Unequivocal HER2 Detection and Quantification
Reliable Detection of HER2 in Using Droplet Digital PCR
- Tab. 1: Performance of two ddPCR platforms in detecting HER amplification. Separate cohorts were tested for HER2 with IHC, FISH, and two different ddPCR platforms, respectively. The thresholds for FISH were calculated separated for each platform, and then then the threshold for the Bio-Rad platform (1.62) was changed to match the threshold for the RainDance platform (1.72). The two systems were in closer agreement if their system-specific thresholds were used.
- Tab. 2: Comparison of results between the two ddPCR platforms. The two platforms produced concordant results among a cohort of 99 patient samples.
- Tab. 3: Performance of ddPCR among IHC equivocal cases. ddPCR was able to produce a definitive result in 102 of 114 cases.
Overexpression of the HER2 gene is predictive of patient response to targeted therapies, but standard HER2 detection methods, immunohistochemistry and fluorescence in situ hybridization, are both technically demanding and often produce equivocal results. Droplet Digital PCR quantification technology, however, may enable a reliable and robust reflex test for HER2 in ambiguous tumor samples.
The over-expression of the human epidermal growth factor receptor 2 (HER2) gene, which occurs in 15-30% of breast cancer cases, is predictive of patients’ response to HER2-targeted therapies, such as trastuzumab and lapatinib [1,2]. Consequently, HER2 expression is analyzed in all breast cancer cases as a standard diagnostic biomarker.
Pathologists test for HER2 protein expression with immunohistochemistry (IHC) and gene amplification with fluorescence in situ hybridization (FISH). The American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) recommends that pathologists perform IHC first, and if the results are inconclusive, then reflex test with FISH .
But the subjective nature of these tests can lead to inconclusive results, with breast cancer patients receiving different interpretations of their HER2 status depending on the lab their samples are sent to . IHC, for instance, produces equivocal results 25-30% of the time . Meanwhile, FISH analyses still yield equivocal results 4% of the time .
IHC and FISH are both technically demanding and can produce unreliable results, primarily because those results are heavily influenced by variations in testing conditions, such as how the samples are fixed and which reagents and assay protocols are used. Furthermore, IHC may lead to inter-observer variability due to disagreement in how to score IHC data [6,7]. Meanwhile, FISH is especially labor-intensive, complicated and time-consuming, which limits the number of patients that a pathologist can test simultaneously .
FISH results can also be indeterminate because the reference standard control for HER2 amplification is not wholly reliable .
FISH assesses HER2 levels by measuring the ratio between the signal intensity of HER2 probes and the signal intensity of probes against the nearby chromosome 17 centromere (CEP17). In HER2 FISH, the level of CEP17 serves as an index of chromosome 17 copy number. However, CEP17 signal intensity can fluctuate due to focal amplification of the centromere, making it an unreliable standard . Increases in CEP17 intensity would result in a reduced HER2-to-CEP17 ratio and mask HER2 amplification.
Because of this ambiguity, the ASCO/CAP guidelines require that a pathologist read 25-100 cells to determine the ratio of HER2 to reference control . Furthermore, they recommend testing with an alternative chromosome 17 probe altogether . Since Droplet Digital PCR (ddPCR) is capable of multiplexing, it is particularly suited for testing with multiple unbiased reference probes such as EFTUD2, thus overcoming the potential challenges associated with the use of CEP17 .
ddPCR was developed for absolute and precise nucleic acid quantification, as well as for rare allele detection, making it a potentially reliable reflex test to IHC and FISH for HER2 detection and quantification. ddPCR involves three simple steps – reaction partitioning, thermocycling, and detection – with minimal hands-on time. Researchers can run 96 samples at once and apply the technique to samples often found in clinical labs, e.g. frozen, formalin-fixed, paraffin-embedded, and plasma DNA samples .
ddPCR is performed by partitioning a PCR reaction into tens of thousands of nanoliter-sized droplets, such that each droplet contains zero or generally no more than a few molecules . A fluorescent probe binds to the target DNA so that when the target DNA molecules are amplified, the droplets containing the target DNA emit a strong “positive” fluorescent signal. At the end of the PCR run, the system counts the number of positive and negative droplets and precisely calculates the quantity of target DNA molecules in the sample . By counting both HER2 and the reference (e.g. CEP17, or better, EFTUD2) gene copies in the same well, ddPCR obviates the need for a standard curve and enables researchers to determine highly precise copy number ratios [11,14].
In one equivalency study, researchers compared the ability of two ddPCR platforms (QX200 Droplet Digital PCR System (Bio-Rad) and RainDrop Plus Digital PCR System (RainDance), to detect HER2 over-expression against a CEP17 probe in 102 samples of invasive breast cancers with IHC and FISH results . Additionally, the researchers tested whether ddPCR could detect HER2 amplification in an independent validation cohort of 114 equivocal HER2 cases that could not be resolved by IHC (IHC 2+) and were later tested with FISH. Although ddPCR is capable of multiplexing with multiple references, which might further improve its performance for this test, this study only used CEP17 as reference so ddPCR could be directly compared to FISH. HER2 amplification using FISH was defined as a HER2/CEP17 ratio of ≥2, based on the ASCO/CAP Guidelines .
Both ddPCR platforms yielded results that agreed with the IHC and FISH results with more than 90% accuracy (tab. 1). They were both at least 75% sensitive and 97% specific. Additionally, both platforms showed very high concordance with each other (at least 97%; tab. 2), suggesting that ddPCR is a robust test for quantifying HER2 in invasive breast cancer tumor samples.
Finally, among a separate cohort of 114 IHC 2+ cases, the RainDance platform successfully determined HER2 status in 102 cases, in concordance with FISH, with 75% sensitivity and 95.3% specificity (tab. 3). Discordant results were found in a few cases tested by ddPCR, which could be due to HER2 status or other sources of error in either FISH or ddPCR.
These data indicate that ddPCR can complement IHC and FISH, potentially serving either as a reflex test or as an alternative to FISH for HER2 diagnosis. However, in either case, it remains to be proven that ddPCR results on equivocal samples is predictive of patient response to therapy.
It is crucial to correctly identify HER2 in cancer patients due to the therapeutic implications of the diagnosis. But the current diagnostic tools, IHC and FISH, could lead to equivocal results. In cases where IHC is inconclusive, there are still issues associated with FISH. ddPCR shows agreement with the standard protocol and offers several advantages that make it a better alternative to these traditional methods. Since ddPCR is capable of multiplexing, one could use multiple controls when testing for HER2 amplification beyond the standard but sometimes unreliable CEP17 marker, such as the more reliable EFTUD2 gene . ddPCR is also more amenable to automation and high-throughput screening, takes less time, and is more objective, making it a potentially more efficient screening tool for HER2. Further studies are required to understand whether ddPCR can be a better predictor of HER2 therapeutic outcome than standard IHC/FISH testing.
We are thankful to Prof. Zunfu Ke, Associate Professor and Associate Chief Physician in the Department of Pathology at The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China, and his team, for performing the research and providing the data included in this article.
1 Bio-Rad Laboratories, Hercules, CA, USA
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