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Trends in lung cancer molecular testing.

Introduction

A 63 year-old female with a 50-pack-year smoking history presented to the emergency room with left arm pain. During the evaluation an approximately 2 cm nodule was discovered in the left lower lobe (LLL). At that time the patient did not consent to biopsy of the nodule. Over the next two years, the nodule increased in size to 2.9 cm with enlarged mediastinal and left hilar lymph nodes and the patient consented to biopsy. Subsequent CT-guided biopsy of the left lung mass demonstrated an adenocarcinoma based on histologic evaluation. The patient had poor pulmonary function tests, was not a surgical candidate, and underwent Stereotactic Body Radiation Therapy (SBRT). Approximately a year later a restaging positron emission tomography (PET) scan demonstrated a LLL lesion and hilar/mediastinal nodes suggesting disease progression. Biopsy of the LLL confirmed an adenocarcinoma and molecular testing for epidermal growth factor receptor (EGFR) gene mutations was requested. Testing revealed a L858R exon 21 EGFR mutation, indicating a potential sensitivity to an EGFR inhibitor therapeutic regimen. (1) The patient was then started on Tarceva (Erlotinib) and 1 year follow-up revealed a stable LLL pulmonary mass without hilar or mediastinal adenopathy consistent with a response to therapy.

Cancer of the lung and bronchus are the leading causes of death in the United States. This is especially true in West Virginia where lung cancer incidence and death rates are among the highest in the nation. (2) The advent of personalized medicine has significantly increased the options for treating patients with lung cancer. Until a few years ago, it was appropriate standard of care for a pathologist to diagnose and group the three most common lung cancers into two categories; non-small cell lung carcinoma (NSCLC, which encompasses both squamous and adenocarcinoma) and small cell carcinoma. This categorization of tumors was sufficient for clinicians to select an appropriate patient therapy. Recent advances in the molecular characterization of NSCLC have improved patient care by identifying specific mutations prior to initiating treatment, so called "companion diagnostic" testing. Appropriate utilization of NSCLC molecular diagnostic testing maximizes the potential efficacy of the chosen treatment and has become the current standard of care. (3)

During the past year, the College of American Pathologists, together in collaboration with the International Association for the Study of Lung Cancer and the Association for Molecular Pathology, issued a white paper that provides recommendations for testing tumors for mutations in the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) genes before prescribing targeted therapies. Among the recommendations made based on expert consensus opinion was an option that both EGFR and ALK testing, in addition to testing on stage IV disease, be performed on patients who present with stage I, II, or III disease. However, the decision on when to perform molecular testing should be made in collaboration with the institutions oncology team. (4) Molecular testing of anatomic pathology specimens should be performed on formalin fixed paraffin embedded (FFPE) tissue that requires no special handling by the clinician performing the biopsy. Once the pathologist renders the histologic diagnosis, residual tissue remaining in the FFPE tissue block is used for molecular testing. (4)

Epidermal Growth Factor Receptor Gene (EGFR)

Epidermal growth factor (EGFR) is the cell surface receptor for epidermal growth factor extracellular protein ligands. (5) Activating EGFR gene mutations have been identified in different histologic types of lung cancer and are most commonly seen in adenocarcinomas (Figure 1a and b) with bronchoalveolar features or those mixed carcinomas with an adenocarcinoma component. EGFR mutations are rare in large cell, squamous and small cell carcinomas. (6) Interestingly, most patients with lung cancers with EGFR mutations are non-smokers (7) and have been noted to be more common in patients of Asian ancestry. (8)

Several distinct EGFR activating mutations have been identified in lung adenocarcinomas. Clinical molecular assays cover wider portions of the EGFR gene, typically exons 18-21. EGFR mutations of exon 19 and L858R in exon 21 are most commonly observed in NSCSC. Although most EGFR mutations are associated with a response to specific tyrosine kinase inhhibitors, some mutations including the T790 and exon 20 mutations are associated with a lack of response. (9) Examples of EGFR receptor inhibitors include erlotinib (Tarceva[R]) and afatanib (Gilotrif[R]). Erlotinib was first approved in 2004 for patients who failed first line treatment with platinum doublet chemotherapy. (10) Later in 2010, the FDA approved erlotinib for maintenance treatment of patients with locally advanced or metastatic NSCLC whose disease had not progressed after platinum based first-line chemotherapy. In May of 2013, it was approved as a first-line treatment in patients with EGFR mutations. A companion diagnostic test for exon 19 deletions and exon 21 (L858R) substitution mutations was approved concurrently (The cobas[R] EGFR Mutation Test). (11)

Afatinib received FDA approval in July 2013 for first-line treatment of patients with metastatic non-small cell lung cancer (NSCLC) whose tumors have EGFR mutations as detected by an FDA-approved test. The therascreen EGFR RGQ PCR Kit (QIAGEN) was approved at the same time for detection of EGFR exon 19 deletions or exon 21 (L858R) substitution mutations. (12)

There are two FDA-approved EGFR mutation assays currently available. In early 2013, Roche's COBAS[R] EGFR mutation test performed on the cobas[R] 4800 automated molecular platform was approved for use as a companion diagnostic test for erlotinib.

The FDA additionally approved erlotinib as a first line treatment in patients who had not received prior treatment for metastatic NSCLC whose tumors have a deletion in exon 19 or 21 as identified by the Roche COBAS[R] EGFR test. (11) The FDA approved the therascreen EGFR RGQ PCR Kit (QIAGEN) test performed on the Rotor-Gene Q real-time polymerase chain reaction (PCR) cycler (Figure 2) as a companion diagnostic test for the drug afatanib in July 2013. (12) Although these assays were developed as companion diagnostics paired with each individual drug, both FDA assays can be used to test tumors prior to using any of the individual drugs. Previous and recent studies have demonstrated both an increased response rate and improved progression free survival with EGFR tyrosine kinase inhibitors compared with chemotherapy in patients with EGFR mutations. (13)

Both of the EGFR assays that have been cleared by the FDA for clinical use are based on PCR in which copies of a DNA target are amplified several orders of magnitude. The PCR primers flank or involve the area of the EGFR gene where mutations occur. The amplification reaction is then monitored in real-time by using fluorescent dyes. The Qiagen therascreen EGFR assay (14) utilizes a specific type of real-time PCR termed "allele-specific PCR". Allele-specific PCR differs from standard real-time PCR in that the primers themselves contain the specific complementary sequence to the relevant area of the EGFR gene, mutation or control. Thus, the primer will only bind and continue the PCR reaction when a specific mutation in the EGFR gene is present in the tumor. This increases the sensitivity of the test because amplification cannot take place unless the tumor carries the target sequence.

The Roche EGFR assay utilizes primers and target specific oligonucleotide probes labelled with fluorescent dyes. These fluorescent dyes also called reporters, are blocked or "quenched" by a molecule that inhibits the fluorescence which is also bound to the oligonucleotide. If the specific mutation is present, the oligonucleotide probe binds to the specific nucleotide sequence and is in-turn cleaved by the DNA polymerase during the real-time PCR reaction. This cleavage of the probe removes the quencher and the reporter's fluorescence can be detected when excited by a specific light frequency. (15)

It is considered the current standard of care to use epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKI) as a first line treatment in patients with activating EGFR mutations. Most national guidelines endorse this approach based on multiple clinical trials that have shown improvement in outcomes including response rate and progression free survival. The use of EGFR TKIs as a second line therapy after chemotherapy has not been compared directly with using these drugs as a first line therapy. There is no definitive survival data showing that one approach is superior to the other. However, most oncologists prefer using EGFR TKIs as a first line treatment due to the favorable side effects profile and improved quality of life. (16)

Anaplastic Lymphoma Kinase Gene (ALK)

ALK mutations were originally described in anaplasic large cell lymphoma, inflammatory myofibroblastic tumors, and more recently in NSCLC. The ALK mutations seen in NSCLC occur as a result of a fusion between echinoderm microtubule-associated proteinlike 4 (EML4) and ALK (Figure 3). They are found in approximately 2-7% of patients with NSCLC. (17) More commonly, patients with ALK rearrangements are younger than typical NSCLC patients and are non- or light smokers. (18) Crizotinib (Xalkori[R]) was the first FDA-approved ALK tyrosine kinase inhibitor for treating locally advanced or metastatic NSCLC. (19) Recent patient studies of ALK mutation positive NSCLC demonstrated a statistically significant improvement in progression free survival in the patients treated with Crizotinib, and response rates for those patients with ALK rearrangements patients was greater than 50%. (20) In April 2014, the FDA granted accelerated approval to Ceritinib (ZYKADIA[R]) for treating patients with ALK-positive, metastatic non-small cell lung cancer (NSCLC) who have had disease progression while on or are intolerant to Crizotinib. (21)

There are two FDA approved assays for ALK testing. The first is a recently approved immunohistochemical stain for the qualitative detection of the (ALK) protein in formalin-fixed, paraffin-embedded (FFPE) tissue (Ventana Medical Systems, Inc). The second detects ALK rearrangements via a fluorescent in-situ hybridization (FISH) assay (Vysis LSI ALK, Abbott). FISH testing utilizes fluorescently labelled oligonucleotide probes that can detect the presence or absence of specific DNA sequences on chromosomes. The Abbott ALK FISH assay uses "break-apart" FISH probes; one green and one orange probe flank the 2p23 area of the ALK gene where ALK translocations are typically found. In tumors with a wild type (normal) ALK gene, the 2 probes both bind to the ALK gene but are in such close proximity to each other that the two fluorescent signals overlap resulting in a single yellow signal. If a chromosomal rearrangement is present at position 2p23 in the ALK gene, the probes bind far enough apart so that both the green and orange signals can be visualized. Deletions in the ALK gene can result in only a single orange or green signal based on the location of the deletion. (22)

Molecular testing for EGFR and ALK mutations has become commonplace for patients with NSCLC with adenocarcinoma features. Recently the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology published guidelines that recommend EGFR and ALK testing for all NSCLC of the lung with an adenocarcinoma component. (4) This would include both pure adenocarcinomas as well as lung tumors with only a component of adenocarcinoma. Although testing for other gene mutations in NSCLC of the lung is not particularly common, other mutations have been identified which could impact current therapies or may lead to additional therapeutic options in the near future.

Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS)

KRAS is a member of the ras gene family and encodes for a GTPase protein involved in multiple signal transduction pathways. (23) In colorectal carcinoma, KRAS mutation testing was the first genetic test employed to guide the treatment of a cancer and is currently approved by the FDA as an indication for monoclonal antibody against EGFR receptors treatment with cetuximab (Erbitux[R]) and panitumumab (Vectibix[R]). (24) In colorectal carcinoma, tumors that have a KRAS genetic mutation resulting in a constitutively activated KRAS protein will continually stimulate the signal transduction pathways downstream of the EGFR. In these cases, targeting the EGFR itself with therapeutic agents will not inhibit the downstream effects and typically results in resistance to cetuximab. (23) Testing for KRAS mutations is typically required prior to treating colorectal cancer patients with cetuximab and panitumumab. (25-26)

Lung tumors with KRAS mutations are seen in approximately 20% of adenocarcinomas, usually occurring in patients who are Caucasian and smokers. Although KRAS mutation testing is not currently standard of care (3) it is considered a poor prognostic marker. (27) Studies regarding personalized therapy options for KRAS mutated lung adenocarcinomas are ongoing and have recently demonstrated possible promising results. (28)

Other Mutations Identified in NSCLC

Proto-oncogene tyrosine-protein kinase ROS (ROS1) is a receptor tyrosine kinase of the insulin receptor family whose mutations were originally described in glioblastoma, a malignant brain tumor. (29) Recently, ROS1 mutations have been described as potential "driver mutations" in that the mutation is positively selected during carcinogenesis and provides a selective advantage to the malignant clone. ROS1 fusion genes are similar to ALK mutations in that both are often found in non- or light smokers with adenocarcinomas presenting at a younger age. Approximately 2% of lung tumors demonstrate ROS1 mutations and tumors with ROS1 fusion genes have been reported to respond to crizotinib (Xalkori[R]) and clinical trials are currently underway. (30)

The mesenchymal-epidermal transition (c-MET) receptor tyrosine kinase is a membrane receptor that is involved in embryonic development and wound healing. (31) In NSCLC, retrospective studies have shown that c-MET gene copy number is a negative prognostic factor. Additionally, c-MET amplification is an important event in acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) in EGFR mutated lung adenocarcinomas. c-MET amplification occurs in up to 20% of NSCLC with acquired resistance to EGFR-TKIs. Ongoing clinical trials will help to determine future roles for c-MET inhibitors as a treatment option. (32-33)

In addition, future and ongoing clinical trials will not be limited to the targetable mutations described above and may involve mutations of other genes including ret proto-oncogene (RET) and HER2/neu. (34) (Table 1) This list of mutations is not all inclusive and other mutations that can impact the therapy of NSCLC will be discovered.

Conclusions

There have been a number of major advances in the molecular characterization of NSCLC over the past decade. This brief case report and review, although not exhaustive, highlights the most common molecular pathways associated with actionable results impacting NSCLC therapeutic options. With the advent of tyrosine kinase inhibitors with efficacy against only specific mutations, detecting these mutations has become increasingly important in the clinical laboratory setting. Identification of EGFR, ALK, and KRAS mutations in NSCLC has already demonstrated clinical utility and has improved patient care. Molecular diagnostics and personalized medicine are rapidly entering clinical arenas, particularly in cancer treatment facilities. As our understanding of the molecular genotyping of NSCLC advances, molecular testing will become a routine part of comprehensive cancer care. The main purpose of this testing in NSCLC and other malignancies is to assure that treatments are targeted to specific mutations, with the ultimate goals of improving therapeutic responses and clinical outcomes.

Matthew B. Smolkin, MD

Dept. of Pathology, WVU Hospital, Morgantown

Mohammed Almubarak, MD

Mary Babb Randolph Cancer Center

WVU School of Medicine, Morgantown

Peter L. Perrotta MD

Dept. of Pathology, WVU Hospital, Morgantown

Corresponding Author: Matthew B. Smolkin, MD, West Virginia University Health Sciences Center, Department of Pathology, Morgantown, WV, 26506. Email: mbsmolkin@hsc.wvu.edu

References

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(7.) Pao W, Miller V, Zakowski M, et al: EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci USA 101:13306-13311,2004.

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(12.) http://www.fda.gov/Drugs/InformationOnDrugs/ ApprovedDrugs/ucm360574.htm

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Table 1. Testing methodologies used to detect gene mutations
in NSCLC lung carcinomas

Gene      Genetic Alteration      FDA Approved     Typical
                                    Companion      Testing
                                   Diagnostic      Method
                                 Test Available?

EGFR    Deletion, insertion,     Yes               PCR
        point mutation

ALK     Translocation            Yes               FISH

Kras    Point mutation           No                PCR

ROS1    Translocation            No                FISH

c-Met   Amplification            No                FISH

Ret     Translocation            No                FISH

Her2    Insertion, duplication   No                PCR

PCR = polymerase chain reaction; FISH = fluorescence in
situ hybridization
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Author:Smolkin, Matthew B.; Almubarak, Mohammed; Perrotta, Peter L.
Publication:West Virginia Medical Journal
Article Type:Report
Date:Sep 1, 2015
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