20q扩增和PI3K/RAS-RAF信号通路基因突变作为结直肠癌肝转移早期诊断的可行性研究

Distant metastases are responsible for the great majority of colorectal cancer (CRC) deaths, mainly due to liver metastases. 40-50% of CRC patients will develop liver metastases after the surgery of primary tumors. Hepatic resection is the only potentially curative treatment for those patients. Therefore, early detection of liver metastases is of the utmost priority and can result in more radical surgery and thus long-term survival. Our recent aCGH （array Comparative Genomic Hybridization）analysis on the primary tumors of Dutch CRC patients showed that a significant chromosome 20q gain was strongly related with the development of liver metastases. Further analysis together with PI3K/RAS-RAF signaling pathway gene mutations (PIK3CA, KRAS and BRAF), revealed that the primary tumors harboring a significant 20q gain but no mutation had a 6-fold increased liver metastases rate (61%) compared to the ones harboring a mutation but no 20q gain (10%). It suggests that analyzing 20q gain and PI3K/RAS-RAF pathway gene mutations in primary tumors can predict risk of liver metastases in CRC patients. However, using aCGH for 20q evaluation is not yet for clinical practice. We propose to use FISH probes on FFPE (formalin-fixed paraffin-embedded) tissue of primary tumors of CRC to analyze 20q aberration. Based on the facts that 20q gain involves the entire chromosome long arm and 20q gain is also the most common chromosome change among Chinese CRC patients, we plan to design several BAC（Bacterial Artificial Chromosome）-DNA probes along 20q arm, in order to approximately map 20q amplicon, and eventually find the most relevant FISH probe(s) related to liver metastases for clinical use. We will study the feasibility of combined detection of 20q gain and PI3K/RAS-RAF pathway gene mutations as an early diagnostic method for liver metastases. Our results will provide evidences for the mechanism of 20q gain and liver metastases development, and new insights in achieving early detection of liver metastases and personalized treatment for Chinese CRC patients.

肝转移是结直肠癌患者死亡的主要原因。40-50%的患者在原发灶切除术后会发生肝转移。肝转移的早期诊断是使患者获得根治性手术得以长期生存的关键。我们新近的研究发现检测原发灶中20q（20号染色体长臂)的扩增和PI3K/RAS-RAF信号通路基因突变可以预测西方患者发生肝转移的风险，其中高危患者肝转移发生率达61%，而低危患者仅为10%。但用aCGH(比较基因组杂交芯片)检测20q扩增不适合临床。本项目拟选择制备多个位于20q的BAC(细菌人工染色体)探针，并结合FISH(荧光原位杂交)技术直接观测原发灶中20q的扩增，从而预测扩增子位置、研发与肝转移相关性最强的FISH诊断探针、探讨联合检测20q扩增和PI3K/RAS-RAF通路基因突变作为中国结直肠癌患者肝转移早期诊断标志物的可行性。为阐明20q扩增机制和肝转移发生机制提供重要理论依据，为实现肝转移早期诊断和个体化治疗提供新的思路和方法。

结直肠癌发病率在世界恶性肿瘤中排名第三位，肝转移是结直肠癌患者死亡的主要原因之一。多种遗传学的改变在结直肠癌的发生发展中起着重要作用，本研究旨在中国结直肠癌患者中检测RAS-RAF/PI3K通路相关基因的主要突变区，以明确其突变频率和突变类型的分布。同时设计多个位于20q的BAC探针，结合iFISH技术了解原发灶中20q的扩增状态，并将上述数据结合患者的临床病理特征和随访资料进行综合分析。为中国结直肠癌患者靶向药物的选择及肝转移早期诊断提供重要依据。.通过分析353例结直肠癌患者KRAS基因2、3、4外显子，NRAS 2和3外显子，BRAF 15外显子和PIK3CA 9、20外显子的突变与临床病理、术后远处转移及生存状况之间的关系。我们发现结直肠癌患者中KRAS、NRAS、BRAF和PIK3CA的突变频率依次为52.7%、3.4%、4.5%和13.1%，其中KRAS 2号外显子之外的RAS基因的突变频率可达到14%。NRAS突变在女性患者中出现的频率高于男性患者；BRAF突变显示与不良的原发灶病理分级、高TNM分期、较深的原发灶浸润程度相关；同时，BRAF突变的患者发生淋巴结转移、术前和术后转移的风险增高；PIK3CA基因突变频率在结肠癌患者中升高。KRAS 2号外显子突变患者的平均年龄高于野生型患者，此类患者发生淋巴结转移的风险也显著高于后者。KRAS 3号外显子突变与低TNM分期、较浅的原发灶浸润程度相关。BRAF突变与患者总体生存率降低显示出强相关性，携带PIK3CA和NRAS突变的Ⅰ-Ⅲ期结直肠癌患者发生术后远处转移的风险显著增加，并能分别作为提示患者远处转移的独立指标。.结直肠癌中20q扩增的片段可能横跨20q12到20qter的区域，3.20q13.2/20p12探针最适合评价肿瘤腺体20q的扩增情况。由于样本量小，尚未发现20q扩增状态与患者临床病理特征、预后及肝转移间存在统计学相关性。.我们的研究数据为预测抗EGFR靶向药物的有效性提供了重要依据，我们发现RAS-RAF/PI3K信号通路相关基因的总突变频率高达62.3%，说明可能有过半的患者并不能真正从靶向治疗中获益。

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