How to Test: Sample Collection

 

Collect and process tissue samples

 

Step 1: Choose an appropriate collection method
Step 1: Choose an appropriate collection method

CHOOSE an appropriate collection method

     

  • Assays may recommend using specific specimen types. For example, some assays have not been validated using cytology samples1,2
  • Collaborate with your multidisciplinary team to ensure appropriate biopsy methods are performed2

     

  • Assays may recommend using specific specimen types. For example, some assays have not been validated using cytology samples1,2
  • Collaborate with your multidisciplinary team to ensure appropriate biopsy methods are performed2

 

Biopsy methods differ in diagnostic yield and adequacy for biomarker testing

BRONCHOSCOPY
± EBUS2

Specimen types

Tissue biopsy

  • Endobronchial biopsy
  • Transbronchial biopsy

Cytology

  • Brushing cytology
  • Washing cytology
  • FNA cytology

 

Diagnostic yield

58% to 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)2,3

Specimen types

Biopsy

  • Tissue biopsy

Diagnostic yield

80% to 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)2-4

Specimen types

Tissue biopsy

  • CNB

Cytology

  • FNA cytology
  • Fluid cytology

Bone

  • Bone biopsy

Diagnostic yield

57% to 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

 

*EBUS-GS and EBUS-TBNA adequacy for biomarker testing ranges from 70% to 95%.

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Step 2: Collect sufficient tissue
Step 2: Collect sufficient tissue

COLLECT sufficient tissue5

 

  • 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 sample2
  • Drawing 2 core needle samples may ensure sufficient tissue is taken6

Some collection methods may be more likely to yield an adequate amount of sample.2 Adequate tissue collection is necessary because:

 

  • Sufficient tissue is required to perform multiple diagnostic tests7
  • Tumor heterogeneity can affect results, as tumors do not uniformly express PD-L1. Depending on sampling, the same tumor may yield positive, intermediate, or negative PD-L1 test results7,8
Collect tissue
Collect tissue

COLLECT sufficient tissue5

  • 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 sample2
  • Drawing 2 core needle samples may ensure sufficient tissue is taken6

Some collection methods may be more likely to yield an adequate amount of sample.2 Adequate tissue collection is necessary because:

 

  • Sufficient tissue is required to perform multiple diagnostic tests7
  • Tumor heterogeneity can affect results, as tumors do not uniformly express PD-L1. Depending on sampling, the same tumor may yield positive, intermediate, or negative PD-L1 test results7,8

Rapid on-site evaluation (ROSE) of tissue quantity and quality by pathology/cytology can ensure sufficient sample is collected.7

Step 3: Choose an appropriate fixative
Step 3: Choose an appropriate fixative

CHOOSE an appropriate fixative

 

  • Unacceptable fixatives may result in a loss of specific staining or alter staining intensity
  • 10% NBF is most commonly recommended for fixation
  • Unacceptable fixatives may result in a loss of specific staining or alter staining intensity
  • 10% NBF is most commonly recommended for fixation
  • Unacceptable fixatives may result in a loss of specific staining or alter staining intensity
  • 10% NBF is most commonly recommended for fixation

Fixative recommendations for PD-L1 assays and guidelines

Fixative recommendations for PD-L1 assays and guidelines

  Ventana PD-L1 (SP263)9,†,‡ Ventana PD-L1 (SP142)1 Dako PD-L1 IHC 22C3 pharmDx10 Dako PD-L1 IHC 28-8 pharmDx11
  • Recommended
  • 10% NBF
  • 10% NBF
  • 10% NBF
  • 10% NBF
  • Acceptable
  • Zinc formalin, Z-5 fixative§
  •  Other fixatives have not been validated
  • Other fixatives have not been validated
  • Other fixatives have not been validated
  • Unacceptable
  • 95% alcohol, AFA, Prefer
  • AFA, Prefer, alcohol-containing fixatives
  • Other fixatives have not been validated
  • Other fixatives have not been validated
Ventana PD-L1 (SP263)9,†,‡
 Ventana PD-L1 (SP142)1
Recommended

10% NBF

10% NBF
Acceptable
Zinc formalin, Z-5 fixative§

Other fixatives have not been validated
Unacceptable

95% alcohol, AFA, Prefer

AFA, Prefer, alcohol-containing fixatives
Dako PD-L1 IHC 22C3 pharmDx10
 Dako PD-L1 IHC 28-8 pharmDx11
Recommended

10% NBF

10% NBF
Acceptable

Other fixatives have not been validated

Other fixatives have not been validated
Unacceptable

Other fixatives have not been validated

Other fixatives have not been validated

†Recommendations are based on validation of other tumor types.9
‡FDA approved for detecting PD-L1 expression in urothelial carcinoma.9
§When used with at least 6 hours of fixation time.9

Step 4: Preserve Tissue Immediately
Step 4: Preserve Tissue Immediately

PRESERVE tissue immediately

 

  • Tissue samples are fragile, and degradation starts upon removal from the body11,12
  • Fix samples immediately, within 30 minutes of acquisition, to preserve tumor characteristics for diagnostic evaluation11,12
  • Tissue samples are fragile, and degradation starts upon removal from the body11,12
  • Fix samples immediately, within 30 minutes of acquisition, to preserve tumor characteristics for diagnostic evaluation11,12
Step 5: Determine optimal fixation methods
Step 5: Determine optimal fixation methods

DETERMINE optimal fixation methods

 

  • Both over- and underfixation can affect biomarker test results and subsequent treatment decisions12,13
  • Both over- and underfixation can affect biomarker test results and subsequent treatment decisions12,13

PD-L1 diagnostic assays recommend fixation1,9-11:

 

Recommendations for processing tissue
Recommendations for processing tissue

||Fixation times may vary by assay.

  • Formalin-fixed tissue must be processed through a series of dehydration steps, followed by paraffin embedding and sectioning4-6,12,13

PD-L1 diagnostic assays recommend fixation1,9-11:

PD-L1 diagnostic assays recommend fixation1,9-11:

 

Optimal fixation
Optimal fixation
Step 6: Process sample
Step 6: Process sample

PROCESS sample

 

  • Formalin-fixed tissue must be processed through a series of dehydration steps, followed by paraffin embedding and sectioning1,9-11,13
  • Formalin-fixed tissue must be processed through a series of dehydration steps, followed by paraffin embedding and sectioning1,9-11,13
  • Formalin-fixed tissue must be processed through a series of dehydration steps, followed by paraffin embedding and sectioning4-6,12,13

FDA-approved assays recommend using1,9-11:

FDA-approved assays recommend using1,9-11:

 

FDA-approved assays recommend using FFPE tissue and 4- to 5- μm sections
FDA-approved assays recommend using FFPE tissue and 4- to 5- μm sections

FDA-approved assays recommend using1,9-11:

 

Recommendations for processing tissue
Recommendations for processing tissue
Step 7: Confirm tumor cell content is sufficient
Step 7: Confirm tumor cell content is sufficient

CONFIRM tumor cell content is sufficient

 

  • Formalin-fixed tissue must be processed through a series of dehydration steps, followed by paraffin embedding and sectioning4-6,12,13

Some PD-L1 diagnostics require a minimum number of viable cells in order to accurately interpret results.

Some PD-L1 diagnostics require a minimum number of viable cells in order to accurately interpret results.

 

Some PD-L1 diagnositics require a minimum number of viable cells in order to accurately interpret results
Some PD-L1 diagnositics require a minimum number of viable cells in order to accurately interpret results

Some PD-L1 diagnostics require a minimum number of viable cells in order to accurately interpret results.

 

Recommendations for processing tissue
Recommendations for processing tissue

Proper sample collection and processing are critical to obtaining accurate biomarker test results.13

 

Turnaround time can impact treatment decisions14

 

Biopsy sample recommend 10 working days from sample receipt
Biopsy sample recommend 10 working days from sample receipt

CAP/IASLC/AMP guidelines recommend a turnaround time of 10 working days from sample receipt at testing laboratory.15

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.15

 

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AFA, alcohol-formalin-acetic acid; AMP, Association for Molecular Pathology; CAP, College of American Pathologists; CNB, core needle biopsy; EBUS, endobronchial ultrasound; FDA, Food and Drug Administration; FFPE, formalin-fixed paraffin-embedded; FNA, fine needle aspiration; IASLC, International Association for the Study of Lung Cancer; NBF, neutral-buffered formalin; PD-L1, programmed death-ligand 1; TTNA, transthoracic needle aspiration.

 

References: 1. Ventana PD-L1 Assay (SP-142) [package insert]. Tucson, AZ: Ventana Medical Systems, Inc.; 2016. 2. Ofiara LM et al. Front Oncol. 2014;4:253. 3. Chen H et al. Cancers (Basel). 2015;7(3):1699-1715. 4. Wu JS et al. Radiology. 2008;248(3):962-970. 5. Ofiara LM et al. Curr Oncol. 2012;19(suppl 1):S16-S23. 6. Kim ES et al. Cancer Discov. 2011;1(1):44-53. 7. Levy BP et al. Oncologist. 2015;20(10):1175-1181. 8. McLaughlin J et al. JAMA Oncol. 2016;2(1):46-54. 9. Ventana PD-L1 (SP263) Assay [package insert]. Tucson, AZ: Ventana Medical Systems, Inc.; 2018. 10. Dako PD-L1 IHC 22C3 pharmDx [interpretation manual]. Santa Clara, CA: Agilent Technologies; 2016. 11. Dako PD-L1 IHC 28-8 pharmDx [interpretation manual]. Santa Clara, CA; Agilent Technologies; 2015. 12. Hammond ME et al. Arch Pathol Lab Med. 2010;134(7):e48-e72. 13. Engel KB, Moore HM. Arch Pathol Lab Med. 2011;135(5):537-543. 14. Lim C et al. Ann Oncol. 2015;26(7):1415-1421. 15. Lindeman NI et al. Arch Pathol Lab Med. 2018;142(3):321-346.

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During ROSE, a pathologist or technologist performs an on-site assessment of biopsy tissue quality and quantity to ensure it is adequate for histological and biomarker testing.7