Test Utilization for Selecting Patients for Targeted Therapies in Non-Small Cell Lung Cancers [Utilization Spotlight]
Since 2012, we have been publishing a Utilization Spotlight in every issue of the Communiqué. Each Spotlight offers a quick view of utilization management best practices in action. This Spotlight is from January 2014.
Several FDA-approved specific inhibitors of certain molecular targets are effective for treating patients with non-small cell lung cancer (NSCLC). Given the available EGFR inhibitors and ALK inhibitors for treatment of NSCLC, patients who have an indication for such therapies should undergo EGFR and ALK testing on their tumors.
Currently, several United States Food and Drug Administration (FDA)-approved specific inhibitors of certain molecular targets are effective for treating patients with non-small cell lung cancer (NSCLC). Specifically, these include the epidermal growth factor receptor (EGFR) inhibitors erlotinib (Tarceva) and gefitinib (Iressa) and the anaplastic lymphoma kinase (ALK) inhibitor crizotinib (Xalkori). Crizotinib has also been shown to be effective in NSCLCs which lack the ALK gene rearrangement, if there is c-ros oncogene 1 (ROS1) translocation. Other drugs are under clinical and preclinical trials seeking FDA approval: rearranged during transfection (RET) inhibitors and mesenchymal-epithelial transition factor (MET) inhibitors, to name a few. Finally, BRAF (v-Raf murine sarcoma viral oncogene homolog B1) inhibitors approved by the FDA for treating advanced melanoma patients who harbor BRAF V600E may also be used for NSCLCs with the same mutation.
Biomarkers for a specific genetic abnormality or aberrant protein expression associated with responsiveness (or resistance) to targeted therapies have routinely been
tested to identify patients for these treatments. Currently the available molecular assays for NSCLC at Mayo Clinic include testing for the EGFR, ALK, ROS1, RET, KRAS and BRAF genes and detection of several others are under development. KRAS (Kirsten rat sarcoma viral oncogene homolog) mutation testing KRASA/KRAS Mutation Analysis, 7 Mutation Panel, Other [Non-Colorectal]) in NSCLC has mainly been used as a surrogate marker to predict the negative response to certain drugs, as well as to predict the negative status for other genetic abnormalities. Practically speaking, the KRAS mutation is mutually exclusive with the EGFR mutation as with the mutations of other driver oncogenes (albeit vanishingly rare exceptions) and has been well documented to be associated with resistance to EGFR inhibitors.
Approximately 85 percent of patients with EGFR mutations respond to EGFR inhibitors approved by the FDA for use in treating patients with NSCLC who have failed to respond to traditional chemotherapy. EGFR inhibitors are also effective as a first-line therapy for NSCLC. Thus, EGFR mutation analysis is used for selecting patients in these clinical contexts. Most molecular diagnostic procedures incorporate polymerase chain reaction (PCR) amplification and bidirectional gene sequencing of exons 18 through 21 of the tyrosine kinase domain of the EGFR gene. Some molecular abnormalities including exon 20 insertions and T790M mutation are associated with the resistance to EGFR inhibitors. Thus, testing the EGFR mutations known for drug resistance as well as responsiveness is important for selecting patients who need to be included or excluded for this costly therapy.
Recently, immunohistochemistry (IHC) has been reported to be helpful when the amount of tissue is insufficient for molecular testing. IHC uses mutation-specific rabbit monoclonal antibodies that will identify two of the most common EGFR mutations—exon 19 deletions and exon 21 L858R mutation—on formalin-fixed paraffin-embedded (FFPE) tissue. EGFR IHC would be informative only when it is positive, however, due to its limited coverage of mutation (detecting exon 19 deletions and exon 21 L858R mutations only) negative results cannot be used to exclude the presence of EGFR mutations. Clinical value of EGFR fluorescence in situ hybridization (FISH) or chromogenic in situ hybridization (CISH) is somewhat controversial at this time.
The EML4-ALK translocation occurs in two to five percent of unselected lung adenocarcinoma patients and is mutually exclusive with KRAS and EGFR mutations, despite rare cases of exceptions documented in the literature. The ALK inhibitor crizotinib is FDA approved for ALK rearranged lung adenocarcinomas with the companion FISH test using a break-apart probe on FFPE tissue. However, ALK IHC can also be used as a screening test given the excellent correlation shown in many studies performed in Europe, Asia, and the United States, including those from Mayo Clinic. Molecular testing guidelines published in 2013 by the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology (CAP/IASLC/AMP) have endorsed screening with ALK IHC as a valid approach. ROS1, originally described in glioblastomas, has been identified as a potential relevant therapeutic target in NSCLCs. Based on recent studies demonstrating the responsiveness to crizotinib in patients with ROS1 rearrangements, ROS1 FISH may be used to select patients for this therapeutic regimen.
BRAF mutations have been reported in three percent of lung adenocarcinoma and occur more commonly in current and former smokers. Several BRAF mutations in NSCLCs include V600E, G469A, and D594G. The incidence of BRAF mutations other than V600E may be significantly higher in NSCLCs than in melanomas. Those with non- V600E BRAF mutations probably will be resistant to BRAF inhibitors according to preclinical data, while eight of 20 patients with BRAF V600E-mutated NSCLC responded to dabrafenib, a BRAF inhibitor, which is currently in phase II clinical trials.
Recently, chromosomal rearrangements of RET have been identified in a very small subset of NSCLCs. The RET gene is mutated in medullary carcinomas of the thyroid, either as somatic (in sporadic cases) or germ line (in hereditary and former smokers. Several BRAF mutations in NSCLCs cases) mutation. A few available agents can effectively block the constitutive activation of the RET kinase. Patients with tumors harboring RET rearrangements may benefit from RET kinase inhibitors.
Assay Development and Method Description
Mayo Clinic’s Molecular Genetics and Cytogenetics laboratories have developed a set of assays to identify several important gene mutations that occur in NSCLC and influence therapy. When used in conjunction with clinical and pathologic information, these assays can assist the clinician in identifying patients with NSCLC that may benefit from targeted therapies with EGFR-tyrosine kinase inhibitors, ALK inhibitors, or RET kinase inhibitors.
Mayo Medical Laboratories offers EGFRX/Lung Cancer, EGFR with ALK Reflex, Tumor. Prior to DNA extraction, a pathologist will review the slides and macrodissect the specimen to enrich for tumor cells. Specimens are subsequently tested by a PCR-based assay which detects 29 mutations within exons 18 through 21 of the EGFR gene. Patients harboring activating mutations in the EGFR gene tend to respond well to the EGFR inhibitors gefitinib and erlotinib. However, following initial clinical response, these patients typically relapse within a year of treatment. In many cases, resistance is caused by an acquired secondary EGFR kinase domain mutation, T790M, a resistance mutation that will reduce the potency of any ATP-competitive kinase inhibitor. This mutation is detected by the EGFRX assay, to ensure that patients with this acquired mutation are not subjected to an ineffective, expensive course of treatment.
If an EGFR mutation is not detected, the specimen will automatically be tested for rearrangements of the ALK locus (FLCA/Lung Cancer, ALK (2p23) Rearrangement, FISH, Tissue) by the FDA-approved companion diagnostic FISH test using the Vysis ALK break-apart FISH probe kit.
While ALK IHC is not included in the EGFRX/Lung Cancer, EGFR with ALK Reflex, Tumor assay, the ALK IHC assay may be requested in conjunction with a pathology consultation (Order 70012/Pathology Consultation and request 82277/Anaplastic Lymphoma Kinase (ALK), Immunostain). ALK IHC has been used as a screening test for Mayo Clinic patients since 2010 with excellent sensitivity and specificity when compared to ALK FISH test results. This comparison has been well documented in the literature. The current molecular testing guidelines by CAP/IASLC/AMP endorsed the ALK IHC as a screening methodology with careful validation. Screening with ALK IHC before FISH would be highly cost effective, given the very low prevalence of ALK positive results at two to five percent in non-selected NSCLC cases. At Mayo Clinic, the ALK IHC assay is performed using a rabbit monoclonal antibody against EML4-ALK mutant protein (clone D5F3. Cell Signaling Technology, Danvers, MA, USA) that has been shown to have near 100% sensitivity and specificity in the literature. Each case is scored as zero (completely negative), one (faint, probably negative), two (weak, equivocally positive), and three (strong, positive). All cases other than score zero (that has been well under 10 percent of all patients tested with ALK IHC) will undergo ALK FISH testing for confirmation.
If the patient specimen is EGFR and ALK negative, testing for RET gene rearrangement or the ROS1 rearrangement may be warranted. FLRET/Lung Cancer, RET (10q11) Rearrangement, FISH, Tissue will identify RET gene rearrangements, and FROS1/Lung Cancer, ROS1 (6q22) Rearrangement, FISH, Tissue is useful for identifying ROS1 gene rearrangements.
Given the available FDA-approved EGFR inhibitors and ALK inhibitors for treatment of NSCLC, patients who have an indication for such therapies should undergo EGFRand ALK testing on their tumors using FFPE tissues. Mayo Medical Laboratories offers EGFRX/Lung Cancer, EGFR with ALK Reflex, Tumor using an algorithmic approach, starting with EGFR mutation analysis followed by ALK FISH if negative for EGFR mutations. ALK IHC is a highly accurate and cost-effective screening method for selecting patients to be tested for ALK FISH. Additional molecular analyses for KRAS, BRAF, ROS1, and RET are offered upon request although currently no therapy is available for these targets, other than within a clinical trial setting, using the agents approved for other tumors or using ALK inhibitor crizotinib for ROS1 rearranged NSCLCs. While the results of these tests may indicate the likelihood of response to targeted therapies, selection of any treatment remains a clinical decision.