- Role in Tumor Cell Survival
- Importance of Testing
- How to Test: Sample Collection
- EGFR Diagnostic Assays
How to Test: Sample Collection
Utilizing tissue and plasma samples can make biomarker testing available for more patients1


Tissue-based testing is the gold standard
for identifying sensitizing EGFR mutations, but plasma may be used if tissue testing is not feasible. Plasma testing may be preferable for patients1,2:
for identifying sensitizing EGFR mutations, but plasma may be used if tissue testing is not feasible. Plasma testing may be preferable for patients1,2:


Who are ineligible for a tissue biopsy due to performance status or tumor location


Who are unwilling to undergo a tissue biopsy


Whose tissue samples are inadequate for molecular testing
Tissue and plasma-based testing methods have different advantages and considerations
Tissue-based testing
Advantages
- Established testing method1
- High sensitivity rates3
- No cell degradation4
Considerations
- Sample heterogeneity may impact patient identification1
- Patient may not be eligible due to performance status or tumor location2
- Complications may develop during the collection process1
Plasma-based testing
Advantages
- Less invasive and fewer limitations1
- Potential for faster turnaround time (~3 days)5
- May save on procedure costs1
Considerations
- Results can be inconclusive due to differences in tumor biology1
- Tumor burden and tumor shedding can influence results1
- DNA may be insufficient for positive identification1
Because plasma testing is less sensitive than tissue testing, negative plasma test results should be retested with tissue.6
Tissue Samples
Collect and process tissue samples


CHOOSE an appropriate collection method
- Assays may recommend using specific specimen types. Collaborate with your multidisciplinary team to ensure appropriate biopsy methods are performed7
- Assays may recommend using specific specimen types. Collaborate with your multidisciplinary team to ensure appropriate biopsy methods are performed7
Biopsy methods differ in diagnostic yield and adequacy for biomarker testing
-
BRONCHOSCOPY
± EBUS7 -
SURGICAL
(eg, mediastinoscopy, thoracoscopy, resection)7,8 -
IMAGE-GUIDED
(eg, TTNA, thoracentesis, bone biopsy)7-9
BRONCHOSCOPY
± EBUS7
Specimen types
Tissue biopsy
- Endobronchial biopsy
- Transbronchial biopsy
Cytology
- Brushing cytology
- Washing cytology
- FNA cytology
Diagnostic yield
58%–94%
depending on if lesion is visible and if biopsy/brushing/washing are combined
Adequacy for biomarker testing
Up to 100%
for endobronchial biopsy
<50%
(in washings)
SURGICAL
(eg, mediastinoscopy, thoracoscopy, resection)7,8
Specimen types
Biopsy
- Tissue biopsy
Diagnostic yield
80%–97%
Adequacy for biomarker testing
- Not well established, but likely adequate (mediastinoscopy)
- 100% in 1 series for medical thoracoscopy
IMAGE-GUIDED
(eg, TTNA, thoracentesis, bone biopsy)7-9
Specimen types
Tissue biopsy
- CNB
Cytology
- FNA cytology
- Fluid cytology
Bone
- Bone biopsy
Diagnostic yield
57%–95%
Adequacy for biomarker testing
Cytology
- Insufficiency rate of 3.7% in 1 series for thoracentesis
- 100% in 1 series in TTNA
Bone
- For bone biopsies, varies by decalcifying agent


COLLECT sufficient tissue10
- Some specimen types are preferable for histology and molecular analysis. For example, a CNB is more likely than FNA to yield an adequate amount of sample7
- Drawing 2 core needle samples may ensure sufficient tissue is taken11
- Some specimen types are preferable for histology and molecular analysis. For example, a CNB is more likely than FNA to yield an adequate amount of sample7
- Drawing 2 core needle samples may ensure sufficient tissue is taken11


Some biopsy samples may not be representative of the overall composition of the tumor, which can compromise clinical decisions.12,13
Rapid on-site evaluation (ROSE) of tissue quantity and quality by pathology/cytology can ensure sufficient sample is collected.14
For a diagnosis of mNSCLC without ROSE, current guidelines suggest using a minimum of 3 transbronchial needle aspiration samples.14


PRESERVE tissue immediately
- Tissue samples are fragile and degradation starts upon removal from the body8,15
- Fix samples immediately to preserve tumor characteristics for diagnostic evaluation8,15
- Tissue samples are fragile and degradation starts upon removal from the body8,15
- Fix samples immediately to preserve tumor characteristics for diagnostic evaluation8,15


CAP/IASLC/AMP Guidelines recommend:
Fix for 6 to 48 hours in 10% NBF.16


PROCESS sample
Sample should be17:
- Embedded in paraffin block
- Cut into 5-µm sections
Sample should be17:
- Embedded in paraffin block
- Cut into 5-µm sections


CAP/IASLC/AMP guidelines recommend:
Fix for 6 to 48 hours in 10% NBF.16


CONFIRM tumor cell content is sufficient
- Tumor cellularity (ie, the relative proportion of tumor and nontumor cells) affects the sensitivity of biomarker testing and may be more important than tumor quantity18
- Most tests require samples with >10% tumor cell content. If the sample is not sufficient, consider tumor enrichment or request a new sample17
- Tumor cellularity (ie, the relative proportion of tumor and nontumor cells) affects the sensitivity of biomarker testing and may be more important than tumor quantity18
- Most tests require samples with >10% tumor cell content. If the sample is not sufficient, consider tumor enrichment or request a new sample17
Turnaround time can impact treatment decisions19


CAP/IASLC/AMP guidelines recommend a turnaround time of 10 working days from sample receipt at testing laboratory.20
Collaboration within the multidisciplinary team can ensure that critical factors, such as specimen type and turnaround time, are communicated for appropriate and timely treatment of patients.16
Plasma Samples
Plasma testing may be helpful when tissue testing is not feasible2


ctDNA may be shed by tumors into the bloodstream. When blood is collected from a patient, ctDNA can be tested for EGFR mutations.1,2
Collect and process plasma samples


COLLECT a blood sample
- ASCO/CAP guidelines recommend collection in cell-stabilizing tubes or EDTA anticoagulant collection tubes21
- ASCO/CAP guidelines recommend collection in cell-stabilizing tubes or EDTA anticoagulant collection tubes21


PROCESS sample as soon as possible
- Guidelines recommend separating blood from plasma as soon as possible, within 6 hours of collection21
- Blood samples are typically processed by filtration or centrifugation21
- Guidelines recommend separating blood from plasma as soon as possible, within 6 hours of collection21
- Blood samples are typically processed by filtration or centrifugation21


STORE the sample
- After processing, isolated plasma can be frozen for storage21
- Avoid multiple freeze-thaw cycles21
- After processing, isolated plasma can be frozen for storage21
- Avoid multiple freeze-thaw cycles21


PROCESS sample as soon as possible
- Guidelines recommend separating blood from plasma as soon as possible, within 6 hours of collection21
- Blood samples are typically processed by filtration or centrifugation21
Turnaround time can impact treatment decisions19


CAP/IASLC/AMP guidelines recommend a turnaround time of 10 working days from sample receipt at testing laboratory.20
Collaboration within the multidisciplinary team can ensure that critical factors, such as specimen type and turnaround time, are communicated for appropriate and timely treatment of patients.16
AMP, Association for Molecular Pathology; ASCO, American Society of Clinical Oncology; CAP, College of American Pathologists; CNB, core needle biopsy; ctDNA, circulating tumor DNA; EBUS, endobronchial ultrasound; EGFR, epidermal growth factor receptor; FNA, fine needle aspiration; IASLC, International Association for the Study of Lung Cancer; mNSCLC, metastatic non–small cell lung cancer; NBF, neutral buffered formalin; NCCN, National Comprehensive Cancer Network; TTNA, transthoracic needle aspiration.
NCCN makes no warranties of any kind whatsoever regarding their content, use or application and disclaims any responsibility for their application or use in any way.
References: 1. Diaz LA et al. J Clin Oncol. 2014;32(6):579-586. doi:10.1200/JCO.2012.45.2011. 2. Bordi P et al. Transl Lung Cancer Res. 2015;4(5):584-597. 3. Ellison G et al. J Clin Pathol. 2013;66(2):79-89. 4. Sholl LM et al. Arch Pathol Lab Med. 2016;140(8):825-829. 5. Sacher AG et al. JAMA Oncol. 2016;2(8):1014-1022. doi:10.1001/jamaoncol.2016.0173. 6. Referenced with permission from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for Non-Small Cell Lung Cancer V6.2018. © National Comprehensive Cancer Network, Inc. 2018. All rights reserved. Accessed September 27, 2018. To view the most recent and complete version of the guidelines, go online to NCCN.org. 7. Ofiara LM et al. Front Oncol. 2014;4:253. doi:10.3389/fonc.2014.00253. 8. Chen H et al. Cancers (Basel). 2015;7(3):1699-1715. 9. Wu JS et al. Radiology. 2008;248(3):962-970. 10. Ofiara LM et al. Curr Oncol. 2012;19(suppl 1):S16-S23. 11. Kim ES et al. Cancer Discov. 2011;1(1):44-53. 12. Piotrowska Z et al. Cancer Discov. 2015;5(7):713-722. 13. Jamal-Hanjani M et al. N Engl J Med. 2017;376(22):2109-2121. 14. Levy BP et al. Oncologist. 2015;20(10):1175-1181. 15. Hammond MEH et al. Arch Pathol Lab Med. 2010;134(7):e48-e72. 16. Lindeman NI et al. Arch Pathol Lab Med. 2013;137(6):828-860. 17. cobas® EGFR Mutation Test v2 [package insert]. Branchburg, NJ: Roche Molecular Systems, Inc.; 2015. 18. Aisner DL et al. Am J Clin Pathol. 2012;138(3):332-346. 19. Lim C et al. Ann Oncol. 2015;26(7):1415-1421. 20. Lindeman NI et al. Arch Pathol Lab Med. 2018:142(3):321-346. doi:10.5858/arpa.2017-0388-CP. 21. Merker JD et al. Arch Pathol Lab Med. 2018. doi:10.1200/JCO.2017.76.8671.
During ROSE, a pathologist or technologist performs an onsite assessment of biopsy tissue quality and quantity to ensure it is adequate for histological and biomarker testing.14
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