*Institute of Pathology, Charité BERLIN, Berlin, Germany; †Institute of Pathology, University of Heidelberg, Heidelberg, Germany; ‡Department of Pathology, Aberdeen University Medical School, Aberdeen, United Kingdom; §Department of Pathology and Cytology, Karolinska University Hospital, Solna, Stockholm, Sweden; ‖Department of Pathology, University Hospital Cologne, Cologne, Germany; ¶Laboratorio de Dianas Terapeuticas, Hospital Universitario Sanchinarro, Madrid, Spain; #Institute of Pathology, Klinikum Großhadern Ludwig-Maximilians-Universität, Munich, Germany; **Institute of Pathology, UZ ANTWERPEN, Edegem, Belgium; ††Institute of Pathology, Klinikum rechts der Isar, TU, Munich, Germany; ‡‡Institute of Pathology, University Hospital Carl Gustav Carus, Dresden, Germany; §§Institute of Pathology, Universitätsspital Basel, Basel, Germany; ‖‖Institute of Pathology, HSK Dr.-Horst-Schmidt-Kliniken GmbH, Wiesbaden, Germany; ¶¶Institute of Pathology, Universitäts Spital Zürich, Zurich, Switzerland; ##Institute of Pathology, Centre Jean Perrin, Clermont-Ferrand, France; ***Patologisk Institut, Århus Universitetshospital, Århus, Denmark; †††Pathology-Berlin, Bioptisches Institut Gemeinschaftspraxis für Pathologie, Berlin, Germany.
J Thorac Oncol. 2014 Nov;9(11):1685-92. doi: 10.1097/JTO.0000000000000332.
Detection of anaplastic lymphoma kinase (ALK)-gene rearrangements in non-small-cell lung cancer (NSCLC) is mainly performed by fluorescence in-situ hybridization (FISH). The question was raised if FISH might be replaced by immunohistochemistry (IHC) in a reliable and reproducible manner across different laboratories.
After calibration of the staining instruments and training of the observers to binary interpretation (positive versus negative), 15 NSCLC were independently tested for ALK protein expression by IHC only in a multicenter setting (16 institutes). Each laboratory utilized the VENTANA ALK-D5F3 IHC assay. As demonstrated by FISH the samples displayed unequivocal ALK break-positivity (6×) and negativity (7×), as well as ALK positive-"borderline" character (2×), which is challenging for FISH diagnosis and thus was RT-PCR-confirmed.
All seven ALK FISH-negative cases were homogenously scored as ALK-IHC negative. All 16 participants scored the two ALK positive-"borderline" samples as unequivocally positive according to their protein expression. Concordant IHC interpretation was also noticed in four of six unequivocal ALK break positive cases. In two of six some observers described a weak/heterogeneous ALK-IHC staining. This would have resulted in a subsequent ALK-testing (FISH/PCR) in a routine diagnostic setting.
This so-called "ALK-Harmonization-Study" shows for the first time that predictive semiquantitative IHC reveals reliable and reproducible results across several labs when methodology and interpretation are strictly defined and the pathologists are uniquely trained. The application of validated ALK IHC assays and its comparison to ALK-FISH is highly needed in future clinical trials. This might answer the question if ALK-IHC cannot only serve as a prescreening tool, but as a stand-alone test at least in cases displaying an unequivocally staining pattern as well as an alternative predictive test in samples with reduced FISH interpretability.
非小细胞肺癌（NSCLC）中检测间变性淋巴瘤激酶（ALK）-基因重排主要通过荧光原位杂交（FISH）进行。人们提出了一个问题，即在不同实验室中，FISH 是否可以通过免疫组织化学（IHC）以可靠且可重复的方式进行替代。
在对染色仪器进行校准并对观察者进行二进制解释（阳性与阴性）的培训后，在多中心环境中（16 个研究所）仅通过 IHC 对 15 例 NSCLC 进行了 ALK 蛋白表达的独立检测。每个实验室均使用 VENTANA ALK-D5F3 IHC 检测试剂盒。根据 FISH 检测，这些样本显示明确的 ALK 断裂阳性（6×）和阴性（7×），以及 ALK 阳性“边缘”特征（2×），这对 FISH 诊断具有挑战性，因此通过 RT-PCR 进行了确认。
所有 7 例 ALK FISH 阴性病例的 ALK-IHC 评分均为阴性。所有 16 位参与者均根据其蛋白表达将 2 例 ALK 阳性“边缘”样本一致评为阳性。在 6 例明确的 ALK 断裂阳性病例中，也注意到了 IHC 解释的一致性。在 6 例中的 2 例中，一些观察者描述了弱阳性/异质性 ALK-IHC 染色。在常规诊断环境中，这将导致随后进行 ALK 检测（FISH/PCR）。
这项所谓的“ALK 协调研究”首次表明，当方法学和解释严格定义且病理学家经过专门培训时，预测性半定量 IHC 可在多个实验室中提供可靠且可重复的结果。在未来的临床试验中，需要应用经过验证的 ALK IHC 检测试剂盒，并将其与 ALK-FISH 进行比较。这可能会回答以下问题，即 ALK-IHC 不仅可以作为一种预筛选工具，而且在显示明确染色模式的病例中以及在 FISH 可解释性降低的样本中作为替代预测性检测，是否可以作为一种独立的检测方法。
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