Lung Cancer Testing: Why FISH for ALK Testing in NSCLC?

Meloni-Ehrig, Aurelia, PhD, DSc
Chief Director,Cytogenetics
AmeriPath
Orlando, FL Also by this Author 

Last year the National Comprehensive Cancer Network (NCCN)1 and the American Society of Clinical Oncology (ASCO)2 issued recommendations for performing gene mutation testing to direct therapy for patients with non-small cell lung carcinoma (NSCLC). Dr. Aurelia Meloni-Ehrig, Chief Director, Cytogenetics, AmeriPath discusses the role of molecular profiling in patient selection for targeted therapies and reviews the tests available in a lung cancer mutation panel.

Epidermal Growth Receptor Factor (EGFR)

EGFR is a gene involved in the development of lung carcinoma, in particular NSCLC,” explains Dr Meloni-Ehrig. “Activation of the EGFR protein via ligand binding stimulates protein tyrosine kinase, which leads to activation of signaling pathways associated with cell growth and survival. A point mutation or deletion of EGFR, generally occurring between exons 18 and 21, can lead to activation of the tyrosine kinase domain.Activating EGFR mutations, which are associated with increased sensitivity to EGFR TKIs, predominate in never-smokers, females, and tumors with adenocarcinoma and non-mucinous histology.1 The most common mutations associated with sensitivity to EGFR TKIs include exon 19 deletions.1 These mutations are associated with response rates of >70% in patients treated with either erlotinib or gefitinib.Other EGFR mutations (eg, T790M and exon 20 insertion), however, are associated with acquired resistance to TKIs.”

Anaplastic Lymphoma Kinase (ALK)

The ALK gene located at 2p23, is a cell-surface molecule that is involved in transmitting signals for cell growth and differentiation.“Rearrangements of the ALK gene through inversion or translocation leads to activation of the TK domain,” notes Dr. Meloni-Ehrig,“and are known to be present in Anaplastic Large Cell Lymphoma (ALCL), inflammatory myofibroblastic tumor, and neuroblastoma. The fusion between echinoderm microtubule-associated protein-like 4 (EML4) and ALK has been identified in a sub-set of patients with NSCLC.” This ALK rearrangement is primarily seen in adenocarcinomas, in younger patients who are never or light smokers, and is estimated to be present in around 4% to 7% of NSCLC.3,4

The FDA has recently approved crizotinib, an ALK inhibitor, for patients with locally advanced or metastatic NSCLC, who have the ALK gene rearrangement (ALK positive). Response rate to the drug has been shown to be 57%, with 72% progression free survival at 6 months.5

KRAS

KRAS has been implicated in the pathogenesis of several cancers and is a significant gene in the pathway of EGFR,” says Dr Meloni-Ehrig. The KRAS gene encodes the KRAS protein, which stimulates signaling pathways downstream from EGFRKRAS mutations lead to a constitutively activated, KRAS protein that continually triggers these downstream signals. Although EGFR TKIs can block EGFR activation, they cannot block the activity of the mutated KRAS protein. Thus, patients with KRAS mutations tend to be resistant to erlotinib and gefitinib. KRAS mutations are more likely found in adenocarcinomas, in patients who are smokers, and in Caucasian patients rather than East Asians and are prognostic for poor survival.

NCCN Guidelines

Current NCCN guidelines recommend that both EGFR and ALK mutation tests are performed to help guide therapy.1 The recommendation applies to adenocarcinomas, large cell carcinomas, and not otherwise specified carcinomas. “Some clinicians also like to test squamous cell carcinoma,” notes Dr. Meloni-Ehrig, “because there are still about 5% patients with squamous cell carcinoma that have either EGFR or ALK mutations, and might benefit from the EGFR or ALK treatment inhibition.”

Additionally, the NCCN guidelines note the association of KRAS mutations with TKI resistance and suggest that KRAS gene sequencing could be useful in identifying patients for TKI therapy.1

Lung Cancer Mutation Panel

“The lung mutation panel is designed specifically for NSCLC,” explains Dr Meloni-Ehrig, “and includes 3 key genes: EGFR, KRAS, and ALK. The EGFR and the KRAS are molecular tests, performed by sequencing, to detect mutations. The ALK is a fluorescence in situ hybridization (FISH) test, that is designed to look for rearrangements involving the ALK gene.”

EGFR, KRAS and ALK mutations are nearly always mutually exclusive which is why there is a strong rationale for including the 3 tests in one panel. Additionally this helps with specimen collection. Testing for each mutation individually necessitates cutting the tissue block, which is very small, several times. Each time you cut, you lose some material. So, it’s preferable to cut the block once and obtain all the sections needed to run the specific tests.”

FISH Testing

Methods for detecting the ALK rearrangements include FISH, PCR, and immunohistochemical (IHC) staining.

“There are key differences between the test methods,” observes Dr Meloni-Ehrig. “The molecular (PCR) test was developed to look specifically for the EML4-ALK fusion in NSCLC. At least 15 fusions involving different exons on the EML4 gene have been described so far. So, when we perform the PCR test looking for a specific fusion and we obtain a negative result, it raises the question: is it because we didn’t have all the primers for all the possible EML4-ALK fusions or is there another ALK rearrangement not involving EML4 present?”?

“The FISH test is the only FDA approved method, and is the test recommended in the NCCN guidelines for detecting an ALK rearrangement in NSCLC. The benefit of the ALK FISH is twofold,” she explains. “Firstly, it doesn’t discriminate between the variant EML4-ALK fusions. It will detect any rearrangement of the ALK gene because the probe uses the so called break-apart strategy. The probe is designed by labeling the 3’ and a 5’ portions of the ALK gene in different colors. If there is no rearrangement, the normal ALK, will appear as a fusion. So, when there is a split, because of a rearrangement, you will see the green and the orange signals separated from each other.”

“The other benefit,” she continues, “is the fact that two other, less common genes are known to form fusions with the ALK gene. Because the FISH employs a “break-apart” strategy, it will identify not only the EML4-ALK fusion, but other kinds of translocations that might be present in NSCLC.”

Additionally, IHC tests, which are used to detect ALK rearrangements in Anaplastic Large Cell Lymphomas, are considered inadequate to detect the majority of ALK rearrangements in lung adenocarcinomas.1

Interpreting FISH Results

“The advantage that cytogenetic specialized individuals have is that, when they look at the various FISH patterns, they are able to visualize what chromosome rearrangement is occurring. You don’t just see fusions and signals,” explains Dr. Meloni-Ehrig. “It is essential to interpret the various patterns carefully to make sure we are giving the right information to the oncologist.”

“In the normal pattern, when ALK is not rearranged, you see two fusions, telling us there are two intact copies of ALK. The typical, positive and straightforward pattern for ALK rearrangement is the presence of one fusion signal for the intact ALK and one green and one red separated from each other for the rearranged ALK. Then there are variations of the abnormal pattern, which gains of multiple fusions in the presence of a split signal or a partial deletion.” It is also important to distinguish between a 3’ and a 5’ partial deletion. Patients with deletion of the 3’ orange signal, which contains the TK domain, will not benefit from the TKI treatment, and this result should be considered negative. Whereas, partial deletion of the 5’ green signal is considered a positive result”

“Sometimes FISH shows gain of one or more fusions without a split signal. What does that mean? Carcinomas in general tend to have more than 46 chromosomes, so there would also be more chromosomes where the ALK gene is located and you might see multiple signals because of that. Obviously, this is also a negative result since no rearrangement of ALK is evident.

References

  1. NCCN Clinical Practice Guidelines in Oncology™. Non-small cell lung cancer. v3.2012. Available at http://www.nccn.org/professionals/physician_gls/f_guidelines.asp.  Accessed May 7, 2012.
  2. Keedy VL, Temin S, Somerfield MR, et al.American Society of Clinical Oncology Provisional Clinical Opinion: Epidermal Growth Factor Receptor (EGFR) Mutation Testing for Patients With Advanced Non–Small- Cell Lung Cancer Considering First-Line EGFR Tyrosine Kinase Inhibitor Therapy.
    http://jco.ascopubs.org/cgi/doi/10.1200/JCO.2010.31.8923. Accessed May 8, 2012     
  3. Shaw et al. J Clin Oncol 2009;27:4247–4253
  4. Solomon et al. J Thoracic Oncol 2009;4:1450–1454.
  5. Kwak EL, Bang Y-J, M.D., Camidge DR et al. Anaplastic Lymphoma Kinase Inhibition in Non–Small-Cell Lung Cancer. N Engl J Med 2010;363:1693-703.