Journal of Thoracic Oncology:
Finding ALK-Positive Lung Cancer: What Are We Really Looking for?
Camidge, D. Ross MD, PhD*; Hirsch, Fred R. MD, PhD*†; Varella-Garcia, Marileila PhD*; Franklin, Wilbur A. MD†
*Division of Medical Oncology and †Department of Pathology, University of Colorado Comprehensive Cancer Center, Aurora, Colorado.
Disclosure: D. Ross Camidge, MD, PhD, serves on the advisory board for Pfizer and has received research funding from Pfizer, Lilly, and Novartis. Fred R. Hirsch, MD, PhD, serves on the advisory board for Pfizer, has received research funding from Ventana and Roche, and holds a patent for EGFR FISH. Marileila Varella-Garcia, PhD, holds a patent for EGFR FISH. Wilbur A. Franklin, MD, holds a patent for EGFR FISH.
Author for correspondence: D. Ross Camidge, MD, PhD, Room ACP 2256, Anschutz Cancer Pavillion, University of Colorado, Aurora, CO 80045. E-mail: firstname.lastname@example.org
The anaplastic lymphoma kinase (ALK) is a transmembrane tyrosine kinase encoded on chromosome 2.1 It was first described as one of the fusion partners within a reciprocal translocation (t(2;5)(p23;q35)) occurring in anaplastic large cell lymphoma.2,3 Activation of ALK through chromosomal rearrangements (inversions or translocations) that fuse various 5′ partners with the 3′ kinase domain of ALK have since been described in several other malignancies, including non-small cell lung cancer.1,4 In lung cancer, the most common 5′ fusion partner for ALK is EML4, but two other fusion partners, KIF5B and TFG, have also been reported.5–7
Interest in this chromosomal arrangement in lung cancer has been piqued by clinical trials of crizotinib (PF-02341066), a dual MET/ALK inhibitor that has shown astonishing clinical activity in patients with ALK rearrangements proven by fluorescence in situ hybridization (FISH) using the Vysis ALK break-apart probe set.8 Registration studies comparing crizotinib to standard chemotherapy in this population are ongoing, and Pfizer has also announced plans to file with the Food and Drug Administration for accelerated approval based on the initial phase I results.
A major limitation, both in implementing these ongoing trials and in getting access to the drug, if it were to be licensed for a molecularly defined indication, is the small proportion of lung cancer patients who have ALK rearrangements. A recent meta-analysis has indicated that only 4% of lung cancers have ALK rearrangements.4 How to best identify this 4% and which ALK testing method should be employed are rapidly becoming crucial issues in the optimal management of patients with lung cancer.
FISH is the current gold standard used as an entry criterion for all the crizotinib trials to date. The break-apart format of the FISH probe allows detection of rearrangements involving the tyrosine kinase domain of ALK independent of the fusion partner or specific breakpoint. The cut point for determination of a positive result has been set for the lung cancer trials at more than 15% of tumor cells positive for split red and green signals (separated by more than two signal diameters) or single red (3′) signals.8,9 Yet, FISH requires specialized laboratory techniques, expert interpretation, and may also be viewed as a relatively expensive screening assay.
A potential alternative to FISH screening is immunohistochemistry (IHC). The ALK protein is expressed at very low levels in most normal tissues, and its presence in a tumor specimen could be sufficiently abnormal as to indicate an oncogenic role in those cells.1 Unlike techniques such as reverse transcriptase polymerase chain reaction, which are fusion partner specific, IHC, similar to the break-apart FISH assay, should detect ALK independent of the fusion partner within any rearrangement. However, as the 5′ fusion partner also contains the promoter for the fusion gene, absolute levels of protein (and the cellular location of the signal) could still vary depending on the 5′ partner. Two studies in this issue of Journal of Thoracic Oncology address the role of IHC in ALK screening lung cancer.10,11 One whole section study performed in the United States and the other a tissue microarray study performed in South Korea compared the results of immunohistochemical staining for ALK with FISH testing. Table 1 summarizes the major points of the two studies. Each group used the intensity and frequency of staining to assign arbitrary scores (0 to 3+) to each specimen. All FISH-positive cases exhibited some level of ALK staining, while all IHC-negative cases were also negative for ALK rearrangements by FISH (100% sensitivity). However, specificity was not as robust. While highly positive IHC cases (3+) were uniformly FISH positive, many cases with lower levels of immunostaining were FISH negative. Each group of investigators conducted their study in slightly different ways. We know, for example, that certain lung cancer subgroups are more likely to have an ALK rearrangement than others. In our recent Colorado series, FISH testing among patients with adenocarcinoma, ≤10 pack-year smoking history, known to be EGFR and KRAS wild-type produced an ALK positivity rate of 45%.9 Yi et al.10 only tested tumor specimens from never smokers with adenocarcinoma, in whom 9.9% were ALK FISH positive. In contrast, Paik et al.11 initially screened all resected non-small cell lung cancer cases, in whom 4.2% were ALK FISH positive. When Yi et al. screened an enriched group, they inevitably increased the positive predictive value of their test while reducing its negative predictive value, something that should be borne in mind when considering applying the same screening data to broader or narrower populations than those within each original study.
Based on the high sensitivity of the applied ALK immunostaining but its moderate specificity, both sets of authors propose two-tier systems for evaluating ALK with an initial immunohistochemical screening step followed by FISH evaluation of 2+, or both 1+ and 2+, IHC-positive cases. Advantages of such a system include the large established infrastructure that would be able to apply immunostaining methods to surgical pathology specimens and a potential large reduction in the number of cases that would require FISH testing. However, such a system would only make sense if (a) the technology was reliable and offered minimal false negatives, and (b) was cost-effective when made truly reliable across multiple service providers. In breast cancer, where a two-tier system for HER2 is currently employed, preanalytic processing procedures, antibody standardization, and reproducibility of scoring between observers have emerged as major issues.12 Preanalytical processing steps in the handling of specimens are only cursorily addressed in the two studies reported in this issue of Journal of Thoracic Oncology. With regard to the choice of antibodies, each study used a different commercially available clone (Table 1). Several studies, in addition to those in this edition of the journal, have recently been published using different anti-ALK antibodies, different comparators, and different scoring systems conducted on differently enriched patient populations.7,8,13–15 In the study by Mino-Kenudson et al.,14 two different anti-ALK antibodies were compared head-to-head, showing significant differences in sensitivity by IHC. The best performing antibody (Cell Signaling clone D5F3), which is currently not commercially available, showed far higher sensitivity and specificity for finding FISH-positive cases than the DAKO ALK1 antibody used by Yi et al. In addition, the experimental antibody showed far higher reproducibility between observers (Kappa score = 0.94) than that demonstrated within the Yi et al. study (Kappa score = 0.55).10,14 The Novocastra antibody used by Paik et al. also showed a high interobserver agreement (Kappa score = 0.92); however, just as with the Cell Signaling antibody, should it become commercially available, a high level of agreement across multiple users and multiple different sites will be required before any given IHC assay could be considered reliable enough for widespread screening.11
When access to a specific drug (crizotinib) is likely to be restricted to those with a defined molecular abnormality, the avoidance of false negatives is imperative. Cut points for submission of a specimen for FISH testing within any proposed two-tier system would need careful consideration. When Yi et al.10 raise the possibility of not FISH testing IHC 1+ patients in their study of never-smoking adenocarcinomas, in which subgroup the ALK positivity rate by FISH was still 5%, they are really suggesting that missing 10% of their total ALK FISH-positive patients (1 of 10) would be acceptable. While any kind of screening is certainly better than no screening, given the overall poor prognosis of lung cancer patients, we would suggest that such a strategy, as it stands, would be less than ideal in the long term.
In addition to trying to develop simpler, quicker, and cheaper screening tests to find the same population as the break-apart FISH probe, other screening techniques, if optimized further, could also offer something else. As only ALK FISH-positive patients currently have access to crizotinib in clinical trials, we do not know whether there are cases that may be positive through other screening techniques, but negative through ALK FISH testing, that may similarly benefit from ALK inhibitor therapy. With the possibility of other ALK screening tests becoming more widely available, in conjunction with greater access to crizotinib, should it gain accelerated approval within the United States, we may well be able to expand the population benefiting from crizotinib through careful exploration of some “atypical” FISH-negative cases in the future.
In conclusion, we believe that both studies in the current issue of Journal of Thoracic Oncology are important contributions to the development of a diagnostic/screening paradigm for ALK in lung cancer. However, it seems clear that IHC cannot fully replace FISH yet nor is a two-tier (IHC then FISH) based screening system currently ready for prime time. Considerable work is still required to assess and standardize the impact of sample preparation, antibody selection, and signal quantification to generate reliable data for any given IHC screening technique on its widespread reproducibility, sensitivity, and specificity. If we really want to aim for a policy of no (true) ALK-positive patient being left behind, we must set our sights high from the outset.
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