Goydos James S, Mann Barbara, Kim Hyunjin J, Gabriel Emmanuel M, Alsina Janivette, Germino F Joseph, Shih Weichung, Gorski David H
Division of Surgical Oncology, UMDNJ-Robert Wood Johnson Medical School, The Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA.
J Am Coll Surg. 2005 Mar;200(3):362-70. doi: 10.1016/j.jamcollsurg.2004.10.032.
It is now known that activating point mutations in components of the mitogen-activated protein kinase pathway commonly occur in melanoma. We previously described a method to detect point mutations in heterogenous tissues containing both wild-type and mutant B-RAF and N-RAS genes by using site-directed mutagenesis to introduce new restrictions sites in the cDNA sequence when the specific point mutations are present. We modified this technique to improve sensitivity and used it to determine the incidence of B-RAF and N-RAS mutations in human melanoma.
We screened 115 melanoma samples for the most common B-RAF and N-RAS mutations found in melanoma using a site-directed mutagenesis-based detection technique. Southern blotting was used to increase sensitivity of the basic system. We also tested this method of genetic mutation detection in fine-needle aspiration specimens and paraffin-embedded tissues.
Sixty-eight samples (20 of 36 primaries, 18 of 27 regional metastases, 16 of 40 nodal metastases, and 9 of 12 distant metastases) harbored the V599E B-RAF mutation (59%), 17 contained a Q61R N-RAS mutation, and 4 contained a Q61K N-RAS mutation. We were able to detect the V599E mutation in genomic DNA from paraffin-embedded melanoma samples and could routinely detect this mutation in fine-needle aspirations of melanoma tumors. This method of detection was sensitive and specific with no false positives.
Activating mutations of B-RAF and N-RAS were present in approximately 60% and 18%, respectively, of samples tested. The site-directed mutagenesis system of mutation detection was both sensitive and specific in detecting these mutations and will likely prove very clinically useful in future studies.
现已明确，丝裂原活化蛋白激酶途径成分中的激活点突变在黑色素瘤中普遍存在。我们之前描述了一种方法，当存在特定点突变时，通过定点诱变在cDNA序列中引入新的限制性位点，来检测同时含有野生型和突变型B-RAF及N-RAS基因的异质组织中的点突变。我们对该技术进行了改进以提高灵敏度，并将其用于确定人类黑色素瘤中B-RAF和N-RAS突变的发生率。
我们使用基于定点诱变的检测技术，对115份黑色素瘤样本筛查黑色素瘤中最常见的B-RAF和N-RAS突变。采用Southern印迹法提高基本系统的灵敏度。我们还在细针穿刺标本和石蜡包埋组织中测试了这种基因突变检测方法。
68份样本（36例原发性肿瘤中的20例、27例区域转移瘤中的18例、40例淋巴结转移瘤中的16例以及12例远处转移瘤中的9例）存在V599E B-RAF突变（59%），17份样本含有Q61R N-RAS突变，4份样本含有Q61K N-RAS突变。我们能够在石蜡包埋的黑色素瘤样本的基因组DNA中检测到V599E突变，并且能够在黑色素瘤肿瘤的细针穿刺样本中常规检测到该突变。这种检测方法灵敏且特异，无假阳性。
在检测的样本中，B-RAF和N-RAS的激活突变分别约占60%和18%。定点诱变突变检测系统在检测这些突变时灵敏且特异，在未来研究中可能会被证明具有非常重要的临床应用价值。
