Biomarker Testing

Lung cancer treatment options for lung cancer patients with advanced-stage (metastatic) non-small cell lung cancer (NSCLC) now include a number of targeted therapiesA type of treatment that uses drugs to attack specific types of cancer cells with less harm to normal cells aimed at particular driver mutationsA change to the DNA of cancerous cells that is considered to have been a cause of the development of the cancer and has helped the cancer cells to grow and several immunotherapiesA type of cancer therapy that uses substances to stimulate or suppress the immune system to help the body fight cancer, infection, and other diseases aimed at a patient's own immune system. Each of these treatments can provide substantial benefits—but not to all patients. For doctors to know whether to prescribe any of these treatments for a lung cancer patient requires a type of testing known as biomarker testing.

Biomarker Testing brochureTo help you understand and share this information, you can request our free booklet that summarizes the detailed information presented here. (Published in January 2018)

Biomarker testing is used among diagnosed advanced-stage lung cancer patients to determine the presence of particular mutations or of a particular protein. It is the first step in precision medicine—ensuring that a patient gets matched to the right treatment at the right time, based on the patient's biomarkerA biological molecule found in blood, other body fluids, or tissues that is a sign of a normal or abnormal process, or of a condition or disease. A biomarker can be a change in DNA (mutation), RNA, or protein status.

This section will help patients:

  • Understand what a biomarker is
  • Learn how biomarkers are used to make lung cancer treatment decisions
  • Understand how biomarker testing is done

For a handy biomarker testing reference, download the flyer below and share it with your family and healthcare providers; the download has useful "quick facts" as well.

Why you should ask your doctor about biomarker testing

What is a biomarker?

A biomarker is any molecule that can be measured in tissues, blood, or other bodily fluids. Presence of a biomarker may be a sign of an abnormal bodily process or condition or a disease. A biomarker may also be called a molecular marker, genotype, or signature molecule.1

Biomarkers can be used to:

  • Determine whether a disease or condition is present
  • Determine how aggressive the disease is
  • Predict how well the body will respond to a treatment for a disease or condition

What is biomarker testing? Why is it important to lung cancer patients?

Biomarker testing (also known as mutation, genomic, or molecular testing) is a way for the healthcare team to gather as much information as possible about a patient's unique lung cancer. It uncovers whether the patient has a treatable driver mutation and establishes the patient's PD-L1Part of the immune system mechanism that keeps T cells from functioning proteinA molecule made up of amino acids that is needed for the body to function properly. Proteins are the basis of body structures, such as skin and hair, and of other substances such as enzymes, cytokines, and antibodies expression level. The results of these tests help determine whether any of the Food and Drug Administration (FDA)-approved lung cancer targeted therapies or a particular biomarker-driven immunotherapy drug is right as part of the patient's treatment plan. Biomarker testing is used to plan these treatments for advanced-stage non-small cell lung cancers. (Note that biomarker testing may also be useful in some situations for early-stage lung cancers.)2

What types of biomarkers are used to determine the best treatment for lung cancer patients?

Two types of biomarkers are currently used to help optimize a lung cancer patient's treatment plan: driver mutations within the cancer's DNAThe molecules inside cells that carry genetic information and pass it from one generation to the next. Also called deoxyribonucleic acid to determine whether a targeted therapy is appropriate and the level of expression of a particular protein in the patient's tumor to determine whether an immunotherapy drug is appropriate.

Driver mutations

All the organs and tissues in our bodies are made up of cells, and each of these cells contains thousands of genesCoded instructions within a cell that control how the cell grows in a systematic and precise way. Genes are made up of DNA, which is a specific code that is use to ultimately make proteins that have specific functions for the cell. It is essential for each gene to have the correct DNA code, or instructions, for making its protein. When the DNA is correct, the protein is able to perform the correct function.3

When a gene has an error in its DNA, it is said to be mutated, or changed. Mutations can be:3

  • Acquired: Also called somatic. Present only in the tumor and not passed on to children
  • Inherited: Present in all cells of the body and passed on to children

Virtually all of the biomarkers that are helpful to making treatment decisions in lung cancer are acquired. Inherited biomarkers are still being researched. In this section, we are only talking about acquired mutations.

Mutations occur often, and normally the body can correct them. However, depending on where in a gene the change occurred, the small change may go undetected by the body and become part of the cell's blueprint. Over time, an accumulation of mutations can result in the formation of a tumor. Mutations that cause cancer are called driver mutations.3

Several types of driver mutations cause cancer. Some of these include:

  • Activating mutation: An activating mutation is a change in the DNA sequence that can cause changes in the protein made by the gene so that the protein is always active, leading to uncontrolled cell growth.4

Examples of activating mutations in lung adenocarcinomaA type of non-small cell lung cancer that usually develops in the cells lining the lungs. It is the most common type of lung cancer seen in non-smokers include an L858R substitution mutation or exon 19 deletion in the epidermal growth factor receptor (EGFR) gene and the V600E mutation in the BRAF gene.4,5

  • Fusion: Fusion, or rearrangement, occurs when a part of one gene fuses with, or attaches to, a part of another gene. The fused gene then produces a unique protein that promotes abnormal, uncontrolled cell growth.6

Examples of fusion genes in lung adenocarcinoma include ALK-EML4 and CD74-ROS1.7,8

  • Amplification: Amplification means that there are many more copies of a gene than normal. The overexpression then leads to increased protein activity and uncontrolled cell growth.6

Examples of genes that can be amplified in lung adenocarcinoma include HER2 and MET.9

  • Deletion: Deletion means part of or the entire gene is missing in the cancer cells. The deletion then leads to reduced levels of protein being produced by the cancer cell and uncontrolled cell growth.6

Examples of deletion genes in small cell lung cancer (SCLC)A fast-growing cancer that forms in tissues of the lung and can spread to other parts of the body. Named "small" for how the cancer cells look under a microscope include TP53 and RB.10

A person's lung cancer may or may not have one of the many known driver mutations. So far, scientists have identified more than 20 different driver mutations sometimes found in NSCLC and SCLC, and they are continuing to look for more.9

These driver mutations are biomarkers that are used in biomarker testing in lung cancer; their presence may determine whether a patient will be prescribed one of several approved targeted therapies or be potentially eligible for a clinical trial for a targeted therapy still in development.

Right now, scientists have the most information about driver mutations in the subtype of NSCLC called lung adenocarcioma. The driver mutations in lung adenocarcinoma that currently have FDA-approved targeted therapy drugs available are EGFR, ALK, ROS1, BRAF V600E, and NTRK1.


Image source: Hirsch F, et al. New and emerging targeted treatments in advanced non-small-cell lung cancer. Lancet. Vol 388. September 3, 2016

Scientists are also making progress in understanding mutations in squamous cell lung cancer, although there are no FDA-approved targeted therapies yet for these.


Image source: Hirsch F, et al. New and emerging targeted treatments in advanced non-small-cell lung cancer. Lancet. Vol 388. September 3, 2016.

Driver mutations in SCLC and other types of lung cancer are also being studied. There are as yet no targeted therapies that are FDA-approved for them.

Immunotherapy Biomarkers

There are several immunotherapy biomarkers; only one, PD-L1, is currently used in the clinic for lung cancer.

  • PD-L1: PD-L1 is a protein biomarker used to determine whether a lung cancer patient is likely to benefit from a treatment with a type of immunotherapy drug called an immune checkpoint inhibitor. Immune checkpoint inhibitors are drugs that help the patient's own immune system fight the cancer. They do this by overriding the immune system's fail-safe mechanisms, which are designed to suppress the immune response at appropriate times to minimize damage to healthy tissue. Patients who have a high level of PD-L1 are more likely to respond to immune checkpoint inhibitors. However, even those with tumors that do not express PD-L1 may respond.11
  • Other immunotherapy biomarkers: While they are not yet used in the clinic for lung cancer, scientists are also studying types of immunotherapy biomarkers other than PD-L1, such as tumor mutational burden (TMB)The total number of mutations (changes) found in the DNA of cancer cells. Knowing the tumor mutational burden may help plan the best treatment. For example, tumors that have a high number of mutations appear to be more likely to respond to certain types of immunotherapy. Tumor mutational burden is being used as a type of biomarker. Also called TMB., CTLA-4 expression, and microsatellite instabilityA change that occurs in the DNA of certain cells (such as tumor cells) in which the number of repeats of microsatellites (short, repeated sequences of DNA) is different than the number of repeats that was in the DNA when it was inherited. The cause of microsatellite instability may be a defect in the ability to repair mistakes made when DNA is copied in the cell. Also called MSI. 12,13

When is biomarker testing appropriate?

Biomarker testing may be appropriate:

  • When the doctors suspect lung cancer and have recommended a biopsy
  • When a patient is already diagnosed with lung cancer
  • When a patient's lung cancer recurs (comes back) after treatment

All patients with a lung cancer diagnosis should discuss biomarker testing with their healthcare team.

For which biomarkers should a patient be tested?

The table below displays guidelines for biomarker testing from the National Comprehensive Cancer Network and the College of American Pathologists (CAP), International Association for the Study of Lung Cancer (IASLC), and Association for Molecular Pathology (AMP) molecular testing guideline partnership):14,15

Type of Lung Cancer

Stage of Lung Cancer

Recommendations for Biomarker Testing

Lung adenocarcinoma

Stage I, II, or III

Testing for the EGFR, ALK, KRAS, ROS1, BRAF V600E, and NTRK1 mutations and PD-L1 protein levels at the time of diagnosis and surgical resection is not always recommended but may be considered. The decision should be made on an individual basis with your doctors.

Stage IV lung adenocarcinoma or lung adenocarcinoma that has recurred or progressed after an initial diagnosis of stage I, II, or III lung cancer in patients who were not previously tested

Tumors should be tested for EGFR, ALK, KRAS, ROS1, BRAF V600E and NTRK1 mutations at the time of diagnosis. Testing for these other biomarkers may be helpful in deciding eligibility for clinical trials: the MET,  RET, and ERBB2 (HER2) mutations and tumor mutational burden (TMB).

Note: Currently, no drug targeting the KRAS mutation has been approved, although there are treatments in development. However, KRAS testing can be informative because cancers with KRAS mutations are very unlikely to respond to targeted therapies for the EGFR mutation.

PD-L1 immunohistochemistryA laboratory method that uses antibodies to check for certain antigens (markers) in a sample of tissue. The antibodies are usually linked to an enzyme or a fluorescent dye. After the antibodies bind to the antigen in the tissue sample, the enzyme or dye is activated, and the antigen can then be seen under a microscope. Immunohistochemistry is used to help diagnose diseases, such as cancer. It may also be used to help tell the difference between different types of cancer. is recommended to determine whether you will benefit from an immunotherapy drug in the first-line (initial treatment) setting.

Squamous cell lung cancer

Stages I, II, and III

  • Currently, biomarker testing is performed only for clinical trials.

Stage IV

  • Currently, biomarker testing is performed only for clinical trials.
  • PD-L1 immunohistochemistry is recommended to determine whether a patient will benefit from an immunotherapy drug in the first-line setting.
  • Testing for EGFR and ALK mutations is recommended ONLY if doctors suspect that the tumor may have lung adenocarcinoma cells (this type of lung cancer is referred to as mixed lung cancer with an adenocarcinoma component).

Small cell lung cancer

All stages

  • Currently, biomarker testing is performed only for clinical trials.

How is tumor tissue collected and processed for diagnosis and biomarker testing?

Biomarker testing requires a sample of the tumor tissue. Doctors most often obtain tumor tissue via a biopsy.

There are many different biopsy techniques that can be used to obtain the tumor tissue. The technique is determined by the location and size of the tumor, as well as the patient's overall health. The patient's healthcare team will discuss the best options with the patient as well as the risks and benefits of the procedures.

Among the tissue collection techniques are bronchoscopyA procedure that uses a bronchoscope to examine the inside of the rachea, bronchi, and lungs. A bronchoscope is a thin, tube-like instrument wit a light and a lens for viewing. It may also have a tool to remove tissue; this tissue can then be checked under a microscope for signs of disease. The bronchoscope is inserted through the nose or mouth, endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA)A procedure in which bronchoscope fitted with an ultrasound device is guided down the trachea; once in place a needle is inserted through the bronchus and into a lymph node to obtain a sample. EBUS-TBNA requires local anesthesia, transthoracic needle biopsyA procedure in which an interventional radiologist inserts a needle into the chest wall to remove fluid or tissue, thoracoscopyA procedure in which a surgeon makes a small incision in the skin of the chest wall and inserts a special instrument with a small video camera on the end to examine the inside of the chest. Samples of tissue are removed for a pathologist to look at under the microscope. Also called VATS, thoracentesisRemoval of fluid from around the lungs through a hollow needle inserted between the ribs, mediastinoscopyA procedure performed under general anesthesia to get a look at the area between the lungs, or mediastinum. A small incision is made above the breastbone, and a thin tube with a lens for viewing and a tool to remove tissue is inserted. The samples are sent to the lab to check for cancer cells, and mediastinotomyA procedure to get tissue from the area between the lungs. An incision, a little larger than that in a mediastinoscopy, is made near the breast bone and between the left second and third ribs in order to reach lymph nodes unreachable by mediastinoscopy.. Regardless of how the tissue is collected, a patient should confirm with the doctors, before the tissue is removed, that adequate tissue will be collected so that all necessary biomarker tests can be performed. Tissue from the tumor is saved for a long time, so that additional testing, if necessary, can be done. For more details about each biopsy technique, read Biopsies.

After the tumor tissue is collected, it is sent to a laboratory for testing. Ideally, comprehensive biomarker testing will be done. In comprehensive biomarker testing, driver mutations in multiple genes are tested for at the same time, rather than sequentially, including not only the ones with approved treatments, but also other known driver mutations. Some of the driver mutations currently without approved treatments may have treatments being tested now or in the near future in clinical trials to which a patient could be matched. An advantage of comprehensive biomarker testing is that when a new mutation target is discovered, it can easily be added to the set of mutations being tested for. Comprehensive biomarker testing is done via a process known as next-generation sequencing, or NGS,

Are multiple biopsies necessary?

Sometimes, an additional biopsy is recommended. This can happen when:15

  • Not enough tissue was obtained during the initial, diagnostic biopsy
  • A targeted therapy that worked well against the lung cancer has stopped working and the cancer has recurred. Testing the resistant cancer for additional mutations that may have evolved or rare changes in histology is indicated to help adjust the patient's treatment plan
  • New drugs are approved for the treatment of lung cancer from which a patient might possibly benefit. The new drug or treatment might require biomarker testing

Therefore, a patient's doctor may recommend additional biopsies and biomarker testing at several points in the treatment process. The ultimate decision to recommend another biopsy depends on the location of the cancer and the patient's health status and lung function.

What is a liquid biopsy? How is it used?

Currently, tissue biopsies are the only way to confirm a diagnosis of lung cancer, and they are the primary way to detect driver mutations or levels ot the PD-L1 protein. However, in certain situations, a patient's doctor may use a liquid biopsy instead of a tissue biopsy, once a diagnosis has already been made via tumor tissue, to decide if certain targeted therapies are right for the patient.

Cells release DNA into the bloodstream; this is called cell-free DNA. Cell-free DNA that is shed by cancer tumor cells is called circulating tumor DNA, or ctDNA. In a liquid biopsy, ctDNA is captured and tested for specific mutations.16

At this time, liquid biopsies may help a patient's healthcare team:16,17

  • Check if the patient's cancer has become resistant to a targeted therapy and decide the next treatment option
  • Follow the patient's response to a particular targeted therapy

If a liquid biopsy test is negative, a tissue biopsy may be recommended. It is important to know that not all cancer cells shed DNA so not all patients can be successfully tested via liquid biopsy.

What information is included in a biomarker testing report?

At the laboratory, the tissue sample collected during the biopsy procedure is tested and analyzed by a pathologistA doctor who identifies diseases by studying cells and tissues under a microscope or with other equipment. Laboratory results from the biopsy are recorded in a report.

The initial report issued by the pathologist, which includes the diagnosis of lung cancer, its histological type, and its stage, is called the pathology report. (In some cases, limited information about the presence or not of several common biomarkers, including most likely EGFR and ALK, may also be included.)

Most often, however, a separate report from a pathologist with information about a more extensive set of biomarker testing results will follow the initial, pathology report. This report is typically called a biomarker testing report (but may also be referred to as a molecular testing report or a genomic testing results report). The biomarker testing report is based on the biomarker testing that occurs after the patient's initial diagnosis of lung cancer. The report contains critical information about a patient's cancer. Specifically, the biomarker testing report indicates whether a patient has biomarkers—either driver mutations or a high level of PD-L1 protein expression—that indicate that a patient may respond to a particular targeted therapy or to an immunotherapy drug, either approved or in clinical development.

The biomarker testing report is usually ready in 10-14 days. Patients should request a copy of the report for their medical records. Sometimes, the report is also available in a patient’s electronic health records, which are accessible through a hospital’s patient portal. The biomarker testing report may be helpful for discussions with the patient’s healthcare team, to pursue a second opinion about the diagnosis and/or the recommended treatment plan, or to pursue a clinical trial. (Note: The tissue samples will also be needed for a second opinion; as mentioned earlier, these are kept for a long time so will be available.)

Based on conversations with several pathologists, these are the top three things to look for in a biomarker testing report:18,19,20

  • Biomarker (mutation or PD-L1 status): This information is on the first page. After the name of each mutation tested for, the report indicates whether or not the mutation is present. In some cases, the report may say “no actionable mutation identified” to indicate that testing did not reveal that the patient has a mutation with an FDA-approved targeted therapy. Likewise, the patient's PD-L1 protein expression level can be found here.
  • Appropriate drug for biomarker: Typically, next to the location of the biomarker status, the report notes the drug or drugs (if multiple options are available) that may be effective for a patient with this particular mutation
  • The pathologist’s contact information: Many patients may not meet their pathologist in person, but that pathologist is available to speak to a patient about the testing results. The pathologist's name and contact information can be found on the bottom of the biomarker testing report, and any questions can be directed to them. Although the pathologist will not respond to questions about a patient's treatment or care plan, the pathologist can address questions about a patient's biomarker status as discussed in the report.

How do the biomarker test results impact treatment?

If an advanced-stage lung cancer patient's tests are positive for the EGFR, ALK, ROS1, BRAF V600E, or NTRK1 mutations, there are FDA-approved targeted therapies for each that may be prescribed. For details about each treatment: what it is, who is eligible for it, how it is administered, etc., read Targeted Therapy.

In addition, a certain level of the PD-L1 biomarker will help the healthcare team determine whether a patient is likely to benefit from an immunotherapy drug. While there are two FDA-approved immunotherapy drugs, one of them is approved only if the PD-L1 protein expression level is high enough. For details about each immunotherapy treatment: what it is, who is eligible for it, how it is administered, etc., read Immunotherapy.

Questions to ask your healthcare team about biomarker testing

Before getting biomarker testing:

  • What are you trying to find with biomarker tests?
  • Have I already had any biomarker tests? Which ones?
  • Who performs these tests?
  • How are the tests performed?
  • Are there any complications from these tests?
  • How long will it take to get the test results?
  • Where can I get more information about biomarker testing?
  • Are there any limitations of biomarker testing?
  • Will insurance pay for these tests?

After getting biomarker testing:

  • What tests were done?
  • What are the results of these tests?
  • How will the results affect my treatment?
  • The test results are negative: should I be retested?
  • The test results are not clear: should I be retested?
  • Are there any medications that target my type of lung cancer?
  • Will I need these tests again? If so, why? When?
  • Are there any clinical trials open to me based on these results?
  • How can I get a copy of my biomarker testing report?

References

  1. NCI Dictionary of Cancer Terms. https://www.cancer.gov/publications/dictionaries/cancer-terms. Accessed August 12, 2019.
  2. Lung Cancer: Metastatic—Non-Small Cell Lung Cancer. NCCN Guidelines for Patients. Version 3.2019. https://www.nccn.org/patient/guidelines/lung-metastatic/. Posted January 18, 2019. Accessed August 12, 2019.
  3. The Genetics of Cancer. Cancer.Net website. https://www.cancer.net/navigating-cancer-care/cancer-basics/genetics/genetics-cancer. Approved March 2018. Accessed August 12, 2019.
  4. Lopes GL, de Queiroz Vattimo EF, de Castro G, Jr. Identifying activating mutations in the EGFR gene: prognostic and therapeutic implications in non-small cell lung cancer. J Bra Pneumol. 2015 Jul-Aug; 41(4): 365-375. doi: 10.1590/S1806-37132015000004531. https://www.ncbi.nlm.nih.gov/pms/articles/PMC4635957/. Accessed August 12, 2019.
  5. Li S, Li L, et al. Coexistence of EGFR with KRAS, or BRAF, or PIK3CA somatic mutations in lung cancer. Br J Cancer. 2014 May 27; 110(11): 2812-2820. doi: 10.1038/bjc.2014.210. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4037826/. Accessed August 12, 2019.
  6. Pao W, Ladany M. Detecting Gene Alterations in Cancers. My Cancer Genome website. https://www.mycancergenome.org/content/page/detecting-gene-alterations-in-cancers. Updated May 27, 2019. Accessed August 12, 2019.
  7. Shaw AT, Solomon B. Anaplastic lymphoma kinase (ALK) fusion oncogene positive non-small cell lung cancer. Uptodate website. https://uptodate.com/contents/anaplastic-lymphoma-kinase-alk-fusion-oncogene-positive-non-small-cell-lung-cancer. Updated June 21, 2019. Accessed August 12, 2019.
  8. Davies KD, Doebele RC. Molecular Pathways - ROS1 Fusion Proteins in Cancer. Clin Cancer Res. 2013 Aug 1; 19(15): 4040-4045. doi: 10.1158/1078-042.CCR-12-2851. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732549. Accessed August 12, 2019.
  9. Hirsch FR, Suda Km, Wiens J, Bunn PA, Jr. New and emerging targeted treatments in advanced non-small-cell lung cancer. Lancet. Sep 3, 2016; 388(10048): 1012-1024. Accessed August 12, 2019.
  10. Byers LA, Rudin CM. Small Cell Lung Cancer: Where Do We Go From Here? Cancer. 2015 Mar 1; 121(5): 664-672. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497465/. Accessed August 12, 2019.
  11. Topalian SL, Taube JM, Anders RA, Patoll DM. Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy. Nat Rev Cancer. 2016 May; 16(5): 275-287. doi: 10.1038/nrc.2016.36. https://www.nbci.nlm.nih.gov/pmc/articles/PMC5381938. Accessed August 12, 2019.
  12. Masucci GV, Cesano A, et al. Validation of biomarkers to predict response to immunotherapy in cancer: Volume 1—pre-analyical and analytical validation. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5109744/. Accessed August 12, 2019.
  13. Clinicaltrials.gov. U.S. National Library of Medicine website. https://clinicaltrials.gov. Accessed August 12, 2019.
  14. Non-Small Cell Lung Cancer: NCCN Evidence BlocksTM. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®). Version 5.2019. Posted June 26, 2019. Accessed August 12, 2019.
  15. Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment with Targeted Tyrosine Kinase Inhibitors. Association for Molecular Pathology website. https://www.amp.org/clinical-practice/practice-guidelines/updated-molecular-testing-guideline-for-the-selection-of-lung-cancer-patients-for-treatment-.... Posted January 2018. Accessed August 12, 2019.
  16. Lovly CM, Berger MF, Vnencak-Jones CL. Circulating DNA. My Cancer Genome website. https://www.mycancergenome.org/content/page/circulating-dna/. Updated April 3, 2019. Accessed August 13, 2019.
  17. Siravegna G, Marsoni S, Siena S, Bardelli A. Integrating liquid biopsies into the management of cancer. Nat Rev Clin Oncol. 2017 Sep; 14(9): 531-548. doi: 10.1038/nrclinonc. https://www.ncbi.nlm.nih.gov/pubmed/28252003. Accessed August 13, 2019.
  18. Interview with Lija Joseph, MD, Lowell General Hospital, Lowell, Massachusetts. July 17, 2019.
  19. Interview with Timothy Craig Allen, MD, JD, FCAP. University of Mississippi Medical Center, Jackson, Mississippi. July 16, 2019.
  20. Interview with Tabetha Sundin, PhD, HCLD (ABB), MB (ASCP). Sentara Healhcare, Norfolk, Virginia. June 27, 2019.

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