Immunohistochemical assays are the current gold standard for evaluating ER, PR, HER2 and Ki67 status in breast invasive carcinoma. The main advantages of IHC for the assessment of these markers are that it is rapid and simple, it can be performed in most pathology laboratories, and (when compared with other assays) it is relatively inexpensive. However, IHC assay reliability has been questioned because alterations during tissue processing, manipulation and fixation, as well as the antibody clone, internal controls and scoring system used may affect the precision of the results. In addition to that, inter-observer variability in interpretation may play also a role as IHC remains a semi-quantitative and non-standardized method[18–20].
The ambiguity encountered in the interpretation of HER2 IHC results especially in cases with HER2 equivocal scores (HER2 IHC =2+) may also represent an issue that most laboratories in resource-constrained settings may not be able to overcome as the gold standard would be, in these cases, FISH for quantifying HER-2 gene amplification. Indeed, FISH has the advantage of being a quantitative method and is considered as the gold standard method for confirming the HER-2 status, not only to resolve IHC 2+ cases, but also for all other cases where it has an excellent correlation with the HER2 IHC results[31, 32] Major disadvantages are that FISH is technically complicated to execute, arduous to establish, has a long run time, and is costly, making it not routinely available in all pathology laboratories worldwide. Moreover, another limitation of this method is that it doesn’t necessarily reflect target protein expression and counting FISH spots is wearisome and can be biased by tumor heterogeneity [9, 19, 20].
Nevertheless, these causes of assay variability may explain the differences in ER, PR, HER2, and Ki67 IHC results in breast carcinomas reported previously.
Currently, treatment of invasive breast carcinoma relies essentially upon ER, PR, HER2 and Ki67 status [33, 34] and accuracy of assays is critical. Hence, to overcome limitations of IHC and HER2 FISH, there have been efforts to establish alternative methods to assess the 4 biomarkers of interest as accurately as possible . One of the options is to use RT-qPCR. Reverse transcription quantitative PCR (RT-qPCR) represents a sensitive, efficient, and reliable approach for analyzing RNA. The initial step in RT-PCR is the production of a single-strand complementary DNA copy (cDNA) of the RNA through the action of the retroviral enzyme, reverse transcriptase, to amplify that part of this cDNA by PCR. RT-PCR is used to analyze differential gene expression or cloned cDNAs. RT-PCR is more sensitive and easier to perform than other RNA analysis techniques. Xpert® Breast Cancer STRAT4 is a real-time RT-qPCR semi-quantitative assay with qualitative cut-off values for Estrogen Receptor (ESR1), Progesterone Receptor (PGR), HER2/ERBB2, and the marker of proliferation Ki-67 (MKi67) mRNAs isolated from FFPE tissues. The test is intended to be used with the GeneXpert® System, which automates RNA isolation and purification from FFPE tissue, as well as amplification and detection of target sequences within the cartridge[18, 37].
In this study, our statistical data demonstrated that the Xpert® Breast cancer STRAT4 closed-system RT-qPCR method shows basically a good concordance rate with IHC+HER2 FISH results.
The concordance between Xpert® Breast cancer STRAT4 and HER2 IHC+HER2 FISH has been evaluated in other studies and varies between 91% and 98%. In the current analysis, our data demonstrate that the Xpert® Breast Cancer STRAT4 assay shows greater than 91% concordance with HER2 IHC+HER2 FISH, suggesting that our results are generally concordant with the previous studies[16–20, 32, 38].
For both ESR1/ER and ERBB2/HER2, data suggested almost perfect agreement between Xpert® Breast Cancer STRAT4 and central IHC (κ "ER"= 0.830 ; κ "HER2"= 0.838), with nearly all of the discordant cases with quantitative dCt values close to the ESR1 and ERBB2 dCt cutoffs, respectively. Our findings are in good agreement with previously reported results: we found overall concordance of 93.50% and 95% for ER and HER2 respectively, and previously published papers have reported values of 97–98% for ER and of 93–97% for HER2[16–18, 20].
The results showed a moderate Kappa correlation agreement for PGR/PR (using PR IHC+ 1%) and MKi67/Ki67 (excluding equivocal cases) between both assays (κ "PR"= 0.565 ; κ "Ki67"= 0.458). Xpert® Breast Cancer STRAT4, however, demonstrated a significant overall concordance with IHC for PGR (83.5%) and MKi67 (81%). The concordance rates observed in other studies vary from 81–92% for PR and from 78–89% for Ki67, in accordance with agreement percentages obtained in our analysis[16–18, 20, 38].
Discordance between assay methods can be attributed to several factors, including the tissue fixation, antibody clone used in IHC, and scoring methods used. Preanalytical factors are essential to monitor and a quality assessment scheme should be put in place in any laboratory routinely performing the assessment of the 4 biomarkers . Particular attention should be paid to the impact of sample handling, time of fixation, duration of tissue fixation, antibody selection, control samples and interpretation of assay on Xpert® Breast Cancer STRAT4 results [8, 10, 11, 14]. In spite of the systematic practice of immunohistochemistry methods, procedural inconsistency remains elevated in clinical settings, leading to interlaboratory and intralaboratory variations and to high false-negative (for ER and PR) and false-positive (for HER2). This inconsistency emphasizes the importance of a standardized retrieval method in the performance of reliable IHC and/ or FISH for the four markers routinely screened in breast cancer diagnosis.
Xpert® Breast Cancer STRAT4 has already been shown to have good agreement with automated semi-quantitative IHC[16–18, 21, 22, 24]. ESR1 and ERBB2 assessments have the highest pertinence in all studies. Comparison between the Xpert® Breast Cancer STRAT4 PGR status and the PR IHC status resulted in more discrepancies. This discrepancy between the two methods could be explained by the fact that total mRNA does not necessarily reflect the total protein and vice versa . Denkert et al. have demonstrated that Ki67 IHC results are greatly variable. For this biomarker, a significant variability in concordance rate has been noted, although this is not unforeseen given the challenges associated with Ki67 IHC evaluation.
Considering our results, as well as other similar results found in other published papers, the Xpert® Breast Cancer STRAT4 assay could be a potential solution to overcome IHC/FISH limitations and may help facilitate access to invasive breast cancer testing in low resource countries. It is a sensitive method that can replace IHC and FISH in remote areas where IHC cannot be performed, because it is a non- operator -dependent technique and does not require an equipped molecular laboratory[9, 11, 16–20, 38].