A comparison of immunohistochemical assays and FISH in detecting the ALK translocation in diagnostic histological and cytological lung tumor material.
BACKGROUND
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.
*Department of Histopathology, Royal Brompton and Harefield NHS Foundation Trust, London; †Centre for Molecular Pathology, The Royal Marsden Hospital, Sutton, Surrey; ‡Department of Oncology, The Royal Marsden Hospital; and §Department of Histopathology, Royal Marsden Hospital, Chelsea, London, United Kingdom.
John
J
Le Quesne
Manisha
M
Maurya
Slaveya G
SG
Yancheva
Mary
M
O'Brien
Sanjay
S
Popat
Andrew C
AC
Wotherspoon
David Gonzalez
DG
de Castro
Andrew G
AG
Nicholson
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000077548:Anaplastic Lymphoma Kinase; D000906:Antibodies; D000077547:Crizotinib; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008175:Lung Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use; D011720:Pyrazoles / Q000627:therapeutic use; D011725:Pyridines / Q000627:therapeutic use; D012372:ROC Curve; D020794:Receptor Protein-Tyrosine Kinases / Q000032:analysis / Q000235:genetics*; D014178:Translocation, Genetic
D000906:Antibodies; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011725:Pyridines; D000077547:Crizotinib; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases