ALK-FISH borderline cases in non-small cell lung cancer: Implications for diagnostics and clinical decision making
Introduction
About 2–7% of non-small cell lung cancer (NSCLC) patients harbor anaplastic lymphoma kinase (ALK) rearrangements [1], [2], [3] that respond to treatment with tyrosine kinase inhibitors (TKI) [4], [5], [6]. Patient selection in the clinical trials that lead to therapy approval by the U.S. Food and Drug Administration (FDA) in August 2011 and the European Medicines Agency (EMA) in October 2012 was based on fluorescence in-situ hybridization (FISH) [5], [6], [7]. Therefore, FISH is regarded as the gold standard to discriminate between ALK-positivity and -negativity necessary for therapy decision.
A tumor cell is considered ALK-positive if split signals (SS) and/or single red signals (SRS) can be detected. SS/SRS are the consequence of a paracentric inversion or translocation in the ALK-gene (chromosome 2) leading to a fusion with the EML4-gene (majority of cases) or with other partners such as KIF5B, TFG and KLC1 [1], [2], [8]. As a result the two adjacent ALK-gene regions covered by two different fluorescent labeled probes rearrange (break), leading to a separation of the two fluorescence dyes (cut-off for a SS: ≥2signal diameters). The wild-type situation (ALK-negative) is indicated by a fused signal (FS), showing (almost) no distance between the two probes. Diagnostic recommendations propose to evaluate 50 tumor cell nuclei and that unequivocal cases (SS/SRS in 5–25 cells) should be evaluated by a second observer. A tumor is considered positive if the (average) SS/SRS rate of the (first and second) cell count reading is at least 15% [8], [9]. Most papers refer to this 15% cut-off for ALK-FISH-positivity [8], [10], [11], [12], [13], [14]. Furthermore, this cut-off is based on the observation that up to 11% of SS may be found in ALK-negative tumors and even in normal lung tissue (technical artifact) [13], [15], [16], [17]. Still, NSCLC diagnosis is mainly performed on small biopsy specimens. These may not contain a sufficient number of tumor cells to always ensure these guidelines can be followed (e.g., less than 50 tumor cells).
The benefit of an ALK-TKI is independent of the total percentage of ALK-altered tumor cells as long as the tumor is “truly” ALK-positive [16]. However, Camidge at al. described that ∼8.5% of NSCLCs show SS between 10% and 15% [18]. This seems highly important as ALK-positive samples (RT-PCR-proven) with SS close to the cut-off are at risk of ALK-FISH-misclassification (borderline group) [19], [20].
To this end we analyzed 753 NSCLCs using ALK-FISH. To investigate the frequency of ALK-borderline cases we put a special focus on samples with ALK-positive tumor cells around the 15% cut-off and performed a correlation with ALK-immunohistochemistry (IHC). Additionally, we systematically evaluated and quantified statistical sampling effects of ALK-FISH-testing in the context of the number of evaluated tumor cells to demonstrate the probabilities of false negative and positive ALK-FISH-classifications.
Section snippets
NSCLC samples
Representative tumor areas of 753 NSCLC resections [499 adenocarcinomas (ADC), 214 squamous cell carcinomas (SCC), 15 large cell carcinomas (LCC), 15 non-small cell lung cancers not otherwise specified (NOS) and 10 adenosquamous carcinomas (ADSC)], collected between 2000 and 2014, were identified and 13 tissue microarrays (TMA) were constructed using two cores for each case (one mm diameter per core). Further details of this cohort with clinicopathological data of 473 samples were already
ALK-FISH of 753 NSCLC and correlation with IHC
Unequivocal ALK-FISH classification was possible in 94.3% (710/753) of the cases with an ALK-FISH-negative status in 93% (700/753) and a positive status in 1.3% (10/753). All of these samples showed SS/SRS in <10% or clearly above 20%. All 710 cases showed concordant negative or positive ALK-IHC results (Table 1). The 700 ALK-FISH negative NSCLC comprised 453 ADC, 208 SCC, 15 LCC, 15 NOS and 9 ADSC. The 10 unequivocal ALK-FISH positive samples were all pure ADC (Table 2). The ALK-FISH pattern
Discussion
We performed ALK-FISH in 753 NSCLC. While the majority of cases could be reliably classified as ALK-positive or negative (unequivocally), 5.7% of the samples showed SS/SRS in a range +/−5% points around the established 15% cut-off value. Our statistical analysis demonstrated that these samples are at risk of misclassification by sampling effects and should be regarded with caution. This might explain the conflicting IHC/FISH results in this range.
In agreement with previous studies [3], [13],
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The authors thank Erika Berg, Stefanie Mende and Hedwig Lammert for their excellent technical assistance. Part of this work was supported by PFIZER within the framework of the FALKE project (detection of ALK-rearrangements by FISH in NSCLC).
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