By: Jennifer H. Freeland
Invalid test results from next generation sequencing (NSG) and other molecular assays negatively impacts patient care. How can we, as histotechnologists, help remedy this? The histology laboratory supports molecular pathology through tissue specimen preparation; and although we have learned over the last decade that certain pre-analytical factors influence the outcome of immunohistochemical assays, we are still learning what factors impact molecular results.
The American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) published guideline documents regarding Her2, estrogen receptor and progesterone receptor which have helped laboratories refine their methods to ensure reproducible and reliable results for immunohistochemistry (IHC), and in situ hybridization (ISH) (5,10,11,12). In 2013, members of CAP, the Association of Molecular Pathology (AMP), and International Association for the Study of Lung Cancer (IASLC) issued guidelines for ALK and EGFR genetic testing in lung cancer (7). In 2018 the three medical societies issued an update on the molecular testing of lung cancers (8).
Targeted therapies that are brought to market rely on the accurate assessment of protein-based biomarkers identified by IHC tests and nucleic acid- based biomarkers (DNA, RNA) identified by ISH, the polymerase chain reaction (PCR), or NGS. In order to ensure that these precious biomarkers are well-preserved, higher quality and consistency in specimen preparation is becoming necessary. For example, ensuring that specimens collected in the OR were fixed in a primary tissue fixative in as minimal a time as possible was considered acceptable practice for the delivery of quality histology specimens (5,10). Other studies suggested that delays to fixation of up to 12 hours didn’t appear to significantly impact antigenicity as detected by IHC (2). Now that we are trying to identify nucleic acid-based biomarkers, we are finding that this time, ischemia time, should be more closely monitored. Particularly for RNA, extended ischemia times for biospecimens has a negative impact on RNA quality, as assessed by qRT-PCR3. It has been recommended by some researchers to try to minimize ischemia time to as little as 30 minutes for optimal biomarker preservation (4).
The guidance documents also refer to selection of tissue fixative and fixation duration. In most cases, it is recommended that tissues are fixed in 10% neutral buffered formalin (NBF) for 6 to 72 hours, depending upon tissue type and size (5,10,11,12). There is a great deal of data available on the performance of formalin-fixed paraffin-embedded (FFPE) tissues with IHC, ISH, PCR, and NGS. This data indicates that NBF has compatibility with IHC; with the suggestions in the guidance documents, specimen preservation has become more standardized, which has enabled more reproducible IHC detection of important protein-based biomarkers.
DNA-based biomarkers, such as mutations, are often tested via ISH, PCR, or by next-generation sequencing. Some mutations are not detectable by routine ISH, such as single nucleotide polymorphisms (SNPs), also referred to as point mutations (10). The oligonucleotide probes are often too large to adequately detect and hybridize a SNP (10). Next generation sequencing, however, can detect many mutation types including SNPs. DNA, while not as robust as protein, can still be relatively well-preserved when using 10% neutral buffered formalin. Formalin cross-links DNA as well as proteins and can also cleave DNA into fragments (8). NGS has been optimized to deal with DNA extracted from FFPE, as it can deal with fragments as short as 300 bps (8). However, it should be noted that molecular assays requiring DNA fragments that are greater than 1000 base pairs (bps) will not typically yield reliable results from FFPE (8).
A tissue’s RNA quality may also be impacted by formalin fixation. In a large comparison study published in the Archives of Pathology and Laboratory Medicine, it was found that formalin-fixed tissues score significantly lower than fresh-frozen tissues for a variety of RNA-quality metrics, including RIN value (RNA quality) and DV200 value (RNA segment length) (1). Tissues were subjected to a formalin-fixation time-course study in which tissues were fixed for 6 to 72 hours (1). When comparing the RNA quality of these tissues, it was noted that RNA segment length appears to maintain consistency through 23 hours of fixation; however, the length of the RNA segment decreases at the 72-hour time point (1). Since length of the RNA molecule is often an indicator of success for downstream RT-PCR and NGS, tissues that have prolonged formalin exposure times may have invalid molecular test results.
Our laboratory conducted a comparison study of fixative types on the impact of NGS for DNA-based biomarkers (6). In this study, methanol-fixed tissues and ethanol-fixed tissues served as controls for nucleic acid quality. These are excellent fixatives for nucleic acids, although not ideal for routine histopathology, H&E, and IHC (6). Other fixatives tested include zinc-formalin and Bouin’s fluid. Generally, Bouin’s fluid-fixed samples performed poorly for all NGS metrics, whereas formalin-fixed tissues performed with acceptable quality (6). An interesting finding was related to Average Base Coverage Depth. These measures discern the average length of the DNA strand. Any strand greater than 2300 base pairs (bps) indicates good quality (6). Less than 2000 bps indicates poor quality (6). As you can see in Figure 1, methanol and ethanol produce good quality DNA results, which is expected from the positive control (6). Formalin / 10% NBF performs with good quality above 2300 bps for the 8 hour and 24-hour time points(6). At the 72-hour time point, the average based coverage depth for formalin-fixed tissue decreases below 2000 bps (6). Our conclusion from this study is that tissues fixed in 10% neutral buffered formalin for up to 48 hours produce the best results for NGS.
Standardization for specimen preparation has been significantly improved over the last decade, and this is especially due to the positive influence of the published guidance documents. The quality and consistency of current IHC and ISH assays is evidence of the positive change. As we continue to learn more about the specimen requirements for NGS and other molecular assays, we may find that the current acceptable standards for pre-analytic factors such as fixation, processing, and specimen handling may need refinement. Ongoing molecular studies and combined histopathology-molecular studies continue to provide evidence that guide these decisions. In the future, refined pre-analytic factors would enable laboratories to minimize invalid molecular results and ensure high precision results for patients.
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2. Engel KB, et al. Effects of preanalytical variables of the detection of proteins by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Archives of Pathology and Laboratory Medicine. 135:537-543. 2011
3. Guerrera F, et al. (2016). The influence of tissue ischemia time on RNA integrity and patient-derived xenografts (PDX) engraftment rate in a non-small cell lung cancer biobank. PLOS One. Doi:10.1371/journal.pone.0145100.
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