In this study, we reaffirmed the potential of the usefulness of ddPCR for the assessment of HER2 gene amplification in breast cancer.
We developed the ddPCR-TCR method in our previous report and showed that HER2 gene amplification assessing in breast cancer using ddPCR was feasible for clinical use [3]. However, in ddPCR-TCR method, the determination of HER2 gene amplification in a tumor was based on the area on the ddPCR-TCR chart on which the case was plotted, making the assessment more qualitative than quantitative.
In the current study, we improved our method by applying a very simple but potent strategy to quantitatively assess HER2 status at the final step of the ddPCR-TCR method. We calculated the unknown ratio of HER2 gene to CEP17 in only tumor cells by combining the ddPCR ratio [R] and TCR [X] and named this calculated HER2/CEP17 ratio of tumor cells as eHER2. This new approach provides us information on HER2 gene amplification of breast cancer specimens using a very simple and cost-effective method with ddPCR.
Our method has several advantages compared with the standard evaluation techniques. IHC and ISH are subjective in determining HER2 status, and special training is necessary for professional pathologists to make an accurate judgment. However, even pathology specialists often have difficulty in judging some equivocal or borderline cases. Our method can obtain accurate and precise data of HER2 gene amplification of a tumor objectively and rapidly, which means that our method can relieve pathologists of a great burden. ddPCR is easy to perform, with only a few manual processes, and the running cost is far cheaper than ISH analyses. ISH is routinely performed for breast cancer specimens in some countries, but not all areas, mainly because of economic reasons. Our method using ddPCR provide information of HER2 gene amplification for breast cancer cases with very low costs, which would help reduce medical spending.
The calculation of eHER2 is the ultimate tool in our method. eHER2 is the estimated HER2/CEP17 ratio of tumor cells using the obtained data of ddPCR ratio and TCR. In this study, we showed a high correlation between ISH ratio and eHER2, and our results indicated that eHER2 is equivalent to ISH ratio. With our method, clinicians and pathologists will be able to obtain the data for the HER2/CEP17 ratio in breast cancers easily and cheaply without the technical needs or costs of performing ISH.
Although ddPCR is a very precise device for DNA amplification measurement, this method has a measuring error to some extent, and this makes it difficult to determine the HER2 status of cases with eHER2 only slightly higher or lower than 2.0. To evaluate such cases with caution, we set the equivocal range as 1.8 ≤ eHER2 ≤ 2.2, or 0.8x + 1 ≤ Rx ≤ 1.2x + 1, based on the determination of HER2 gene amplification with ISH in the first ASCO/CAP guidelines for breast cancer [2]. One factor that hinders the accuracy ddPCR analysis is the heterogeneity of HER2 expression and/or HER2 gene amplification within a tumor. Wang et al. evaluated HER2 gene amplification with ddPCR, specifically for the HER2 equivocal cases, and showed high accuracy and usefulness of ddPCR. Still, they also considered that the presence of intratumoral heterogeneity of HER2 would make the evaluation challenging [11]. Therefore, it seems reasonable to set the equivocal range in our method. Such cases within this range may well be determined their HER2 status comprehensively considering the results of conventional tools such as IHC and ISH. However, we speculate that this range could be set much narrower, considering the potential of ddPCR.
The effect of the error also depends on the TCR of each case; the influence of error is relatively small if TCR of a specimen is high enough, while range of the error of calculated eHER2 can be larger as TCR gets smaller. Based on this observation, we set another equivocal range: 1.2x + 0.8 ≤ Rx ≤ 0.8x + 1.2. This second equivocal range is graphically symmetrical about a point with the first equivocal range (Fig. 2). The eHER2 of cases within the second equivocal range can be calculated as far away from the true HER2/CEP17 of tumor cells, so we need to be careful when determining the HER2 status of a case. The eHER2 of cases with very low TCR but out of the second equivocal range may also be far from the true HER2/CEP17 but is still useful in HER2 status determination. For example, a case with relatively low TCR (e.g. 0.2) but with very high ddPCR ratio (e.g. over 2.0) would be HER2 positive without doubt, and vice versa.
By setting these two equivocal ranges, we succeeded in improving sensitivity and specificity of our method. The ranges were quite similar to the equivocal area on ddPCR-TCR method in our previous study [3], and eight cases within the previous equivocal area were also included in our new equivocal ranges. One of the goals of our method is to accurately select HER2-positive cases and thus to determine appropriate candidates of anti-HER2 therapy. To increase the precision of our method, we need to determine more optical equivocal ranges in the future.
Most of the cases, except for cases within the equivocal ranges, showed strong concordance between conventional ISH ratio and our established eHER2, however one case (case number 36 in Table 2 and Fig. 4) showed discrepant results between the two. In this case, the ISH ratio was lower than 1.8 while eHER2 was higher than 2.2. HER2 gene amplification was calculated with FISH at the time of diagnosis and no longer observable because of degraded fluorescence, so we re-examined this sample with DISH. Five slides were granted to evaluate the quality of the DISH exam, and we found instability of CEP17 signals among the slides, indicating that the quality of FFPE specimen may be deficit owing to insufficient fixation or degradation of the tissue. The re-examined result of the HER2/CEP17 ratio with DISH was 1.56, which was similar to the value using FISH (1.70), but these ratios may be underestimated. The IHC staining results from this case also showed some discrepancies. One pathologist diagnosed this case as HER2 with an IHC score 3+, because complete circumferential membrane staining could be seen for at least 10% of tumor cells, but another pathologist diagnosed as HER2 score of 2 + since membrane staining was not sufficient to determine as completely circumferential. These discrepant diagnoses of IHC between the two pathologists are likely owing to the low quality of the specimen. With our method using ddPCR, HER2/CEP17 ratio can be measured accurately unless the DNA of tumor cells is severely damaged, and at this point our method is still advantageous over conventional assays. Clinically, this patient did not receive anti-HER2 therapy after surgery, but fortunately no sign of recurrence has been seen over these six years.
Our method represents a novel strategy to evaluate HER2 status. Although this technique may be unusual for pathologists, who are trained to determine HER2 status by observing specimen, clinicians can easily integrate eHER2 into daily medical practice, as are trained in examinations that yield numerical values and ranges, such as blood tests. However, while IHC and ISH can be visually observed repeatedly and are preserved with prepared slides, the calculated ddPCR ratio, TCR and eHER2 are only calculated data, and these data must be managed carefully to avoid problems, such as patient mix-ups.
In this study, we focused on quantifying HER2 status determination and established the calculated value of eHER2, which has the potential to replace conventional HER2 examinations. One of the purposes of quantification in determining HER2 status is to simplify the evaluation for clinicians and pathologists to recognize the degree of HER2 gene amplification. However, our final goal is to use eHER2 as predictive factor of anti-HER2 therapy and a prognostic factor. Xu et al. evaluated the association between disease-free survival and HER2 amplification level by ISH in a meta-analysis but concluded that HER2 amplification level is not a prognostic factor for HER2-positive breast cancer with trastuzumab-based targeted therapy [12]. We could not find a large-scale study that indicates the relationship between the rate of HER2/CEP17 with ISH and the effectiveness of anti-HER2 antibody. Also, there has been no report showing the relationship between HER2 status examined with ddPCR and the therapeutic effect of anti-HER2 therapy in breast cancer, including the assessment of pathological complete response rates after preoperative chemotherapy. The direct target of currently marketed anti-HER2 drugs is indeed overexpressed-HER2 protein, not amplified-HER2 gene. However, ASCO/CAP guidelines [2, 5, 10] treat IHC and ISH equally as methods for HER2 test in breast cancer; thus, we developed a technique to digitally assess HER2 gene amplification as a novel HER2 testing tool, which makes better use of the characteristics of ddPCR. In the future, it would be necessary to select cases that are preferable to receive intensive anti-HER2 therapy, e.g. trastuzumab plus pertuzumab, in the adjuvant setting for early HER2-positive breast cancer, and eHER2 may play an important role in these cases. Additional studies including more cases are required, and future research should analyze whether eHER2 is a really useful predictive or prognostic marker for HER2-positive breast cancer patients.
In conclusion, we evolved the strategy for the ddPCR-TCR method from our previous report and proposed eHER2 as a new HER2 determination tool in breast cancer. We succeeded in showing a strong correlation between ISH ratio (HER2 gene to CEP17) and eHER2, and we demonstrated that eHER2 has the potential to replace the conventional HER2 examination methods. A larger scale study is needed to apply our method in clinical use.