A Comparison of Immunohistochemical Assays and FISH in Detecting the ALK Translocation in Diagnostic Histological and Cytological Lung Tumor Material

Abstract

Introduction:Detection of the ALK rearrangement in a solid tumor gives these patients the option of crizotinib as an oral form of anticancer treatment. The current test of choice is fluorescence in situ hybridization (FISH), but various cheaper and more convenient immunohistochemical (IHC) assays have been proposed as alternatives.Methods:Fifteen FISH-positive cases from patients, seven with data on crizotinib therapy and clinical response, were evaluated for the presence of ALK protein using three different commercially available antibodies: D5F3, using the proprietary automated system (Ventana), ALK1 (Dako), and 5A4 (Abcam). A further 14 FISH-negative and three uncertain (<15% rearrangement detected) cases were also retrieved. Of the total 32 specimens, 17 were excisions and 15 were computed tomography-guided biopsies or cytological specimens. All three antibodies were applied to all cases. Antibodies were semiquantitatively scored on intensity, and the proportion of malignant cells stained was documented. Cutoffs were set by receiver operating curve analysis for positivity to optimize correct classification.Results:All three IHC assays were 100% specific but sensitivity did vary: D5F3 86%, ALK 79%, 5A4 71%. Intensity was the most discriminating measure overall, with a combination of proportion and intensity not improving the test. No FISH-negative IHC-positive cases were seen. Two FISH-positive cases were negative with all three IHC assays. One of these had been treated with crizotinib and had failed to show clinical response. The other harbored a second driving mutation in the EGFR gene.Conclusions:IHC with all three antibodies is especially highly specific (100%) although variably sensitive (71%-86%), specifically in cases with scanty material. D5F3 assay was most sensitive in these latter cases. Occasional cases are IHC-positive but FISH-negative, suggesting either inaccuracy of one assay or occasional tumors with ALK rearrangement that do not express high levels of ALK protein.

A comparison of ALK gene rearrangement and ALK protein expression in primary lung carcinoma and matched metastasis

“…The overall agreement in the 68 samples between FISH and IHC was 88%, with a sensitivity of 71% and a specificity of 100%. This observation is in keeping with the reported high specificity of the D5F3 antibody . A relatively low ALK IHC sensitivity in our study might also be related to the fact that we used archival tissue.…”

“…[2][3][4] The Vysis LSI ALK break-apart fluorescence in-situ hybridization (FISH) probe kit (Abbott Molecular, Des Plaines, IL, USA) was used to identify NSCLC with ALK rearrangements in the first clinical trials, and therefore the US Food and Drug Administration (FDA) approved it as the first companion diagnostics for the detection of ALK rearrangements in the United States. 5 Other methods for detection of ALK rearrangement status reported extensively in the literature include immunohistochemistry (IHC), [6][7][8][9][10][11][12][13] reverse transcription-polymerase chain reaction (RT-PCR) 14,15 and, more recently, next-generation sequencing. 10,16,17 In 2013, the College of American Pathologists (CAP), the Association for Molecular Pathology (AMP) and the International Association for the Study of Lung Cancer (IASLC) guideline for molecular testing in lung adenocarcinoma recommended ALK FISH testing as a standard method for detection of ALK rearrangements.…”

Lung Carcinoma Predictive Biomarker Testing by Immunoperoxidase Stains in Cytology and Small Biopsy Specimens: Advantages and Limitations

“…6,7,17,26,47,48 Clone 5A4, which is as reliable as D5F3 in the detection of ALK rearrangement, is used more commonly in Europe. 17,49 The performance of ALK IPOX in cytology specimens (formalin-fixed cell blocks) thus far is similar to that in surgical specimens. Additional validation studies are needed.…”

Updates in the approach to intrathoracic sarcomas

“…The first consideration is whether this represents a neoplasm or a reactive process, and this can usually be determined with routine morphological investigation. There are areas where even this can be problematic, such as distinguishing an inflammatory myofibroblastic tumour (IMT) from organizing pneumonia or obstructive changes in the lung, and immunohistochemistry for anaplastic lymphoma kinase (ALK) may be of use in such a situation, followed by confirmatory molecular cytogenetic testing, although not all cases of IMT harbour ALK rearrangements . In such situations, reporting should be pragmatic, and the most appropriate management, which may be excision, should be decided upon.…”