Dacic, Sanja MD, PhD; Nikiforova, Marina N. MD
HISTOLOGY OF LUNG CARCINOMA AND MOLECULAR TESTING
Large number of studies has shown that lung carcinoma histology is an important component of treatment decisions, for reasons of safety or efficacy.1–5 Use of pemetrexed and bevacizumab is restricted to nonsquamous cell carcinoma. Pemetrexed showed superior efficacy in patients with nonsquamous cell histology in both first-line and second-line settings, whereas bevacizumab showed life-threatening toxicity in patients with squamous cell histology.6,7 Therefore, a large emphasis is given to morphologic subtyping of non–small cell lung carcinoma (NSCLC). If NSCLC cannot be subclassified on morphology alone, a limited panel of immunohistochemical markers (eg, TTF-1 for adenocarcinoma; p40 for squamous cell carcinoma; synaptophysin/CD56 for neuroendocrine carcinomas) should be used. Using this diagnostic approach the number of cases classified as NSCLC, NOS should not be >5%. Histologic subtyping is still important in triaging tumor samples for appropriate molecular testing.
Recently published College of American Pathologists (CAP)/IASLC/AMP guideline for molecular testing in lung carcinoma recommended EGFR and ALK testing for adenocarcinomas and mixed lung cancers with an adenocarcinoma component (eg, pleomorphic carcinoma, carcinosarcoma, adenosquamous).2–4 Large cell carcinomas with adenocarcinoma immunophenotype should also be subjected to recommended molecular testing in lung adenocarcinoma. EGFR and ALK testing are not recommended for squamous cell carcinoma or small cell carcinoma. A spectrum of potentially targetable driver mutations and copy number changes in squamous cell carcinoma has been recently published.8 Currently, there is no guideline for routine clinical molecular testing in squamous cell carcinoma outside of clinical trials.
PRESENT STANDARD OF CARE MOLECULAR TESTING
Positive Predictors of Response to EGFR-TKI
EGFR-TKIs are associated with a relatively low toxicity when compared with platinum-based chemotherapies. It was believed for many years that these therapies can be given to patients based on their clinical characteristics regardless of mutation status. The Iressa Pan-Asia Study trial showed that EGFR mutation-negative patients treated with first-line gefitinib had shorter progression-free survival than those who received carboplatin-paclitaxel.9 This trial suggested that it is harmful to treat patients who do not harbor EGFR mutations with first-line EGFR TKIs and that tumor genotype should determine the most appropriate first-line treatment.
Activating somatic mutations in the exons 18-21 of the tyrosine-kinase domain of EGFR have been shown to best correlate with a high likelihood of response to EGFR-TKI.10–12 They occur in approximately 10% white and 30% Asian patients with lung adenocarcinoma, usually women and never smokers. Therefore, EGFR mutation testing in adenocarcinoma or other types of NSCLC with adenocarcinoma component (eg, adenosquamous carcinoma, sarcomatoid carcinoma) is recommended. The 2 most common mutations in-frame deletions in exon 19 and L858R point mutations in exon 21 account for 90% of all EGFR mutations. Other less common mutations associated with response have also been reported (Table 1). Many initial trials with EGFR-TKI used Sangers sequencing for assessment of tumor genotype. As molecular testing for EGFR-TKI has not been standardized for almost a decade, many laboratories developed various mutation detection methods which were not used in trials. The most recent CAP/IASLC/AMP guideline for molecular testing recommended that laboratories can choose any assay that has sensitivity or specificity for detection of EGFR mutations equal or better than Sangers sequencing.2–4
Positive Predictors of Response to ALK Inhibitors
ALK rearrangements occur in approximately 4% of lung adenocarcinoma patients, usually younger, never smokers with clinically advanced disease.13–15 The majority of the ALK rearrangements within lung adenocarcinoma result from an interstitial deletion and inversion in chromosome 2p and result in the EML4-ALK fusion gene product. The oncogenic activity of this fusion product has been effectively blocked by small-molecule inhibitors of the ALK. On the basis of demonstrated efficacy and safety in phase 1 and 2 studies, crizotinib was granted accelerated approval by the Food and Drug Administration (FDA) for the treatment of advanced, ALK-positive NSCLC.16 Different assays have been used for assessment of ALK rearrangement including fluorescence in situ hybridization (FISH), reverse tanscriptase-polymerase chain reaction, and immunohistochemistry, and showed similar predictive performance.16–18 However, FDA approved the ALK Break Apart FISH Probe Kit (Abbott Molecular, Des Plaines, IL) as a companion diagnostic for targeted therapy with crizotinib in lung cancers. This recommendation pretty much restricted the ALK testing to FISH assays in the USA. It is felt that reverse tanscriptase-polymerase chain reaction for ALK is not suitable for clinical testing because of a large number of ALK fusion products and uncertainty about their predictive significance. Furthermore, RNA-based assays on formalin-fixed paraffin-embedded tissue could be challenging. Therefore, the CAP/IASLC/AMP guideline also recommended ALK-FISH assay as a standard of care.
The typical signal pattern for ALK-rearranged adenocarcinoma is a “split signal pattern” with an orange/green fusion signal, 1 separate orange and 1 separate green signal (Fig. 1). ALK gene is considered rearranged if a gap between separated green and red signals is >2 signal diameters and if this pattern is observed in ≥15% of analyzed nuclei. ALK-FISH can be very heterogenous and other patterns can be observed including extra isolated 3′ALK signal, additional fusion signals from polysomy, and more complex ALK rearrangements.
NOT STANDARDIZED OTHER CONSIDERATIONS FOR MOLECULAR TESTING
Clinical Testing for EGFR-TKI Resistance
There are 2 major types of resistance to EGFR-TKI—primary and secondary (acquired). Somatic mutations in the gene involved in the EGFR signaling cascade (KRAS, HER2, BRAF, PI3K, LKB1, SHP2) which are mutually exclusive with EGFR mutations are considered to represent a primary mechanism of resistance to EGFR-TKI. The possible exceptions are mutations in the PI3K gene. Development of new therapies targeting the downstream RAS effector pathways PI3K/AKT/mTOR and RAS/RAF/MEK will likely require knowledge about mutational status of these genes, but currently there is no guideline for their implementation in clinical practice outside of clinical trials.
Approach to secondary (acquired) resistance in patients treated with EGFR-TKI is different. The mechanisms of resistance are illustrated in Figure 2. The most common mechanism of resistance is EGFR-TKI mutation T790M, caused by a single-base substitution C to T, at nucleotide 2369.19 This mutation is found as a second mutation on the EGFR allele harboring the initial “sensitizing” EGFR mutation. This mutation can be rarely found in pretreatment specimens, usually in cases of familial lung cancer. The CAP/IASLC/AMP guideline strongly recommended development and implementation of sensitive assays for this mutation. It was also recommended that if in-house assays cannot be developed, a send out of cases to qualified laboratories should be considered.
The second most common mechanism of acquired resistance is amplification/polysomy of the MET gene.20 Currently, there is no recommendation for assessment of MET in EGFR-TKI-treated patients. In contrast, new drugs targeting against the MET kinase receptor or its ligand are in clinical trials with exploration of various assays such as FISH and IHC.21 Similarly, testing for other less common mechanisms of resistance such as HER2 amplification are not standardized or recommended. A subset of adenocarcinoma treated with EGFR-TKI will undergo morphologic transformation into small cell lung carcinoma or sarcomatiod carcinoma which could be assessed on hematoxylin and eosin alone.22,23 Therefore, it would be a good practice to obtain a tumor sample in patients with acquired EGFR-TKI resistance that would allow morphologic and potential genotypic assessment of the tumor.
Clinical Testing for Acquired Resistance to ALK Inhibitors
Similar to EGFR-TKI, acquired resistance to ALK inhibitors represents a major obstacle to successful treatment.24,25 Several mutations and ALK fusion gene amplification have been reported, but currently none of them have been recommended for routine testing.
Targeted therapies for many biomarkers (eg, BRAF, HER2) are in development, and their implementation in the clinical practice is pending (Table 2). However, requests for 2 biomarkers, ROS1 and RET rearrangements are increasing.
Chromosomal rearrangements involving the ROS1 gene can be identified in about 2% of lung adenocarcinoma occurring in young, never smokers patients.26–28 Lung adenocarcinomas with RET fusions showed response to FDA-approved RET-TKIs (cabozantinib and vandetanib) and to ALK/MET inhibitor crizotinib. Furthermore, there are several commercially available FISH probes that could be easily implemented in the clinical practice. Although there is no guideline for clinical testing for ROS1, because of availability of FDA-approved drugs and an increasing demand for the assessment of ROS1 rearrangements, laboratories should strongly consider implementation of FISH testing for ROS1 rearrangements.
RET rearrangement, a hallmark of radiation-induced thyroid cancer, has been reported to occur in 1% of lung adenocarcinoma patients. Patients with this rearrangement tend to be younger and never smokers.27,29–31 The somatic RET fusion genes, mostly recognized as CCDC6-RET (PTC1) and NCOA4-RET (PTC3), are associated with sporadic and radiation-induced papillary thyroid cancer. Clinically available TKIs such as sunitinib, sorafenib, and vandetanib target RET kinase activity, suggesting that another subset of NSCLC may be treatable with a kinase inhibitor. The possibility of association between RET rearrangement in lung adenocarcinoma patients with a history of therapeutic radiation for breast carcinoma or mediastinal lymphomas has been recently suggested (S. Dacic et al., unpublished observations, 2013). In vitro studies with cell lines also suggested that RET fusions may represent a genetic mechanism of radiation-induced lung adenocarcinoma.
POTENTIAL ROLE OF IMMUNOHISTOCHEMISTRY
Many clinical laboratories lack technical expertise for molecular assays. Furthermore, molecular assays are expensive comparing to other standard assays such as immunohistochemistry which is performed and readily interpreted by surgical pathologists. Although not recommended as a screening method for gene mutations or gene rearrangements, immunohistochemistry may be considered in cases with insufficient tumor tissue for molecular assays or in cases with technically suboptimal molecular assays.17,18,32–36 Summary of antibody characteristics potentially used as a selection criteria or screening tools for targeted therapies is shown in Table 3.
FUTURE OF MOLECULAR TESTING IN LUNG CANCER
Introduction of next-generation sequencing (NGS)37 technology enabled high-throughput detection of multiple genetic alterations in both constitutional and cancer genomes. NGS offers simultaneous sequencing of thousands to millions of short nucleic acid sequences in a massively parallel way.38,39 It provides clear advantages over the conventional sequencing technique, such as Sanger sequencing, by allowing to sequence large regions of the genome at lower cost and with higher sensitivity. It can be used for whole-genome sequencing, exome sequencing, transcriptome sequencing (mRNA sequencing), and targeted sequencing of multigene panels. Sequencing of subset of genes or genetic regions is performed either by hybrid capture approach or by amplification-based target-enrichment methods.35 It is a high-resolution, cost-effective method to interrogate patient samples for disease-specific genotype variants. It offers the distinct advantages of rapid laboratory turnaround time along with the generation-limited sequencing data that can be rapidly processed and analyzed without burdensome storage requirements. This is in contrast to whole-genome and exome sequencing that generates vast amounts of information but requires complex, costly, and time-consuming processing and analysis. Whereas large-scale analyses are essential for the discovery projects, it is more than likely that targeted panels will offer further advance in routine clinical molecular diagnostics.
Several commercial NGS platforms [Ion Torrent Personal Genome Machine (PGM, Life Technologies Inc., Grand Island, NY) and the MiSeq instrument (Illumina Inc., San Diego, CA)] are currently available for targeted sequencing and the technology will continue to develop (Table 4). They utilize different chemistries, including semiconductor sequencing and sequencing-by-synthesis technology, respectively. Semiconductor sequencing detects the protons released as nucleotides are incorporated during synthesis of clonally amplified DNA and a signal is proportional to the number of incorporated bases.40 Sequencing-by-synthesis approach is utilizing fluorescently labeled reversible-terminator nucleotides on clonally amplified DNA templates immobilized on the surface of a glass flowcell.37 They share similar performance characteristics providing up to 200 bp sequencing read length, 98% to 99.5% sequencing accuracy, adequate sequence depth or “coverage” (>500× for multigene panels), and total sequence output up to 1 to 10 Gb (Gigabases). Importantly, they provide high speed of sequencing (5 to 24 h) and the sequencing is less expensive compared with the larger sequencing platforms. The cost can be even further reduced by molecular barcoding of tumor samples which allows multiplexing of multiple samples in one run for high-throughput. Finally, targeted NGS sequencing instruments provide simplified and user-friendly bioinformatics analysis.
Targeted NGS is beginning to be implemented in the clinical laboratory practice. In oncology, it is applied for detection of individual mutations in cancer-related genes that may assist in cancer diagnosis, have prognostic value, or used for prediction of response to targeted therapy.38,41–43 It can be performed on formalin-fixed paraffin-embedded and freshly collected tissue specimens and on small fine-needle aspiration biopsies.40 Either commercially available panels (eg, AmpliSeq cancer panel, Life Technologies Inc., and TruSeq Cancer panel, Illumina) that offers sequencing for thousands of mutations in 50 cancer-related genes or custom NGS panels can be successfully used (Fig. 3).44
The implementation of NGS technology in a clinical laboratory is complex and requires significant expertise in clinical, technical, and bioinformatics aspects of sequencing. The laboratories should strictly apply all quality measures for NGS test development, validation, and quality assurance under guidance of the Clinical Laboratory Improvement Amendments and CAP regulations.
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