This month we will continue discussing the common barriers to biomarker testing for cancer patients in the community.
As you may recall, these are the top 10 barriers that I’ve seen to biomarker testing in the community:
- High cost of testing.
- Long turnaround time for results.
- Limited tissue quantity.
- Preanalytical issues with tissue.
- Low biomarker testing rates.
- Lack of standardization in biomarker testing.
- Siloed disciplines.
- Low reimbursement.
- Lengthy complex reports.
- Lack of education on guidelines.
As I mentioned last month sample quantity and quality are both important when considering biomarker testing. We covered tissue quantity issue last month; let’s move on to tissue quality issues. I will focus on what happens to the tissue prior to testing being performed, called the preanalytical phase of testing. We now know that preanalytical variables can alter protein structure, DNA, and RNA (1). This can alter the results that are used to diagnose and treat patients. Despite the impact to downstream assays, this area is typically poorly controlled. Some of the sources of variance due to preanalytical processes include procurement, fixation, processing, and specimen storage (1).
ROSE: I covered rapid onsite evaluation (ROSE) during procurement last month with regards to tissue quantity. Having a pathologist evaluate both tissue quality and sufficiency during the biopsy is invaluable. For molecular analysis, areas of necrosis should be avoided.
Time to get specimen into fixation (cold ischemic time): Tissue begins to degrade as soon as it is removed from the body. The amount of time between removing tissue from the body and fixing it is referred to as the cold ischemic time. This time should be as short as possible. Less than 1 hour is recommended if molecular studies are going to be performed on the specimen (1). Tissue begins degrading immediately and this degradation can affect the results of biomarker test such as IHC & FISH (1). I’ve seen cases where the biopsy was collected, but due to a busy day in the OR, it was not sent to pathology immediately. This can cause the tissue to be so degraded that testing cannot be performed. Unfortunately, sometimes we don’t know there was a fixation issue until the molecular assay fails repeatedly. This is an expensive way to determine something went wrong in the preanalytical phase.
For small biopsies such as core needle biopsies or even fine needle aspirates, one way to decrease time to get the specimen into fixative is by having the fixative in the suite where the biopsy is collected so that it is put into fixative immediately. This won’t work for large pieces of tissue that may need to be dissected for optimal fixation penetration. For these biopsies, diligence needs to be taken to get the biopsy to the laboratory as soon as possible. Multidisciplinary communication will help ensure the hand-off occurs in a timely manner. Specimens may need to be dissected for optimal fixation penetration. Other specimens may need to be decalcified prior to fixation.
Decalcification: Most decalcification processes with formic acid have negative downstream effects on molecular testing. We have about a 50% success rate of PCR actually being able to amplify DNA after standard decal due to degradation. The solution to this is to use EDTA-based decalcification; however this could lengthen the fixation time drastically. Some new gentle decals are on the market that could be used without increasing the turnaround time.
Proper Fixative: We also need to ensure specimens are fixed in the appropriate fixative. The most common and widely accepted fixative is 10% neutral buffered formalin (NBF). DNA yield and some downstream biomarker tests are negatively impacted if unbuffered formalin is used (1). Tissue fixation in alcohol such as 70% ethanol may be even better than NBF if the downstream assay involves DNA extraction; however ethanol fixation may negatively impact IHC or FISH assays (2).
Time in Fixative: Studies show that the appropriate amount of time a biopsy needs to be in fixative is about 6-72 hours (1). The wide range of times is due to the range of specimen sizes, it is generally accepted that formalin penetrates tissue at the rate of 1 mm/hour (3). Tissue may need to be dissected to ensure complete fixation within a reasonable time. If the tissue is not fixed appropriately, the tissue will degrade. However, too much time in fixative can lead to degraded DNA.
Downstream biomarker assays such as FISH, PCR, and ISH can be affected if the fixative time is greater than 72 hours (1). Small community sites that do not have someone processing specimens over the weekend may need to adjust their biopsy schedules on Fridays to ensure specimens do not sit in fixative for more than 72 hours. Unfortunately, unless the specimen is a breast biopsy, in which CAP requires fixation times to be documented and controlled, fixation times are rarely documented and controlled. This is an area we could all improve on. It would be nice to know how long a specimen was stored when we are troubleshooting a downstream assay.
The impact of processing variables on biomarker testing has not been well published. One variable that has been called out as having a negative effect on PCR is mixing beeswax with paraffin wax. Only pure paraffin wax should be used. Other changes to the processing schedules should be performed with coordination between departments. If the anatomic pathology laboratory makes changes to their process, a verification study on the impact to the downstream assays should be performed. How extensive that study is should be determined by the medical director.
As long as the specimen was properly fixed, FFPE block storage is normally fairly hearty. The literature recommends blocks that are used for molecular analyses be less than 10 years old (1). Although I wouldn’t recommend using blocks older than 10 years, cases positive for some biomarkers are rare, so during our validations some blocks used were over 10 years old, and the downstream PCR-based assays still worked. However, I have no way to know if the DNA yield was compromised.
Unlike FFPE blocks, long-term storage of FFPE slides does affect downstream testing (4). Room-temperature storage seems to be worse than refrigerated storage of slides. Slides should be used for IHC and biomarker testing relatively quickly (< 30 days).
Unfortunately I cannot cover all sources of preanalytical error thoroughly in a 1,000 word blog post, but hopefully this sparks your interest enough to check out the references where the authors give a more detailed explanation of preanalytical issues.
- Bass BP, Engel KB, Greytak SR, Moore HM. A review of preanalytical factors affecting molecular, protein, and morphological analysis of formalin-fixed, paraffin-embedded (FFPE) tissue: how well do you know your FFPE specimen? Arch Pathol Lab Med. 2014;138:1520-30.
- Lindeman NI, Cagle PT, Aisner DL, Arcila ME, Beasley MB, Bernicker EH, et al. Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors: Guideline From the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology. Arch Pathol Lab Med. 2018;142:321-46.
- Howat WJ, Wilson BA. Tissue fixation and the effect of molecular fixatives on downstream staining procedures. Methods. 2014;70:12-9.
- Economou M, Schoni L, Hammer C, Galvan JA, Mueller DE, Zlobec I. Proper paraffin slide storage is crucial for translational research projects involving immunohistochemistry stains. Clin Transl Med. 2014;3:4.
-Tabetha Sundin, PhD, HCLD (ABB), MB (ASCP)CM, has over 10 years of laboratory experience in clinical molecular diagnostics including oncology, genetics, and infectious diseases. She is the Scientific Director of Molecular Diagnostics and Serology at Sentara Healthcare. Dr. Sundin holds appointments as Adjunct Associate Professor at Old Dominion University and Assistant Professor at Eastern Virginia Medical School and is involved with numerous efforts to support the molecular diagnostics field.