The (Con-) Fusion in ALK Diagnostics: When Food and Drug Administration-Approved Algorithms Fail.

TO THE EDITOR: The recent study by Cai et al showed that in non–small-cell lung cancer (NSCLC), intratumoral heterogeneity of ALK-rearranged and ALK/EGFR-coaltered lung adenocarcinoma is not a rarity but occurs frequently in the daily practice. We appreciate this study and the authors’ comments because they address an important and underestimated problem. If read in deep detail, the study would lead one to conclude that the present algorithms for identifying ALK-positive carcinoma fail. To date, despite the increasing availability of immunohistochemistrybased stainings used in addition to or instead of fluorescence in situ hybridization (FISH) methods, FISH methods, including Food and Drug Administration–approved assays, are still widely used. Thus, it is still assumed that a minimum of 10% to 15% of tumor cells must test positive before the sample is diagnosed as ALK positive. However, the current study contradicts this assumption by showing that within a single tumor, there may be regions that fulfill the requirements for ALK positivity, whereas other regions are clearly negative. For this reason, any previous cutoff values become obsolete because one cannot exclude that a small diagnostic biopsy containing percentages below these values are indeed negative, and because even a single positive cell could be a part of the heterogeneity or could be the single cell that is selected during the upcoming therapies. Moreover, even if the entire tumor could be diagnostically investigated by an ALK screening— an issue that in practice can never be performed because further histologic analyses are required and not all slides can be used for FISH—there remains a chance that metastases of the tumor bear different mutation profiles. The study addresses an important topic that we previously discussed, namely, that the combined mutations in NSCLC are not rare exceptions but daily reality. Thereby, in addition to EGFR/ALK combinations, KRAS/ALK comutations can occur. During routine diagnostics, we identified two patients eligible for crizotinib therapy because they carried a combinedALK andROS translocation. The first patient was a 79-year-old Caucasian male with an approximately 5–pack-year smoking history. The patient had a primary NSCLC and tested negative for mutations of the genes KRAS, BRAF, and EGFR. The analysis of the tumor infiltrates identified two regions that tested positive for ROS mutations by FISH analysis but negative for ALK mutations, whereas in a third tumor region, the opposite was observed (ie, ALK positivity with negative results forROSmutations). The second patient was a 63-yearold Caucasian female with a cumulative smoking history of 60 packyears. Shewas diagnosed in a late stage and already suffered frombrain and liver metastases, but because of a positive result for a combined ALK and ROS mutation in the entire primary tumor (Fig 1), she received crizotinib therapy. Although it was expected that in this double-eligible patient the crizotinib therapy should have had a positive effect, no treatment success was observed, and the patient died shortly thereafter. In light of the study by Cai et al, these cases make the diagnostic situation even more complicated. At the stage of molecular diagnostics, it is unclear which cell populations of a heterogeneous tumor are most likely to grow further and become therapy resistant or, vice versa, which parts of the tumor are sensitive to a planned and eligible therapy. As soon as a mixed population of tumor cells occurs, it must be taken into account that standard therapy regimens may not be the optimal choice and the first-line-second-line concept of tumor therapy is at least questionable for those patients. Moreover, the tumor heterogeneity would be one explanation for the high rate of crizotinib nonresponders because it remains to be investigated whether this nonresponse can be reduced to ALKnegative cells or whether distinct ALK mutations/variants display an altered response to crizotinib. It is still not known which variants of an ALK/EML4 fusion respond to crizotinib therapy, and the study of Cai et al exacerbates this problem.