高分辨熔解曲线分析技术用于癌组织融合基因快速检测的研究

Gene fusion is that two broken gene fused to a new one, which often leads to the abnormal activation of oncogene. It has been more and more clear that gene fusions have an important role in the pathogenesis of carcinoma, besides haematological disorders and sarcoma. Cancer cells with fusion gene indicates the sensitivity to a kinase inhibitor. The patients with lung cancer harbored EML4-ALK or ROS1 fusion genes have good response to kinase inhibitor crizotinib. Therefore, it is necessary to detect fusion gene in cancer tissues fast and sensitivly in order to make the effective treatment. Fusion genes are difficult to detect for the huge variation types, while it is more difficult to detect in cancer tissues. Because normal cells would be unavoidable to mix with cancer cells in cancer samples either from surgery or from biopsy. Sometimes, the percentage of cancer cells is very low. If the detection method of gene fusion is not sensitive enough, there would be false negative results. Several techniques including fluorescence in situ hybridization (FISH), the methods based on RT-PCR or real time RT-PCR, next generation sequencing, and so on, are popular, but they all have their own shortcomings, such as time and money wasting and unknown fusion gene type unable to be detected. In this study, we will establish a fast and sensitive method to detect fusion genes in cancer tissues based on high resolution melting analysis, which is a fast, sensitive, low contamination gene variance detection method and can be used to scan unknown gene mutations. According to the huge variance of fusion gene sequence, we will design a snapback primer to amplify the single strand cDNA with RNA template including all kinds of variance sequence. The tail of snapback primer is used as a probe to hybridize in the inner of single strand cDNA. When detecting the unknown fusion gene type, the probe is complement with the common sequence of all variance sequence. When detecting the specific fusion gene type, the probe is complement with the specific fusion sequence. LCGreen plus is used as fluorescence dye and melting curves of the hybridization in the inner molecule by the probe of snapback primer will be optimized and made as a series of positive standards. Then we can tell if there is fusion gene or what kind of type fusion gene by the shape of melting curves and the type of snapback primer. The new established method and FISH will both be used to detect EML4-ALK and ROS1 fusion genes in lung cancer tissues, and the results will be proved by sequencing, in order to test the clinical feasibility of the new methods in fusion gene detection.

融合基因参与恶性肿瘤发生、发展，已经成为肿瘤治疗的分子靶点。检测融合基因对指导肿瘤分子靶向药物治疗具有重要临床意义。但融合基因序列变化多，癌组织标本常混杂过多正常组织细胞等问题使癌组织融合基因较难检出，现有检测方法都有各自不足，限制了临床广泛应用。本项目拟依据融合基因序列一端相对保守，另一端变化较大的结构特点，设计与保守端互补，并含有杂交序列的回弹引物，以RNA为模板扩增包含全部融合位点的单链cDNA片段，杂交序列与单链产物分子内杂交，生成局部双链，以饱和双链荧光染料LCGreen Plus为指示剂，利用高分辨熔解曲线分析快速、敏感、低污染、可检测未知基因变异的优点，依据回弹引物杂交区位置的不同和熔解曲线形状改变实现未知和已知融合位点融合基因的检测。应用新方法和FISH法分别检测肺癌中EML4-ALK和ROS1融合基因，并以测序证实，以评价验证新方法快速、敏感检测融合基因的临床可用性。

分子靶向治疗在非小细胞肺癌治疗中获得良好收益依赖于分子靶向药物靶点准确检测。目前常见的分子靶向药物靶点主要有两类，一类是EGFR、KRAS基因的点突变和缺失突变，另一类是ALK融合基因。点突变和缺失突变相对容易检测，检测方法较成熟，但实体瘤融合基因检测仍是基因检测的难点。融合基因变化多，只ALK融合基因的伴侣基因就有多种，同一融合伴侣基因的断裂位点也各不相同。多家报道显示不同融合类型对靶向药物敏感性，获得性耐药产生和患者预后都有不同。临床常用ALK融合基因检测方法如荧光原位杂交和免疫组织化学方法的共同缺点是检测周期长，价钱高，不能区分融合类型。目前融合类型区分仍依赖于测序。因此，建立快速，敏感可用于临床的融合基因检测及分型方法具有重要临床意义。本研究建立了基于高分辨熔解曲线分析的ALK融合基因快速检测方法，能够在一个反应管内同时检测至少20种融合类型ALK融合基因，并建立新的高分辨熔解曲线分析方法，结合关键参数，实现至少20种融合类型的分型。成功实现了对临床石蜡组织标本的检测，检测准确率达100% (47/47),测序证实分型准确率为100% （7/7）。样品cDNA浓度低至1ng/uL仍可检出，且实现融合基因分型。建立融合基因阳性对照标准品合成方法，合成肺癌常见融合基因类型36种，包括ALK，ROS1和RET融合基因，和用于EGFR拷贝数检测内对照的EGFR和参考基因融合的基因片段。本研究结果实现了ALK融合基因快速检测，且可以推广应用于多种类型标本，其他融合基因的快速检测。本研究开发了新的高分辨熔解曲线分析方法，用于区分序列不同的核酸片段，实现了不依赖于测序的核酸片段数字化区分。本研究建立的融合基因标准品构建方法，能够灵活构建任意二个或多个基因接合的融合基因，不仅可以用于融合基因的检测分析，还可推广应用至基因定量检测，如拷贝数分析。在样品质量检测、试剂盒质控，平行分析，仪器间平行分析等方面具有重要推广应用价值。