RNAscope-based HER2 mRNA detection shows high concordance with fluorescence in situ hybridization in invasive breast carcinoma: a retrospective study

Introduction

Breast cancer (BC) is the most prevalent cancer among women and the second leading cause of death in this population (1). Invasive ductal carcinoma (IDC) is the most common subtype of BC, typically managed through multidisciplinary approaches in clinical practice (2-4). The human epidermal growth factor receptor 2 (HER2) gene, located in the q21 region of chromosome 17, encodes a transmembrane receptor protein with tyrosine kinase activity that plays a crucial role in the signal transduction of cell growth and differentiation (5). The expression status of HER2 is independent of tumor size, lymph node metastasis, age, and the expression of progesterone receptor (PR) and estrogen receptor (ER) (6). HER2 is a significant prognostic factor for IDC and represents one of the most promising targets for therapeutic intervention (7). Research has demonstrated that HER2-targeted therapies, such as trastuzumab, pertuzumab, and trastuzumab-metformin conjugates, exhibit substantial therapeutic efficacy (1,8). The administration of HER2-targeted drugs is contingent upon the HER2 status of the tumor (9). Therefore, accurate assessment of HER2 status in IDC is of paramount clinical importance.

Currently, immunohistochemistry (IHC) is commonly used in clinical practice to detect HER2 status in IDC due to its simplicity and low cost. Fluorescence in situ hybridization (FISH) is typically employed when IHC results are uncertain, particularly for IHC 2+ results, to assess HER2 gene amplification for retesting and confirmation (10). Most HER2 statuses classified as IHC 2+ can be determined by FISH (11). However, discrepancies between IHC and FISH results can occur, and the interpretation of these results remains controversial (12). Studies have indicated that RNAscope can serve as an effective supplementary examination method alongside IHC and FISH in IDC (13). When HER2 status is classified as IHC 2+ or when FISH results are uncertain, RNAscope detection offers distinct advantages (14). Additionally, RNAscope detection may play a significant role in evaluating HER2 messenger RNA (mRNA) status (15). Therefore, this study aims to investigate the consistency between RNAscope and FISH in assessing HER2 status among IHC 2+ IDC cases, and to explore whether RNAscope may serve as a complementary diagnostic tool. As a retrospective, single-center analysis, this work is intended to be hypothesis-generating, with the goal of providing preliminary insights for further clinical evaluation. We present this article in accordance with the STROBE reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2486/rc).

Methods

Study design and patient selection

A retrospective analysis was conducted on 104 cases of IDC diagnosed through routine pathology at The First Affiliated Hospital of Harbin Medical University from January 2020 to January 2024. Initially, the expression of HER2 protein was assessed using IHC. Subsequently, cases with an IHC score of 2+ were selected using a random number method. The HER2 status and expression levels were further evaluated using RNAscope and FISH to analyze the consistency between the two methods. Finally, next-generation sequencing (NGS) was performed to determine HER2 gene expression in cases with inconsistent results from the previous methods. The study design is illustrated in Figure 1. Inclusion criteria included: (I) postoperative pathological diagnosis of IDC; (II) availability of IHC and FISH test results; (III) completeness and usability of specimens. Exclusion criteria included: (I) prior cancer treatments before surgery (hormone therapy, chemotherapy, targeted therapy, or radiation therapy); (II) incomplete clinical data. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The First Affiliated Hospital of Harbin Medical University (approval No. 202128). All participants were informed about the research protocol and provided written informed consent to participate in the study. All specimens and data were anonymized.

Figure 1 Flow chat of the study design. FISH, fluorescence in situ hybridization; HER2, human epidermal growth factor receptor 2; IDC, invasive ductal carcinoma; IHC, immunohistochemistry; NGS, next-generation sequencing.

Detection methods & interpretation techniques

IHC for HER2

The collected 4 µm thick tissue sections were stained using the Ventana Benchmark Ultra fully automated immunohistochemistry analyzer (Roche) in accordance with standardized procedures to evaluate the expression of HER2 protein. The interpretation of IHC results was performed following the 2018 ASCO/CAP HER2 testing guidelines (16). According to the expression levels, which range from low to high, there are four grades of HER2 IHC: 0, 1+, 2+, and 3+. IHC 3+ is defined as HER2 positive, while IHC 0 and IHC 1+ are classified as HER2 negative. IHC 2+ is categorized as HER2 uncertain.

FISH for HER2

The PathVysion HER2 DNA Probe Kit (acquired from Abbott Laboratories, USA) was employed for FISH detection in accordance with standard procedures. The CytoVision DM6000B fluorescence microscope system (obtained from Leica, Germany) facilitated the observation of FISH results and image acquisition. The primary process involved counting the copy numbers of the HER2 gene (red) and CEP17 (green) in at least two invasive tumor regions and in over 20 cell nuclei, respectively. In cases with ambiguous FISH results (as detailed below), the counting was repeated following the aforementioned method: if the HER2/CEP17 ratio exceeds 2.0 or the average HER2 copy number per tumor cell is ≥6.0, and the HER2/CEP17 ratio is below 2.0, the HER2 status is classified as positive. Conversely, if the HER2/CEP17 ratio is below 2.0 and the average HER2 signal per cell ratio is less than 4.0, the HER2 status is deemed negative. When the HER2/CEP17 ratio is below 2.0 and the average HER2 signal per cell ratio is between 4.0 and 6.0, the 2018 guidelines classify it as positive (if IHC 3+) or negative (if IHC 0, 1+, or 2+). Furthermore, if the HER2/CEP17 ratio exceeds 2.0 while the average HER2 signal per cell is below 4.0, the 2018 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines define the HER2 status as positive (if IHC 3+) or negative (if IHC 0, 1+, or 2+) (3,17).

RNAscope in situ hybridization for HER2 mRNA

An RNAscope FFPE 2.0 kit (Advanced Cell Diagnostics, Newark, NY, USA) was utilized to detect HER2 mRNA expression from archived specimens. FFPE tissue sections, measuring 4 µm in thickness, were pretreated through heating and protease digestion prior to hybridization with a target probe specific for HER2, comprising a 20-probe pair set that targets the HER2 mRNA sequence. The sections were then placed in a hybridization oven (Advanced Cell Diagnostics) for the hybridization process. Diaminobenzidine (DAB) was employed for color development, and hematoxylin was used for counterstaining. Two sections served as positive controls using the housekeeping gene UbC probe, while a negative control was established with the bacterial gene DapB to assess background staining. mRNA staining was evaluated across the entire section under a 20× objective lens, with signal quantification performed in 100 cancer cells under a 40× lens. HER2 expression was scored according to the guidelines provided in the RNAscope FFPE Assay Kit: no staining (score 0); staining that was difficult to discern under a 40× objective lens in more than 10% of tumor cells (score 1); staining that was challenging to observe under a 20× objective lens but clear under a 40× objective lens in over 10% of tumor cells (score 2); staining that was hard to see under a 10× objective lens but evident under a 20× lens in more than 10% of tumor cells (score 3); and staining that was easily observable under a 10× objective lens in over 10% of tumor cells (score 4). A score of 4 was categorized as indicative of HER2 over-transcription. Based on the kit instructions, the scoring criteria were adjusted according to the HER2 expression results, differentiating between HER2 negative scores of 0–2 and HER2 positive scores of 3–4 (18). RNAscope scoring was independently performed by two experienced pathologists who were blinded to the corresponding IHC, FISH, and NGS results. In cases of discrepant interpretation, a consensus was reached through joint review. Inter-observer agreement was assessed during the initial evaluation to ensure scoring consistency.

NGS for HER2

NGS was performed on patients whose results from FISH, IHC, and RNAscope were inconsistent. A standard procedure was employed for tissue genomic DNA extraction using the column extraction method to obtain total DNA from formalin-fixed paraffin-embedded (FFPE) tissue sections. The breast tumor fraction was extracted utilizing the QIAamp DNA FFPE Tissue Kit (Qiagen, Venlo, The Netherlands) in accordance with the manufacturer’s instructions. Following quantitative and qualitative analysis of the extracted DNA, a Covaris LE220 (Covaris, Inc., Woburn, MA, USA) was utilized to fragment the genomic DNA for the construction of a DNA library (200–250 bp). Probe-targeted capture technology was employed to enrich the target DNA fragments. The library was constructed according to the instructions provided with the Illumina paired-end sequencing library kit. After quantification using a quantitative kit, the Illumina HiSeqX-10 platform (Illumina, Inc., San Diego, CA, USA) was used to sequence the exons and adjacent intron regions of the target gene. The Illumina processing software facilitated image analysis and error estimation to generate the raw data. Data were analyzed using the GATK pipeline for alignment, variant calling, and copy number variation (CNV) analysis. HER2 gene amplification was defined as a copy number ≥6, and overexpression was inferred based on elevated read depth and consistency with RNA in situ hybridization results. Quality control included a minimum sequencing depth of 500× and tumor content ≥20%. These criteria were applied uniformly across the five discordant cases.

Statistical analysis

Data analysis was conducted using SPSS version 27.0 (IBM, Armonk, NY, USA). Continuous data that met the criteria for normal distribution, as determined by the Kolmogorov-Smirnov test, were expressed as means ± standard deviations (SD). Categorical data were presented as counts and percentages [n (%)]. The κ consistency test was employed to assess the agreement among different methods. A two-sided P value of less than 0.05 was considered statistically significant.

Results

IHC detection of HER2

A total of 104 patient specimens were included in this study. According to the 2018 ASCO/CAP HER2 detection guidelines, the IHC results were interpreted as follows: IHC 0/1 (n=43, 41.3%), IHC 2+ (n=35, 33.7%), and IHC 3+ (n=26, 25.0%) (Figure 2).

Figure 2 HER2 results detected by the IHC method. (A) Hematoxylin-eosin staining of primary invasive ductal breast cancer at 400× magnification; (B) IHC at 400×. The brown staining indicates positive HER2 protein expression, with approximately 20–30% of the cell membrane exhibiting weak positive and incomplete staining, corresponding to IHC 1+. Alternatively, if less than 30% of the cell membrane shows negative or non-staining, this is categorized as IHC 0; (C) IHC at 200×. The brown staining indicates positive HER2 protein expression, with about 30% of the cell membrane demonstrating weak to moderate intensity and incomplete staining, corresponding to IHC 2+. (D) IHC at 200×. The brown staining indicates positive HER2 protein expression, with more than 80% of the cell membrane exhibiting strong positive and complete staining, corresponding to IHC 3+. HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry.

RNAscope and FISH detection of HER2 (IHC interpreted as IHC 2+)

A total of 35 cases with an IHC score of 2+ for IDC were collected, all of which were female with a mean age of 61.91±5.41 years. In the FISH analysis, 23 cases (65.7%, 23/35) were negative, while 12 cases (34.3%, 12/35) were positive. In the RNAscope analysis, 18 cases (51.43%, 18/35) were negative, and 17 cases (48.57%, 17/35) were positive (Table 1).

Consistency analysis of RNAscope and FISH detection of HER2 (IHC interpreted as IHC 2+)

The overall consistency rate between RNAscope and FISH detection of HER2, where IHC was interpreted as IHC 2+, was found to be 85.7% [30 out of 35 cases, kappa =0.672, 95% confidence interval (CI): 0.445–0.899] (see Table 2). The detailed analysis is as follows: (I) 12 cases (34.3%, 12/35; numbered 3, 7, 11, 16, 18, 21, 22, 23, 26, 27, 31, 32) were positive in FISH detection, and RNAscope detection also yielded positive results, achieving a consistency rate of 100%; (II) among the 23 cases that tested negative by FISH (65.7%, 23/35), RNAscope detected negative results in 18 cases (78.26%, 18/23; numbered 2, 4, 5, 6, 8, 9, 10, 12, 13, 15, 17, 19, 20, 25, 28, 30, 34, 35), which aligned with the FISH results. Conversely, 5 cases (21.74%, 5/23; numbered 1, 14, 24, 29, 33) that tested positive with RNAscope were inconsistent with the FISH test results.

Using FISH as the reference standard, additional diagnostic performance metrics were calculated for RNAscope in the IHC 2+ cohort (n=35). RNAscope demonstrated a sensitivity of 100% (12/12), a specificity of 78.3% (18/23), a positive predictive value (PPV) of 70.6% (12/17), and a negative predictive value (NPV) of 100% (18/18).

NGS testing

There were five cases (numbers: 1, 14, 24, 29, 33) with inconsistent results between RNAscope and FISH detection of HER2, where IHC was interpreted as IHC 2+. NGS was performed to assess their gene expression (Figure 3). Among these, one case (20%, 1/5, number: 14) was negative and consistent with FISH, while four cases (80%, 4/5, numbers: 1, 24, 29, 33) showed consistency with RNAscope, all of which were positive.

Figure 3 NGS was performed to assess gene expression in cases where the detection of HER2 with the methods of RNAscope and FISH (interpreted as IHC 2+ by IHC) were inconsistent. (A) IHC, 400×: ~30% of tumor cell membranes show weak-to-moderate incomplete brown staining, interpreted as IHC 2+ (equivocal). (B) FISH, 1,000×: HER2 (red) and CEP17 (green) signals yield a HER2/CEP17 ratio of 1.81 and average HER2 copy number between 4.0 and 6.0, interpreted as FISH-negative. (C) RNAscope, 200×: sparse or absent brown punctate signals in tumor cells, interpreted as RNAscope-negative. (D) RNAscope, 400× (different case): abundant brown punctate signals in tumor cells, interpreted as RNAscope-positive. Panels C and D represent RNAscope-negative and RNAscope-positive staining patterns, respectively. CEP17, Chromosome 17 Centromere Probe; FISH, fluorescence in situ hybridization; HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; NGS, next-generation sequencing.

Discussion

According to the ASCO/CAP 2018 and Chinese 2019 HER2 testing guidelines (17,19), accurate assessment of HER2 status in BC (including IDC) is essential for selecting targeted therapies and predicting prognosis (9,20). Both guidelines recommend initial HER2 evaluation by IHC, with further confirmation by FISH in equivocal cases (IHC 2+). FISH, which typically uses dual-probe systems including HER2 and CEP17, is considered more objective and less affected by sample pre-processing. It has thus become a widely adopted method to resolve ambiguous HER2 results (3). However, the classification of certain IHC 2+ cases remains controversial (3,4).

RNAscope has been tested and validated across various diseases (21-25). The detection results obtained through RNAscope are consistent and reproducible, and it can identify noncoding RNA21 that IHC cannot detect. Given these characteristics, RNAscope has been proposed as a potential complementary method rather than a replacement for established HER2 testing modalities. Thus, this study aims to explore the consistency of RNAscope and FISH results in detecting IDC, and to investigate whether RNAscope can serve as an auxiliary tool for determining HER2 status in IDC cases classified as IHC 2+, thereby providing a new perspective for clinical practice.

In this study, IHC was initially employed to assess HER2 status. A total of 35 IHC 2+ samples were randomly selected and numbered using a random number method. RNAscope and FISH were utilized for detection. The consistency rate between the two detection methods was found to be 85.7% (30/35, κ=0.678, 95% CI: 0.425–0.872), indicating a strong agreement between RNAscope and FISH. The ASCO/CAP clinical practice guidelines were applied to interpret the results. Five samples exhibited inconsistent results between RNAscope and FISH detection of HER2 (with IHC interpreted as 2+), and these were subsequently subjected to NGS. Among these, one case (20%, 1/5, sample number: 14) was negative and consistent with FISH results, while four cases (80%, 4/5, sample numbers: 1, 24, 29, 33) were consistent with RNAscope, all of which tested positive. These findings suggest that RNAscope may potentially identify a subset of HER2-positive cases among FISH-negative samples. This observation is supported by recent studies. For example, a 2024 study using RNAscope in 526 BC cases demonstrated a statistically significant correlation between HER2 mRNA levels and protein expression by IHC (P<0.0001), and showed that responders to trastuzumab deruxtecan (T-Dxd) had significantly higher HER2 mRNA levels than non-responders (6.4±8.2 vs. 2.6±2.2 dots/cell, P=0.030) (26). Another study found that among 88 HER2 non-amplified BCs, approximately 58.8% of tumors classified as IHC 0/ultralow were reclassified as HER2-low by mRNA analysis, indicating that RNA-based assays may detect HER2 expression not captured by conventional IHC alone (27). However, given that NGS validation was performed in only five discordant cases, no definitive conclusions regarding the comparative sensitivity of RNAscope and FISH can be drawn. Accordingly, these results should be interpreted with caution due to the limited statistical power.

Importantly, according to the 2018 ASCO/CAP guidelines, tumors classified as IHC 2+ and FISH-negative are considered HER2-negative for clinical decision-making. In this context, RNAscope-positive/FISH-negative cases identified in the present study are not intended to alter HER2 status classification under current guidelines. Rather, RNAscope findings should be interpreted as supplementary and exploratory information that may provide additional biological insight in equivocal cases.

In conclusion, this exploratory study demonstrates a high level of agreement between RNAscope and FISH in IHC 2+ cases. RNAscope may serve as a complementary diagnostic approach, particularly in cases with ambiguous HER2 status. However, this study acknowledges several limitations. Although the IDC samples were initially diagnosed via IHC, they were subsequently assigned random numbers for retesting with FISH and RNAscope, in accordance with the latest ASCO/CAP clinical practice guidelines issued in 2018 (3). This randomization was intended to minimize potential biases arising from interpretation criteria, sampling variability, tumor heterogeneity, and other influencing factors. Nonetheless, the inherent limitations of retrospective research data remain. Additionally, the small sample size poses a risk to the statistical validity of the findings. Furthermore, RNAscope detection is subject to technical limitations; it necessitates high-quality samples and requires researchers to possess advanced skills in sample processing and result analysis, which could potentially introduce clinical risks associated with testing methods.

Conclusions

RNAscope demonstrates substantial agreement with FISH in IHC 2+ IDC and may serve as a complementary, hypothesis-generating adjunct for HER2 assessment in ambiguous cases, pending further validation in larger, prospective studies.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2486/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2486/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2486/prf

Funding: This work was supported by the Natural Science Foundation of Heilongjiang Province for Distinguished Young Scholars of China (No. JC2018022), the Scientific Research Innovation Fund of The First Affiliated Hospital of Harbin Medical University (No. 2023M09), and the Fundamental Research Funds for the Provincial Universities in Heilongjiang Province (No. 2024-KYYWF-0154).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2486/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Ethics Committee of The First Affiliated Hospital of Harbin Medical University (No. 202128). All participants were informed about the study protocol and provided written informed consent to participate.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Trimboli RM, Giorgi Rossi P, Battisti NML, et al. Do we still need breast cancer screening in the era of targeted therapies and precision medicine? Insights Imaging 2020;11:105. [Crossref] [PubMed]
2. Senkus E, Kyriakides S, Ohno S, et al. Primary breast cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2015;26:v8-30. [Crossref] [PubMed]
3. Cardoso F, Costa A, Senkus E, et al. 3rd ESO-ESMO International Consensus Guidelines for Advanced Breast Cancer (ABC 3). Ann Oncol 2017;28:16-33. [Crossref] [PubMed]
4. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines). Breast Cancer. Version 2.2022. Fort Washington: National Comprehensive Cancer Network; 2022.
5. Mitri Z, Constantine T, O'Regan R. The HER2 Receptor in Breast Cancer: Pathophysiology, Clinical Use, and New Advances in Therapy. Chemother Res Pract 2012;2012:743193. [Crossref] [PubMed]
6. Stocker A, Trojan A, Elfgen C, et al. Differential prognostic value of positive HER2 status determined by immunohistochemistry or fluorescence in situ hybridization in breast cancer. Breast Cancer Res Treat 2020;183:311-9. [Crossref] [PubMed]
7. Waks AG, Winer EP. Breast Cancer Treatment: A Review. JAMA 2019;321:288-300. [Crossref] [PubMed]
8. Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 2021;71:209-49. [Crossref] [PubMed]
9. Wang WJ, Lei YY, Mei JH, et al. Recent progress in HER2 associated breast cancer. Asian Pac J Cancer Prev 2015;16:2591-600. [Crossref] [PubMed]
10. Sun Y, Chen C, Zhang X, et al. High Neutrophil-to-Lymphocyte Ratio Is an Early Predictor of Bronchopulmonary Dysplasia. Front Pediatr 2019;7:464. [Crossref] [PubMed]
11. Grimm EE, Schmidt RA, Swanson PE, et al. Achieving 95% cross-methodological concordance in HER2 testing: causes and implications of discordant cases. Am J Clin Pathol 2010;134:284-92. [Crossref] [PubMed]
12. Bogdanovska-Todorovska M, Petrushevska G, Janevska V, et al. Standardization and optimization of fluorescence in situ hybridization (FISH) for HER-2 assessment in breast cancer: A single center experience. Bosn J Basic Med Sci 2018;18:132-40. [Crossref] [PubMed]
13. Anderson CM, Zhang B, Miller M, et al. Fully Automated RNAscope In Situ Hybridization Assays for Formalin-Fixed Paraffin-Embedded Cells and Tissues. J Cell Biochem 2016;117:2201-8. [Crossref] [PubMed]
14. Wang Z, Portier BP, Gruver AM, et al. Automated quantitative RNA in situ hybridization for resolution of equivocal and heterogeneous ERBB2 (HER2) status in invasive breast carcinoma. J Mol Diagn 2013;15:210-9. [Crossref] [PubMed]
15. Bernet L, Martinez Benaclocha M, Castera C, et al. mRNA in situ hybridization (HistoSonda): a new diagnostic tool for HER2-status in breast cancer-a multicentric Spanish study. Diagn Mol Pathol 2012;21:84-92. [Crossref] [PubMed]
16. Wolff AC, Hammond MEH, Allison KH, et al. Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Focused Update. J Clin Oncol 2018;36:2105-22. [Crossref] [PubMed]
17. Coleman MP, Quaresma M, Berrino F, et al. Cancer survival in five continents: a worldwide population-based study (CONCORD). Lancet Oncol 2008;9:730-56. [Crossref] [PubMed]
18. Recommended by Breast Cancer Expert Panel. Guideline for HER2 detection in breast cancer, the 2019 version. Zhonghua Bing Li Xue Za Zhi 2019;48:169-75. [Crossref] [PubMed]
19. Wang H, Wang MX, Su N, et al. RNAscope for in situ detection of transcriptionally active human papillomavirus in head and neck squamous cell carcinoma. J Vis Exp 2014;51426. [Crossref] [PubMed]
20. Bingham V, Ong CW, James J, et al. PTEN mRNA detection by chromogenic, RNA in situ technologies: a reliable alternative to PTEN immunohistochemistry. Hum Pathol 2016;47:95-103. [Crossref] [PubMed]
21. Bingham V, McIlreavey L, Greene C, et al. RNAscope in situ hybridization confirms mRNA integrity in formalin-fixed, paraffin-embedded cancer tissue samples. Oncotarget 2017;8:93392-403. [Crossref] [PubMed]
22. Vassilakopoulou M, Togun T, Dafni U, et al. In situ quantitative measurement of HER2mRNA predicts benefit from trastuzumab-containing chemotherapy in a cohort of metastatic breast cancer patients. PLoS One 2014;9:e99131. [Crossref] [PubMed]
23. Tong Y, Chen X, Fei X, et al. Can breast cancer patients with HER2 dual-equivocal tumours be managed as HER2-negative disease? Eur J Cancer 2018;89:9-18. [Crossref] [PubMed]
24. Yu H, Batenchuk C, Badzio A, et al. PD-L1 Expression by Two Complementary Diagnostic Assays and mRNA In Situ Hybridization in Small Cell Lung Cancer. J Thorac Oncol 2017;12:110-20. [Crossref] [PubMed]
25. Sheffield BS, Fulton R, Kalloger SE, et al. Investigation of PD-L1 Biomarker Testing Methods for PD-1 Axis Inhibition in Non-squamous Non-small Cell Lung Cancer. J Histochem Cytochem 2016;64:587-600. [Crossref] [PubMed]
26. Li X, Lee JH, Gao Y, et al. Correlation of HER2 Protein Level With mRNA Level Quantified by RNAscope in Breast Cancer. Mod Pathol 2024;37:100408. [Crossref] [PubMed]
27. Baez-Navarro X, van Bockstal MR, van der Made A, et al. A Comparison Between Immunohistochemistry and mRNA Expression to Identify Human Epidermal Growth Factor Receptor 2-Low Breast Cancer. Arch Pathol Lab Med 2025;149:635-42. [Crossref] [PubMed]