title: ALK-FISH borderline cases in non-small cell lung cancer: Implications for diagnostics and clinical decision making.
abstract: BACKGROUND
  Fluorescence in-situ hybridization (FISH) for the detection of ALK-rearrangements in non-small cell lung cancer (NSCLC) is based on at first sight clear cut-off criteria (≥15% of tumor cells) for split signals (SS) and single red signals (SRS). However, NSCLC with SS-counts around the cut-off may cause interpretation problems.
  
  
  METHODS
  Tissue microarrays containing 753 surgically resected NSCLCs were independently tested for ALK-alterations by FISH and immunohistochemistry (IHC). Our analysis focused on samples with SS/SRS in the range between 10% and 20% (ALK-FISH borderline group). To better understand the role of these samples in routine diagnostics, we performed statistical analyses to systematically estimate the probability of ALK-FISH-misclassification (false negative or positive) for different numbers of evaluated tumor cell nuclei (30, 50, 100, and 200).
  
  
  RESULTS
  94.3% (710/753) of the cases were classified as unequivocally (<10% or ≥20%) ALK-FISH-negative (93%; 700/753) or positive (1.3%; 10/753) and showed concordant IHC results. 5.7% (43/753) of the samples showed SS/SRS between 10% and 20% of the tumor cells. Out of these, 7% (3/43; ALK-FISH: 14%, 18% and 20%) were positive by ALK-IHC, while 93% (40/43) had no detectable expression of the ALK-protein. Statistical analysis showed that ALK-FISH misclassifications occur frequently for samples with rearrangements between 10% and 20% if ALK-characterization is based on a sharp cut-off point (15%). If results in this interval are defined as equivocal (borderline), statistical sampling-related ALK-FISH misclassifications will occur in less than 1% of the cases if 100 tumor cells are evaluated.
  
  
  CONCLUSIONS
  While ALK status can be determined robustly for the majority of NSCLC by FISH our analysis showed that ∼6% of the cases belong to a borderline group for which ALK-FISH evaluation has only limited reliability due to statistical sampling effects. These cases should be considered equivocal and therapy decisions should include additional tests and clinical considerations.
authors.affiliation: Institute of Pathology, Charité Universitätsmedizin, Charitéplatz 1, 10117 Berlin, Germany.
authors.forename: Maximilian
authors.initials: M
authors.lastname: von Laffert
authors.affiliation: Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany.
authors.forename: Albrecht
authors.initials: A
authors.lastname: Stenzinger
authors.affiliation: Institute of Pathology, Charité Universitätsmedizin, Charitéplatz 1, 10117 Berlin, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Hummel
authors.affiliation: Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany.
authors.forename: Wilko
authors.initials: W
authors.lastname: Weichert
authors.affiliation: Institute of Pathology, Charité Universitätsmedizin, Charitéplatz 1, 10117 Berlin, Germany.
authors.forename: Dido
authors.initials: D
authors.lastname: Lenze
authors.affiliation: Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany.
authors.forename: Arne
authors.initials: A
authors.lastname: Warth
authors.affiliation: Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Penzel
authors.affiliation: Institute of Pathology, Vivantes Klinikum Berlin, Rudower Straße 48, 12351 Berlin, Germany.
authors.forename: Hermann
authors.initials: H
authors.lastname: Herbst
authors.affiliation: Institute of Pathology, Johannes Wesling Klinikum Minden, Hans-Nolte-Straße 1, 32429 Minden, Germany.
authors.forename: Udo
authors.initials: U
authors.lastname: Kellner
authors.affiliation: Institute of Pathology, Charité Universitätsmedizin, Charitéplatz 1, 10117 Berlin, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Jurmeister
authors.affiliation: Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Schirmacher
authors.affiliation: Institute of Pathology, Charité Universitätsmedizin, Charitéplatz 1, 10117 Berlin, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Dietel
authors.affiliation: Institute of Pathology, Charité Universitätsmedizin, Charitéplatz 1, 10117 Berlin, Germany. Electronic address: frederick.klauschen@charite.de.
authors.forename: Frederick
authors.initials: F
authors.lastname: Klauschen
mesh_terms: D000077548:Anaplastic Lymphoma Kinase; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis* / Q000235:genetics*; D000066491:Clinical Decision-Making; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results; D014178:Translocation, Genetic
chemical_list: C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases
keywords: Anaplastic lymphoma kinase (ALK); Fluorescence in-situ hybridization (FISH); Immunohistochemistry (IHC); Non-small cell lung cancer (NSCLC); Statistical analysis