Correlation of Damage Formation in the p53 gene with Therapeutic Effectiveness Assayed by Multiplex Long Quantitative PCR

Abstract

肺癌在台灣地區有相當高的死亡率，其主要原因為患者對於放射線治療或化學治療常常會產生抗性；但由於癌症治療效性不易從病人之臨床病徵上預測，因此，發展一套可以預測病人治療結果的分子指標是相當重要的。此外，在前人的研究中指出p53基因的變異與癌細胞抗藥性的產生有關；再加上病人血液細胞中DNA的變異與組織中癌細胞DNA的變異相關。所以，本研究利用雙片段定量聚合酵素連鎖反應(Multiplex long quantitative polymerase chain reaction, Multiplex long QPCR)的方法，藉由DNA模板上的損傷會抑制PCR反應時DNA聚合酵素的聚合能力，反應完成後比較有傷害與無傷害的DNA反應效率，即可得到DNA的傷害程度的原理，來偵測化療之肺癌病人週邊血液淋巴球細胞之p53基因的傷害敏感性，並分析其和化療結果的關係。實驗的數據顯示，29位化療無效的病人週邊血液淋巴球細胞中，其p53基因對於30 J/m2的紫外線照射之後，有呈現DNA傷害的人數比例為83%(24/29)；而在15位化療有效的病人其p53基因呈現傷害的人數比例則較低(67%，10/15) (P=0.23)。在化療無效且有DNA傷害的病人當中，平均DNA傷害的程度為0.20±0.17/p53分析片段；而化療有效的病人當中，其平均傷害的程度則為0.16±0.17/ p53分析片段(P=0.89)。雖然化療的結果與兩個族群間DNA傷害的程度沒有顯著的差異，但是，可以看出在化療無效的病人中，其DNA傷害的程度有高於化療有效病人的趨勢。因此，利用Multiplex long QPCR的方法來偵測病人血液中的DNA傷害程度，在未來經過更多樣本的確認後，似乎可以作為預測病人治療結果的指標。Lung cancer is the leading cause of cancer deaths in Taiwan. In the radiotherapeutic and chemotherapeutic modalities used to treat lung cancer, the great majority of patients will relapse with a tumor that is largely refractory to further treatment. Therefore, the development of methods for predicting resistance of cancer cells is important. In addition, previous research has found that there is a correlation between damage sensitivity and drug resistance of cancer cells, and a correlation between the DNA damage in the patients' blood cells and cancer cells of the tissue. The current investigation optimized the multiplex long quantitative polymerase chain reaction (QPCR) to measure the formation of ultraviolet (UV)-induced DNA lesions in specific genes such as the p53 tumor suppressor gene in the blood lymphocytes from chemotherapeutic patients and analyzed the correlation of damage formation with the therapeutic effectiveness. The average damage incidence was 0.20±0.17 in the 29 non-effective chemotherapeutic lung cancer patients, whereas the average damage incidence was 0.16±0.17 in the 17 effective chemotherapeutic patients. In addition, the proportion of patients with DNA damage was 83% (24/29) and 67% (10/15) for non-effective and effective groups, respectively. Although the preliminary data of multiplex long QPCR showed that the damage formation by UV in the p53 gene of lymphocytes from lung cancer patients with different chemo-effectiveness were not significantly different, there was a tendency of higher damage formation in the non-effective lung cancer patients than the effective patients. Therefore, the present study demonstrates that the measurement of DNA damage sensitivity by the multiplex long QPCR assay in lymphocytes may offer a valid estimate of the therapeutic effectiveness of cancer patients. This rationale, however, needs to be strengthened by further investigations in a larger cohort of patients.