How to Test: Sample Collection

Utilizing tissue and plasma samples can make biomarker testing available for more patients1

Using tissue and plasma to detect EGFR mutations
Using tissue and plasma to detect EGFR mutations

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:

Using tissue and plasma to detect EGFR mutations
Using tissue and plasma to detect EGFR mutations

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

Using tissue and plasma to detect EGFR mutations
Using tissue and plasma to detect EGFR mutations

Who are unwilling to undergo a tissue biopsy

Using tissue and plasma to detect EGFR mutations
Using tissue and plasma to detect EGFR mutations

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

Step 1: Choose collection method
Step 1: Choose collection method

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

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
Step 2: Collect tissue
Step 2: Collect tissue

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
Tumor heterogeneity in tissue biopsy
Tumor heterogeneity in tissue biopsy

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

Step 3: Preserve tissue
Step 3: Preserve tissue

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
Stopwatch icon
Stopwatch icon

CAP/IASLC/AMP Guidelines recommend:

Fix for 6 to 48 hours in 10% NBF.16

Step 4: Process tissue sample
Step 4: Process tissue sample

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
Stopwatch icon
Stopwatch icon

CAP/IASLC/AMP guidelines recommend:

Fix for 6 to 48 hours in 10% NBF.16

Step 5: Confirm tumor cell content
Step 5: Confirm tumor cell content

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

10 working days icon
10 working days icon

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 shed by tumors into the bloodstream
ctDNA shed by tumors into the bloodstream

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

Step 1: Collect blood sample
Step 1: Collect blood sample

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 blood sample
Process blood sample

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
Step 3: Store the sample
Step 3: Store the sample

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
Step 2: Process blood sample
Step 2: Process blood sample

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

10 working days icon
10 working days icon

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.