| Summary: | 博士 === 國立陽明大學 === 臨床醫學研究所 === 89 === ABSTRACT
Cancer has been the first killer of ten leading causes of death in Taiwan since 1982. Understanding of molecular mechanisms of carcinogenesis, sensitive methods for early diagnosis, and novel effective treatment are among the most important subjects of medical research. Nasopharyngeal carcinoma (NPC) is a common cancer in southern China. The conventional treatment for NPC is radiotherapy. Because of recent advances in radiation oncology, locoregional control has been greatly improved. The failure in treatment is due to distant metastasis predominantly. How to overcome the problem of distant metastasis is the critical step to further increase cure rate of NPC. The major works of this thesis include (1) molecular detection of distant metastasis, and (2) improvement of therapeutic management.
Cancer metastasis involves many processes and cancer cells should enter circulation before they can establish a successful metastatic colony. Early detection of disseminated cancer cells in the peripheral blood is a simple, non-invasive assay and has been vigorously investigated during recent years. Although NPC is a tumor with highly metastatic tendency, molecular detection of micrometastasis has rarely been reported. By meticulous design of primer pairs of Cytokeratin 19 (CK-19) and a verification system, we have first established a nested reverse-transcriptase polymerase chain reaction (RT-PCR) system and tested 95 patients with previously untreated NPC and 45 healthy volunteers. Under our nested RT-PCR experimental conditions, 36.4% (32/88) clinically non-metastatic and 57.1% (4/7) metastatic NPC patients had CK-19 mRNA in their blood. For clinically non-metastatic (M0) NPC patients, there was no statistical correlation between the detection rate of circulating CK-19 transcripts and clinical T-stage, N-stage, and overall stage. After a short follow-up period, we found that the clinical significance of circulating CK-19 mRNA remains unclear and should be further studied. Because the reported sensitivity of one blood sampling and one enzyme RT-PCR test is low in the literature, we collected blood samples from clinically metastatic (M1) NPC patients and investigated the effect of blood sampling and effect of different Taq DNA polymerase. Our data demonstrated that multiple blood sampling or using multiple enzymes for RT-PCR detection of tumor cells in single-point blood sample can enhance the sensitivity in the diagnosis of metastasis in NPC patients. These approaches may be used for the early detection of circulating tumor cells in non-metastatic cancer patients.
NPC is a tumor of epidermoid origin with endemic distribution and closely associated with Epstein-Barr virus (EBV). It has been shown that EBV RNA, DNA, and protein can be found in NPC cells, but rarely detected in infiltrate lymphocytes or adjacent normal tissue. Thus, we try to evaluate whether the EBV DNA can serve as a tumor marker for NPC detection or not. Using primers specific to EBV nuclear antigen 1 (EBNA-1) and a nested PCR technique, we found that the positive rates of EBNA-1 in NPC patients (88/124=71%) were significantly higher than that in healthy volunteers (16/114=14%). After adequate follow-up, the presence of EBV DNA in peripheral blood cells is proved as a novel and important risk factor for patients with NPC that indicates a significantly higher risk of developing distant metastasis as well as a lower survival rate. In order to differentiate the origin of EBV DNA, we isolated the B cells from the whole blood cells and performed nest PCR. We observed that the major source of viral DNA detected in peripheral blood cells was circulating tumor cells, not EBV-infected B cells. By PCR and direct sequencing, we found there were several point mutations of EBNA-1 gene in fresh frozen NPC tumor cells and peripheral blood cells. A complete match pattern of sequence variation between NPC tumor cells and peripheral blood cells further support that EBV in peripheral blood cells arise from disseminated tumor cells.
In the second part of this study, we made an effort to improve the treatment outcomes of advanced NPC, which has caused little progress during the past years. Because NPC is a radio- and chemosensitive tumor, we first inserted two courses of appropriate concomitant chemotherapy into conventional radiotherapy program. Then, we shortened the radiotherapy time from 7-8 weeks to 6 weeks according to the recent principle of radiobiology. Our data reveal that both conventional radiotherapy and partially hyperfractionated accelerated radiotherapy plus concurrent chemotherapy are both feasible and effective, with acceptable toxicities. We further conducted a phase III randomized trial to compare concurrent chemoradiotherapy versus radiotherapy alone for advanced NPC. Recent analyses show that the nasopharynx disease-free survival is significantly better in the combined therapy group but the metastasis-free survival and overall survival have little difference. Most patients failed due to distant metastasis. In a prospective non-randomized trial, we tested the role of post-radiation adjuvant chemotherapy for very advanced NPC patients and used a novel drug delivery mode- weekly 24-hr continuous infusion of cisplatin, 5-FU and leucovorin (PFL). After adequate follow-up, we observed that PFL adjuvant chemotherapy significantly reduce the rate of distant metastasis and prolong survival of very advanced NPC patients. Based on the above studies and our clinical experience, we propose a simple but new staging system that will more precisely reflect the prognosis than the present AJCC/UICC staging system.
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