Optimizing the detection of lung cancer patients harboring anaplastic lymphoma kinase (ALK) gene rearrangements potentially suitable for ALK inhibitor treatment

Abstract

Purpose: Anaplastic lymphoma kinase (ALK) rearrangements, associated with sensitivity to an experimental ALK/MET inhibitor, occur in 3% to 5% of non-small cell lung cancers. Intratumoral fluorescence in situ hybridization (FISH) heterogeneity has been reported. We explored the heterogeneity basis, the requirements for accurately determining ALK FISH positivity, and the effect of enriching the tested population using clinical and molecular factors.

Experimental design: Lung cancer patients were screened by ALK and MET FISH and for EGFR and KRAS mutations.

Results: Thirteen ALK-positive cases were identified from 73 screened patients. Gene copy number increases occurred together with classic rearrangements. All positive cases were adenocarcinomas, 12 were EGFR/KRAS wild-type, and 1 had a coexistent EGFR exon 20 mutation. No association with MET amplification occurred. ALK positivity was associated with <10-pack-year smoking status (P = 0.0004). Among adenocarcinomas, without KRAS or EGFR mutations, with <10-pack-year history, 44.8% of cases were ALK positive. ALK FISH positivity was heterogeneous, but mean values in tumor areas from ALK-positive patients (54% of cells; range, 22-87%) were significantly higher than in adjacent normal tissue or tumor/normal areas from ALK-negative patients (mean, 5-7%). Contiguous sliding field analyses showed diffuse heterogeneity without evidence of focal ALK rearrangements. One hundred percent sensitivity and specificity occurred when four or more fields (∼60 cells) were counted.

Conclusions: Intratumoral ALK FISH heterogeneity reflects technique, not biology. The clinical activity of ALK/MET inhibitors in ALK-positive patients probably reflects ALK, but not MET, activity. Prescreening by histology, EGFR/KRAS mutations, and smoking status dramatically increases the ALK-positive hit rate compared with unselected series.

Fig. 1

NSCLCs hybridized with the ALK break-apart FISH probe (Abbott Molecular). Native ALK status (indicated by yellow arrows) shows fusion of the probes adjacent to the 3′ and 5′ ends of the gene, labeled, respectively, with red and green fluorophores. Rearranged ALK is indicated by the presence of split 3′ (red arrows) and 5′ (green arrows) signals, including single red signals. A, ALK FISH-negative specimen. B, “classic” ALK FISH-positive specimen showing split red and green signals. C, ALK FISH-positive specimen showing two or three copies of single red signals per cell. D, ALK FISH-positive specimen showing evidence of gene copy number increase with clusters of multiple single red and single green signals (double arrows).

Fig. 2

Box-and-whisker plot of ALK-positive and ALK-negative samples in tumor and adjacent normal areas. Seventeen ALK-positive and 15 ALK-negative patients were analyzed to determine the % of ALK-positive cells from various tumor and adjacent normal areas from each individual patient. P values were computed by Welch two-sample t test. The box portion of the box-and-whisker plot includes 50% of the data. The bold line in the box represents the median of the data, and the borders of the box represent the lower quartile (25% percentile) and the upper quartile (75% percentile) of the data. The upper and lower whiskers represent the maximum and minimum % of ALK-positive cells.

Fig. 3

Scatter plot of patient number [adenocarcinomas of known EGFR and KRAS status (marked)] versus smoking status in pack-years. The vertical line separates ALK-positive from ALK-negative cases. ALK FISH positivity was statistically significantly enriched among never/light smokers (<10 pack-years; P = 0.0004, Fisher’s exact test; proportion positive among never/light smoking adenocarcinomas = 13 of 33, 39%; proportion positive among those with >10–pack-year smoking history = 0 of 22).