed good efficacy in treatment of nasopharyngeal cancer

Introduction: Prognosis of patients with metastatic or recurrent nasopharyngeal cancer (NPC) is usually poor. Despite some good response to chemotherapy by cisplatin-containing regimen, long term remission in NPC patients is not common. Response rate to second line drug after failing cisplatin is poor. Heat shock protein-90 (HSP-90) is a molecular chaperone essential to the folding of a wide variety of oncogenic client proteins. Inhibition of HSP-90 can lead to a combinatorial blockage of signal transduction leading to cancer cell death. Here, we reported a successful preclinical study showing good efficacy of an anti-HSP-90 drug, AUY922 in treating NPC in cell culture in vitro and xenograft model in vivo in nude mice. Experimental Design: WST assay was used to determine the growth inhibitory dose of AUY922 in 3 NPC cell lines (C666, HONE1 & CNE2) and 2 cisplatin resistant NPC cell lines (HONE1-EBV-Cisp-Res & HK1-LMP1-Cisp-Res). Subcutaneous NPC xenografts was treated by AUY922 i.p. to examine tumor growth inhibition. DNA flow cytometric analysis (DNA-FCM) and Yo-Pro staining were carried out to analyze the effect of AUY922 in cell cycle progression and apoptosis. RT-PCR and Western blotting were performed to examine the effects of AUY922 in regulating Epstein-Barr virus (EBV) gene transcripts and kinase pathway phosphorylation respectively. Results: AUY922 (at GI-50 of 22 nM for C666) could effectively induce tumor growth inhibition in all the 3 NPC cell lines with 300-900 and 180-2700 folds of higher sensitivity than cisplatin or 5-FU. NPC xenograft regression was accomplished by AUY922 treatment at 35-65 mg/kg alone with additive effect when combined with cisplatin. Importantly, AUY922 was also growth inhibitory in the 2 cisplatin resistant NPC cell lines in vitro or in vivo hinting its potential use in the clinical setting in patients refractory to cisplatin. By DNA-FCM, AUY922 could induce prominent G2 cell cycle phase arrest and sub-G1 apoptotic peak at 60 nM (with low level of early apoptosis by YoPro staining). >95% NPC patients in Hong Kong are EBV positive. AUY922 can dramatically induce the expression of 2 EBV transcripts - BZLF1 (for viral lytic cycle) and LMP2A (associated with cytotoxic T cell lysis) indicating its potential in facilitating tumor lysis through these processes. Western blot delineated substantial regulation of phosphorylation in AKT, mTOR, GSK-3-beta, NF-KappaB, IKK-alpha/beta and IKB-alpha pathways but not in CREB, p38 and PARP pathways. Conclusion: The findings of this preclinical study clearly showed the good potential of this HSP90 inhibitor drug, AUY922 in NPC treatment. Ethics approval is pending to initial clinical trials of AUY922 in patients with metastatic/locally recurrent NPC in Hong Kong. Citation Format: Timothy T.C. Yip, Roger K.C. Ngan, Wai-Tong Ng, Lewis T.C. Chan, William C.S. Tai, Wai-Wai Cheng, Victor W.S. Ma, Kwok-Wai Lo, Ya-Ping Li, Michael B.H. Yang, Eric C.H. Wong, Brigette B.Y. Ma, Nai-Ki Mak, George S.W. Tsao, Maria Li-Lung. Preclinical study of HSP-90 inhibitor drug, AUY922 showed good efficacy in treatment of nasopharyngeal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1734. doi:10.1158/1538-7445.AM2015-1734

Abstract LB-182: Serum amyloid A is elevated in serum of lung cancer patients with poor prognosis

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Discussion Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-182.