ALK基因重排：多中心试验结果

GeneDiagnostic, ZytoVision

Anaplastic lymphoma kinase(ALK)gene rearrangement in non-small cell lung cancer(NSCLC):

Results of a multi-centre ALK-testing

V Laffert M,Warth A,Penzel R,et al.

BACKGROUND:The reliable identification of non-small cell lung cancers(NSCLC)with chromosomal breaks in the gene of the anaplastic lymphoma kinase(ALK)is crucial for the induction of therapy with ALK-inhibitors.In order to ensure a reliable detection of ALK-breaks by means of fluorescence in situ hybridization(FISH)testing,round robin tests are essential.In preparation of a nation (German)-wide round robin test we initiated a pre-testing phase involving8experts in FISH-diagnostics to identify NSCLC cases(n= 10)with a pre-tested ALK-status.In addition,ALK immunohistochemistry(IHC)was performed to assess ALK protein expression. MATERIAL AND METHODS:Sections derived from a tissue microarray,each consisting of3cores from10NSCLC cases,were independently tested for ALK protein expression by IHC and genomic ALK-breaks by FISH involving8institutes of pathology.Based on a pre-screening,5cases were identified to be clearly ALK-break negative,whereas the remaining5cases were ALK-break positive including one case with low percentage(20%)of positive cells.The latter had been additionally tested by RT-PCR.

RESULTS:The5unequivocal ALK-break negative NSCLC were almost consistently scored negative by means of FISH and IHC by all8experts.Interestingly,4of the5cases with pre-defined ALK-breaks revealed homogenous FISH results whereas IHC for the detection of ALK protein expression showed heterogeneous results.The remaining case(low number of ALK-break positive cells)was scored negative by3experts and positive by the other5.RT-PCR revealed the expression of an EML4-ALK fusion gene variant1. CONCLUSION:ALK-break negative NSCLC cases revealed concordant homogeneous results by means of FISH and IHC(score0-1) by all8experts.Discordant FISH results were raised in one ALK-break positive case with a low number of affected tumor cells.The remaining4ALK-break positive cases revealed concordant FISH data whereas the ALK-IHC revealed very diverse results.The cases with concordant FISH results provide an excellent basis for round robin ALK-FISH testing.As long as standardized ALK-IHC protocols are missing,ALK protein expression cannot by regarded as the method of choice for identification of patients eligible for treatment with ALK inhibitors.

ALK基因重排：多中心试验结果

背景：

用可靠的手段确认非小细胞肺癌（NSCLC)ALK基因断裂对于选择ALK阻断剂治疗是致关重要的。为了确认采用FISH手段诊断ALK断裂的可靠性，需要做一系列的验证（round robin test)。为做全国（德国）范围内的系列验证，我们在此启动了一个预试验，内容是要求8位FISH诊断专家来确认NSCLC病例（n=10)的ALK状态（原先已经做过相应检测）。此外，ALK IHC用于评估ALK的蛋白表达水平。

材料与方法：

以10例NSCLC病例的组织制作成的每例三芯点组织芯片为样本，由8家病理科分别采用IHC方法检测ALK蛋白表达和采用FISH方法(ZytoVision,德国蔡图微）检测ALK基因断裂。基于实验前筛选，5例为明确的ALK阴性，5例为ALK阳性，其中1例为以低百分比（20%）阳性细胞数确定的阳性标本，并经RT-PCR进一步确认。

结果：5例ALK断裂阴性的NSCLC标本经8个病理专家采用FISH和IHC方法检测评分的结果几乎一致为阴性。有意思的是，原选定为ALK阳性的5例样本中，有4例FISH检测结果一致，但是经IHC检测ALK蛋白表达的结果却各不相同。余下的1例（ALK阳性细胞值较低）有3位专家判定为阴性，其余5为判定为阳性，RT-PCR结果显示引物1EML4-ALK基因融合。

结论：

8位病理专家对ALK阴性的NSCLC样本的FISH和IHC（0-1）结果评判表现出一致性。4例ALK阳性标本的FISH评判结果一致，另1例ALK阳性样本FISH评判结果不一致，其原因是由于阳性细胞值低造成的；而IHC检测显示出了不同的结果。这些样本的FISH结果一致性为ALK FISH系列检测提供了良好的基础。只要ALK IHC操作未规范化，ALK蛋白表达的检测结果就不能作为确定ALK抑制剂治疗的标准。

点评：建议IHC ALK用作筛查，FISH ALK用于确诊；严重病情情况下即使IHC ALK阴性，也可FISH ALK复查，因为IHC 约有4%的假阴性（Savic S et al.2015).