How to Test: Sample Collection

 

Proper tissue collection and processing can ensure accurate biomarker testing

 

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

CHOOSE an appropriate tissue collection method

     

  • Some PD-L1 diagnostic assays may recommend using specific specimen types. Communication with your multidisciplinary team can ensure appropriate biopsy methods are performed1-6

 

Collection recommendations from PD-L1 assays

Collection recommendations from PD-L1 assays

 

  • TURBT
  • Cystectomy
  • Core needle biopsy
  • Cytology
  • Decalcified bone
  • Decalcified tissue
Preferred sampling procedures2,3
  • TURBT
  • Cystectomy
  • Core needle biopsy
Unsuitable samples4-6
  • Cytology
  • Decalcified bone
  • Decalcified tissue




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

  • Sufficient tissue is required to perform a routine diagnostic work-up and molecular testing7
  • 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 results7-9
Step 2: Preserve tissue
Step 2: Preserve tissue

  

PRESERVE tissue immediately

  • Minimize cold ischemia time, eg, the time between tissue removal and fixation.10 Tissue degrades from the moment surgery interrupts blood supply to the tumor until tissue fixation10
  • Minimize cold ischemia time, eg, the time between tissue removal and fixation.10 Tissue degrades from the moment surgery interrupts blood supply to the tumor until tissue fixation10

 

Include the time of sample acquisition and the time of fixation on the record of submission to the pathology department.10

Include the time of sample acquisition and the time of fixation on the record of submission to the pathology department.10

 

Tissue should be fixed as soon as possible, within 1 hour of acquisition.10,11,*

*Examination of cold ischemia time of the Ventana PD-L1 (SP263) Assay did not establish any conditions from 0 to 24 hours that were unfavorable.10,11

 

Include the time of sample acquisition and the time of fixation on the record of submission to the pathology department.10

 

Step 3: Choose a fixative agent
Step 3: Choose a fixative agent

CHOOSE an appropriate fixative agent

  • Unacceptable fixatives may result in a loss of specific staining or alter staining intensity.12 10% NBF is most commonly recommended for fixation4-6,13
  • Unacceptable fixatives may result in a loss of specific staining or alter staining intensity.12 10% NBF is most commonly recommended for fixation4-6,13

Fixative recommendations for PD-L1 assays and guidelines

Fixative recommendations for PD-L1 assays and guidelines

  Ventana PD-L1 (SP263)13 Ventana PD-L1 (SP142)2,4 PD-L1 IHC 22C3 pharmDx5,† PD-L1 IHC 28-8 pharmDx6,†, ‡ ICUD-EAU Recommendations11,§
  • Recommended
  • 10% NBF
  • 10% NBF
  • 10% NBF
  • 10% NBF
  • 10% NBF
  • Acceptable
  • Zinc formalin, Z-5 fixative||
  •  Zinc formalin
  • Other fixatives have not been validated
  • Other fixatives have not been validated
  • 4% paraformaldehyde 
  • Unacceptable
  • 95% alcohol, AFA, Prefer
  • AFA, Prefer fixative, alcohol-containing fixatives, Z-5 fixative
  • Other fixatives have not been validated
  • Other fixatives have not been validated
  • Not discussed
Ventana PD-L1 (SP263)13
Ventana PD-L1 (SP142)2,4
Recommended

10% NBF

10% NBF

Acceptable

Zinc formalin, Z-5 fixative||

Zinc formalin

Unacceptable

95% alcohol, AFA, Prefer

AFA, Prefer fixative, alcohol-containing fixatives, Z-5 fixative

PD-L1 IHC 22C3 pharmDx5,†
PD-L1 IHC 28-8 pharmDx6,†, ‡
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

ICUD-EAU Recommendations11,§
Recommended

10% NBF

Acceptable

4% paraformaldehyde 

Unacceptable

Not discussed

Recommendations are based upon validation of other tumor types.5,6
FDA approved for detecting PD-L1 expression in NSCLC and melanoma tissues.6
§Guidelines on screening, diagnosis, and molecular markers in bladder cancer.11
||When used with at least 6 hours of fixation time.13

Step 4:  Determine fixation methods
Step 4:  Determine fixation methods

DETERMINE optimal fixation methods

  • Both over- and underfixation can affect biomarker test results and subsequent treatment decisions10,12
  • Both over- and underfixation can affect biomarker test results and subsequent treatment decisions10,12

PD-L1 diagnostic assays recommend fixation4-6,13:

PD-L1 diagnostic assays recommend fixation4-6,13:

Fixation recommendations for PD-L1 testing in urothelial carcinoma
Fixation recommendations for PD-L1 testing in urothelial carcinoma

#Fixation times may vary by assay. The ICUD-EAU recommends fixation for 12 to 24 hours for smaller samples, such as those obtained by TURBT.11

#Fixation times may vary by assay. The ICUD-EAU recommends fixation for 12 to 24 hours for smaller samples, such as those obtained by TURBT.11

Step 5: Process tissue
Step 5: Process tissue

PROCESS tissue based on testing instructions

  • Formalin-fixed tissue must be processed through a series of dehydration steps, followed by paraffin embedding and sectioning4-6,12,13
  • 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 using4-6,13:

FDA-approved assays recommend using4-6,13:

 

Recommendations for processing tissue
Recommendations for processing tissue
  • 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 using4-6,13:

FDA-approved assays recommend using4-6,13:

 

Recommendations for processing tissue
Recommendations for processing tissue
Step 6: Store tissue
Step 6: Store tissue

STORE tissue for potential future use

  • PD-L1 testing in clinical trials used either freshly biopsied tissue or archival paraffin-embedded tissue14-16
  • PD-L1 testing in clinical trials used either freshly biopsied tissue or archival paraffin-embedded tissue14-16

Paraffin embedding allows for long-term storage of formalin-fixed tissue samples.12

Paraffin embedding allows for long-term storage of formalin-fixed tissue samples.12

 

Tissue storage recommendations
Tissue storage recommendations
Step 7: Confirm tumor cell content
Step 7: Confirm tumor cell content

CONFIRM tumor cell content is sufficient

Some PD-L1 diagnostics require a minimum number of viable cells in order to accurately interpret results.5,6,13

 

Some PD-L1 diagnostics require a minimum number of viable cells in order to accurately interpret results.5,6,13

Recommended minimum of viable cells
Recommended minimum of viable cells

Proper sample collection and processing are critical to obtaining accurate biomarker test results.12

AFA, alcohol-formalin-acetic acid; CAP, College of American Pathologists; FDA, Food and Drug Administration; FFPE, formalin-fixed paraffin-embedded; ICUD-EAU, International Consultation on Urologic Disease-European Association of Urology Consultation on Bladder Cancer; IHC, immunohistochemistry; NBF, neutral buffered formalin; NSCLC, non–small cell lung cancer; PD-L1, programmed death-ligand 1; TURBT, transurethral resection of bladder tumors.

 

References: 1. Hicks DG et al. Arch Pathol Lab Med. 2008;132(8):1226-1227. 2. Ventana PD-L1 Assay (SP-142) [interpretation guide for urothelial carcinoma]. Tucson, AZ: Ventana Medical Systems, Inc; 2016. 3. Powles T et al. Nature. 2014;515(7528):558-562. 4. Ventana PD-L1 Assay (SP-142) [package insert]. Tucson, AZ: Ventana Medical Systems, Inc; 2018. 5. Dako PD-L1 IHC 22C3 pharmDx [interpretation manual]. Carpinteria, CA: Dako North America Inc; 2015. 6. Dako PD-L1 IHC 28-8 pharmDx [product information]. Carpinteria, CA: Dako North America Inc; 2015. 7. Levy BP et al. Oncologist. 2015;20(10):1175-1181. 8. McLaughlin J et al. JAMA Oncol. 2016;2(1):46-54. 9. Drake CG et al. J Clin Oncol. 2016;34(26):3115-3116. 10. Hammond ME et al. J Clin Oncol. 2010;28(16):2784-2795. 11. Kamat AM et al; International Consultation on Urologic Disease-European Association of Urology Consultation on Bladder Cancer 2012. Eur Urol. 2013;63(1):4-15. 12. Engel KB, Moore HM. Arch Pathol Lab Med. 2011;135(5):537-543. 13. Ventana PD-L1 (SP263) Assay [package insert]. Tucson, AZ: Ventana Medical Systems, Inc; 2018. 14. Massard C et al. J Clin Oncol. 2016;34(26):3119-3125. 15. Rosenberg JE et al. Lancet. 2016;387(10031):1909-1920. 16. Sharma P et al. Lancet Oncol. 2016;17(11):1590-1598. 17. Centers for Medicare & Medicaid Services. http://www.ncleg.net/documentsites/committees/PMC-LRC2011/December%205,%202012/CLIA%20Regulations%20Record%20Retention%20%20(42CFR%C2%A7493.1105).pdf. Accessed March 15, 2018. 18. College of American Pathologists. http://www.ncleg.net/documentsites/committees/PMC-LRC2011/December%205,%202012/College%20of%20American%20Pathologist%20Retention%20Policy.pdf. Updated March 2010. Accessed March 12, 2018.