ALK FISH Assays for Non-small Cell Lung Cancer Treatment Selection: Scoring Issues and Probe Design

ALK FISH Assays for Non-small Cell Lung Cancer Treatment Selection: Scoring Issues and Probe Design Helen J. Lawce and Susan Olson Abstract Lung cancer is becoming more treatable with new pharmaceuticals. There is a growing interest in FISH testing to determine which drugs to use on lung cancer patients for the best results, with a concurrent need for laboratories to provide ALK FISH testing for nonsmall-cell lung cancer. However, the test has some unique difficulties and requires a great deal of experience to interpret in some cases. A three-color probe may help make the ALK FISH test more reliable. Glossary Adenocarcinoma: malignant tumor of a gland Carcinoma: a cancer arising in the epithelial tissue of the skin or of the lining of the internal organs Companion diagnostic: a diagnostic test linked to a specific, known therapeutic drug to provide information to enable treatment decisions Large-cell carcinoma: a microscopically identified variant of certain cancers, for example lung cancers, in which the abnormal cells are particularly large Squamous-cell carcinoma: a carcinoma that arises from squamous epithelium tinib : denotes a tyrosine kinase inhibitor Lung cancer kills 1.4 million people worldwide with 160,000 of these deaths occurring in the United States. It is the leading cause of cancer deaths in the United States, exceeding the mortality of breast, prostate, and colon cancer combined. Tobacco smoking causes the majority of lung cancers. However, there are a growing number of lung cancers in individuals who have never smoked ( lifetime never smokers ), with a frequency of approximately 10-15% of all lung cancers. Lung carcinoma in nonsmokers is usually attributed to genetic factors, aging, radon gas, asbestos, and/or air pollution. Most primary lung cancers are carcinomas that are derived from epithelial cells, and the main categories are small-cell lung carcinoma ( oat cell cancer ) and non-small-cell lung carcinoma (NSCLC). The three types of NSCLC are adenocarcinoma, squamous-cell carcinoma, and large-cell carcinoma. Each type of lung cancer grows and spreads differently and may be treated differently. For the last decade, tyrosine kinase inhibitors (TKIs) have been used to treat late stage lung cancers. Tyrosine kinases (TKs) are enzymes that are responsible for the activation of signal transduction through phosphorylation of various proteins. Tyrosine kinases (TKs) include the BCR/ABL fusion protein in CML, for which the TKI imatinib (Gleevec) was developed. Shortly after the FDA approved Gleevec, the TKIs gefitinib (Iressa), and erlotinib (Tarceva) were developed against carcinogenic mutations leading to overexpression of the TK EGFR (epidermal growth factor receptor, also called erbb1) for the treatment of advanced lung tumors. ALK (anaplastic lymphoma kinase) is a TK that was originally discovered because of its involvement in a translocation t(2;5) (ALK/NPM [nucleophosmin] fusion) in anaplastic large cell lymphoma. The ALK gene is rearranged, mutated or amplified in several types of tumor besides lymphoma including neuroblastoma and NSCLC. In 2007, Soda et al. reported that an inversion on the chromosome 2 short arm (Figure 1) caused a fusion of ALK with EML4 (echinoderm microtubule-associated protein-like 4) inhibiting apoptosis and inducing cell proliferation. Recent studies have indicated that the ALK-EML4 gene fusion is present in 2-7% of all NSCLCs in the United States, with a higher frequency in nonsmoking patients. Patients with this gene fusion are typically younger non-smokers who do not have mutations in either the EGFR gene or in the K-Ras gene. Lung cancer patients whose tumors carry the ALK rearrangement respond well to the TKI crizotinib (Xalkori). The recently published College of American Pathologists guidelines recommend fluorescent in situ hybridization (FISH) testing for ALK rearrangements in adenocarcinomas and mixed lung cancers with an adenocarcinoma component, but not for other types of lung cancer such as squamous, small cell, or large cell carcinoma with no evidence for adenocarcinoma differentiation (Lindeman et al., 2013). The requirement for this new assay to determine treatment has translated into a growing need for FISH testing. Often, EGFR mutation testing is being done at the same time or before the FISH testing and so the CAP guidelines cover both tests. The FDA has approved the Abbott Molecular Laboratories ALK Break Apart FISH assay as a companion diagnostic for selection of patients to be treated with the FDA-approved ALK TKI, crizotinib. It consists of a Spectrum Orange-labeled 300-kb probe on the telomeric side of ALK and a Spectrum Greenlabeled 442-kb probe on the centromeric side (Figure 2). If the ALK region is not rearranged, it will appear as a fused orange and green signal. Due to the high ploidy of many lung tumors, there may be multiple fused signals in a nucleus with no rearrangements (Figure 3a). If the ALK region has been translocated or inverted, it will appear as separated orange and green signals separated by more than two signal widths apart (Figures 3b,c). Often there 66

2p 42,250,020 42,413,192 29,269,144 29,997,936 2p with location of EML4 and ALK relative to each other a) 2p b) Inversion Fusion of EML4/ALK: a. Wild type b. Inversion with fusion of ALK/EML4 Figure 1. Diagram of the EML4/ALK inversion rearrangement on chromosome 2. Multiple different EML4-ALK translocation variants have since been identified that contain various truncations of EML4 but always contain the tyrosine kinase domain of ALK, starting at exon 20. The most common (~70%) translocations in NSCLC involve EML4 exon 13 (variant 1) or EML4 exon 6 (variant 3a/b; 3b contains an additional 33-bp due to alternative splicing). Reprinted by permission from Atlas Genet Cytogenet Oncol Haematol July 2009; Perner S, Wilbertz T, Stiedl AC, Rubin MA. EML4 http://atlasgeneticsoncology.org//genes/eml4id44353ch2p21.html is loss of the green signal resulting in a single orange signal instead of the classic split orange and green signals, and these variant patterns are considered positive because the orange signal is on the kinase coding side of the ALK gene. The native fused orange/green signal may be missing but presence of an orange and green signal is enough to call a cell positive for a rearrangement (Figure 3d). For ALK rearrangements, because there are several partners besides EML4, a break-apart FISH probe design is the strategy of choice. Other partners for ALK include KIF5B (kinesin family member 5B on chromosome 10) and TFG (TRK-fused gene, chromosome 3). Because the ALK/EML4 rearrangement is often caused by a small paracentric inversion of 2p21/2p23, the split signals do not always move very far apart, making the interpretation of the FISH more difficult than other break-apart probe rearrangements. Further confounding the interpretation is that tumors may be multiclonal, heterogeneous, and mixed with stromal elements so that the numbers of cells exhibiting a clear split signal can be proportionally small. Depending upon multiple factors, both preanalytical (necrosis, fixation times, age of slides or tissue blocks) and analytical (e.g., enzymatic overdigestion), signals from tumors without rearrangements can separate almost as much as those harboring an inversion. An unusual feature of this rearrangement is the variability of the EML4 breakpoint, with at least 11 variants (Figure 4). The variant breakpoints can cause differences in the distance that the orange and green signals separate from each other (Wallandar et al., 2012). Wallander et al. (2012) hybridized ALK to tumors with different EML4 variants detected with multiple methods including PCR, and they report that for EML4 variant 3a/b, there was concordance 67

Telomere 2p23 Region Centromere LSI ALK Dual Color, Break Apart Rearrangement Probe 2 Figure 2. The Abbott Molecular Laboratories FDA approved ALK break-apart probe design, reproduced by permission of Abbott Molecular Laboratories. Figure 3. ALK patterns in adenocarcinoma of lung. A. No rearrangement of ALK but extra signals for ALK. B. ALK is split (positive cells) but signals do not separate very far since the rearrangement, a paracentric inversion, is within the same arm of chromosome 2. C. ALK is split (positive cells) and signals are widely spaced. Most likely these cells are carrying an interchromosomal exchange. D. ALK is split, leaving only a normal fusion with a single orange signal (left) or no normal fusion with only an orange and green signal (right). Both are considered positive for a scorable rearrangement by FDA guidelines. 68

variant 1 EML4 ALK variant 2 variant 3a variant 3b variant 4a variant 4b linker of 11 bp variant 5a variant 5b Intron 19 of ALK Figure 4. Some of the 11 (and growing in number) known EML4 breakpoint variations and fusion isoforms (numbers = exon number). Variant 1: exon 1-13 (EML4) + exon 20-29 (ALK) Variant 2: exon 1-20 (EML4) + exon 20-29 (ALK) Variant 3a: exon 1-6a (EML4) + exon 20-29 (ALK) Variant 3b: exon 1-6b (EML4) + exon 20-29 (ALK) Variant 4a : exon 15 (EML4) + exon 20-29 (ALK) Variant 4b : exon 14 (EML4) + linker of 11bp + exon 20-29 (ALK) Variant 5a : exon 2 (EML4) + exon 20-29 (ALK) Variant 5b : exon 2 (EML4) + intron 19 (ALK) + exon 20-29 (ALK) Reprinted by permission from Atlas Genet Cytogenet Oncol Haematol July 2009; Perner S, Wilbertz T, Stiedl AC, Rubin MA. EML4 http://atlasgeneticsoncology.org//genes/eml4id44353ch2p21.html between scorers on the positive samples, but for variant 1, only 1 tumor out of 9 was scored positive by all three readers using a 40% cutoff. Variant 1 is the most common (Perner et al., 2009). Therefore, it can be difficult to score ALK FISH assays, and there is need for experience with many normal and abnormal samples to gain familiarity with and accuracy in scoring them. The CAP guidelines suggest that a pathologist not only mark the tumor area for the technologist to score, but that they either do the scoring or check the slides after the technologist scores them. No matter who scores the slides, the recommendations suggest that the best strategy is to have two scorers check each sample, and that they are trained in the appearance of tumor nuclei so they do not inadvertently score normal stroma, lymphocytes or neutrophils. Again, it is not unusual for two scorers to find discordant results due to the difficulties inherent in the test (Wallender et al., 2012). The original trials in which ALK FISH was shown to predict treatment response determined that the cutoff for interphase positive FISH findings for split signals was 15% or more (Camidge et al., 2010; Rodig et al., 2009), and that cutoff is still being used in most laboratories. The original researchers did not address the findings of the single orange signal patterns with loss of the green. However, the Abbott insert does include the variant signal patterns and uses the 15% cutoff value for all positive findings (split orange and green or loss of green with extra orange signals). For cases of ALK interchromosomal translocations, the split signals are easily scored due to the distance between the chromosomes involved. For inversions, where artifactual split signals are difficult to distinguish from true split signals, the FDA method encourages the use of the cutoff value of 15% to interpret the findings, with the artifactual split signals falling below 15% 69

2p23.2-23.1 D651941 ALK ~729kb D25213 SHGC-144348 EML4 ~160kb SHGC-57909 ~410kb ~493kb ~623kb ~437kb Figure 5. Cymogen Dx 4 color ALK-EML4 probe design, reprinted by permission. The ALK-EML4 del-tect Four Color direct labeled FISH probe: the two probes flanking the ALK gene are a telomeric CYMO-AQUA ALK probe ~410 kb in size and a centromeric CYMO-ORANGE probe ~493 kb in size. The two probes flanking the EML4 gene are a telomeric CYMO-GREEN probe ~623 kb in size and a centromeric CYMO-RED probe ~437 kb in size. Figure 6. Cymogen Dx 4 color ALK-EML4 probe hybridized to normal tonsil cell. in this paradigm. Nonetheless, even with two or more scorers, the findings can appear equivocal. In response to the inherent difficulties scoring ALK breakapart probes CymoGen Dx (New Windsor, New York) developed a unique four-color probe design for the EML4/ALK rearrangement. Their probe consists of two colors flanking each gene (Figures 5, 6). The ALK is aqua, and the ALK (centromeric) is orange, so if the ALK (aqua) is rearranged with a different chromosome, it will simply separate from the orange, leaving the EML4 fusion intact. If there has been an inversion, the aqua ALK will form a fusion with either the green or the red that flank the EML4, and sometimes with both the green and the red signals, depending on which EML4 breakpoint is involved (Figures 7). A single ALK aqua is also considered a positive finding with the four-color probe, just as it would be in the Abbott probe design with the single ALK signal in orange. This probe design is elegant and informative. However, with four colors, it is difficult to interpret against a polyploid background with overlapping signals and truncation artifact. For a day-to-day clinical test, it may be more feasible to create a combination of the two approaches, reducing the design to three colors. In order to make a standard, red or orange/green break-apart probe more sensitive for the inversion rearrangement, it seems reasonable to add an aqua probe that spans the EML4 gene region. For equivocal split signals, the aqua can be used to determine a true split signal pattern. If it splits and partially fuses with the ALK (orange), it would be consistent with an inversion. If the EML4 aqua did not split or juxtapose with the ALK signal, it would indicate that no inversion had taken place. There is a three-color break-apart commercial probe available with this design including an EML4-spanning aqua component from the German company Zytovision. Ideally, the aqua component of the probe could be made available as an additive to the standard two-color probe set from Abbott Molecular Laboratories for investigating equivocal cases. Such a design would make the ALK assay for NSCLC much more sensitive and specific, and if it were available for the FDA-approved probe it would allow for better accuracy in choosing treatment regimens using this companion diagnostic. Because of the unusual characteristics of the ALK- EML4 rearrangement with the complications of potentially heterogeneous/necrotic/truncated/mucinous tumors that may 70

Figure 7. Cells from the same tumor hybridized with: A. the FDA approved Abbott Molecular Laboratory ALK probe and B. the 4-color Cymogen ALK-EML4 probe set. have faint or loose signals, the two-color ALK break-apart probe set has a fair probability of a false positive or negative result. It is critical to be sure we are doing our best for these patients and the health care community. References Camidge DR, Kono SA, Flacco A, Tan AC, Doebele RC, Zhou Q, Crino L, Franklin WA, Varella-Garcia M. Optimizing the detection of lung cancer patients harboring anaplastic lymphoma kinase (ALK) gene rearrangements potentially suitable for ALK inhibitor treatment. Clin Cancer Res. 2010; 16(22):5581-5590. Lindeman, N, Cagle, P, Beasley, M, Dhananjay, A, Dacic, S, Giaccone, G, Jenkins, R, Kwaitkowski, D, Saldivar, J, Squire, J, Thunnissen, E, Ladanyi, M. Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors. Guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Arch Pathol Lab Med, February 12, 2013. Perner S, Wilbertz T, Stiedl AC, Rubin MA. EML4 (echinoderm microtubule associated protein like 4). Atlas Genet Cytogenet Oncol Haematol. July 2009. http://atlasgeneticsoncology.org/genes/ EML4ID44353ch2p21.html Rodig SJ, Mino-Kenudson M, Dacic S, Yeap BY, Shaw A, Barletta JA, Stubbs H, Law K, Lindeman N, Mark E, Janne PA, Lynch T, Johnson BE, Iafarate AJ, Chirieac LR. Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population. Clin Cancer Res. 2009; 15(16):5216-5223. Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, Fujiwara S, Watanabe H, Kurashina K, Hatanaka H, Bando M, Ohno S, Ishikawa Y, Aburatani H, Niki T, Sohara Y, Sugiyama Y, Mano H. Nature. 2007 Aug 2;448(7153):561-6. Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, Fujiwara S, Watanabe H, Kurashina K, Hatanaka H, Bando M, Ohno S, Ishikawa Y, Aburatani H, Wallander ML, Geiersbach KB, Tripp SR, Layfield LJ. Comparison of reverse transcription-polymerase chain reaction, immunohistochemistry, and fluorescence in situ hybridization methodologies for detection of echinoderm microtubule-associated proteinlike 4-anaplastic lymphoma kinase fusion-positive non-small cell lung carcinoma: implications for optimal clinical testing. Arch Pathol Lab Med. 2012 Jul; 136(7):796-803. Wallander ML, Geiersbach KB, Tripp SR, Layfield LJ. Comparison of reverse transcription-polymerase chain reaction, immunohistochemistry, and fluorescence in situ hybridization methodologies for detection of echinoderm microtubule-associated proteinlike 4-anaplastic lymphoma kinase fusion-positive non-small cell lung carcinoma: implications for optimal clinical testing. Arch Pathol Lab Med. 2012 Jul;136(7):796-803. Helen J. Lawce and Susan Olson Oregon Health & Science University Knight Diagnostic Cytogenetic Laboratories lawceh@ohsu.edu 71