The immunohistochemical (IHC) or fluorescence/chromogenic in situ hybridization (FISH/CISH) assays for assessing HER2 are now recommended by the American Society of Clinical Oncologists and the College of American Pathologists, but there are an increasing number of published studies describing alternative diagnoses at the molecular level. Inspired by these studies, we established a laboratory-developed test (LDT) to analyze HER2 status not only at the gene expression level but also at the gene copy number. A precise copy number calculation was fulfilled including the Control Genomic DNA of known concentration, which allowed subsequent assay validation at the DNA level. The results were reported according to the concordant results of the DNA and RNA approaches. By comparing with IHC determination, completely identical results were found in ten blank samples, which underlines the legitimacy of molecular biological approaches in this diagnostic field. An equivocal sample that was positive by IHC and qPCR was found to be negative by the FISH and so it may change the choice of personalized medicine. The topic of this short communication will hopefully contribute to allowing IVD-certified diagnostics based on the HER2 gene expression profile or copy number to be tested in the Czech Republic as well.

3rd Faculty of Medicine Charles University 100 00 Prague Czech Republic

Department of Anatomical Pathology University Hospital Bulovka 180 81 Prague Czech Republic

Laboratory of Clinical Genetics University Hospital Bulovka 180 81 Prague Czech Republic

The Central Laboratories University Hospital Bulovka 180 81 Prague Czech Republic

Zobrazit více v PubMed

Gheni N., Westenberg D. Quantitative Real-Time PCR Assay with Immunohistochemical Evaluation of HER2/Neu Oncogene in Breast Cancer Patients and Its Correlation with Clinicopathological Findings. Indian J. Pathol. Microbiol. 2020;63:123–128. doi: 10.4103/IJPM.IJPM_136_19. PubMed DOI

Pinto A., Matos J., Pereira T., Silva G., André S. S-phase Fraction, Lymph Node Status and Disease Staging as the Main Prognostic Factors to Differentiate between Young and Older Patients with Invasive Breast Carcinoma. Oncol. Lett. 2022;24:329. doi: 10.3892/ol.2022.13449. PubMed DOI PMC

Jones A. Combining Trastuzumab (Herceptin®) with Hormonal Therapy in Breast Cancer: What Can Be Expected and Why? Ann. Oncol. 2003;14:1697–1704. doi: 10.1093/annonc/mdg483. PubMed DOI

Shou J., Massarweh S., Osborne C.K., Wakeling A.E., Ali S., Weiss H., Schiff R. Mechanisms of Tamoxifen Resistance: Increased Estrogen Receptor-HER2/Neu Cross-Talk in ER/HER2-Positive Breast Cancer. J. Natl. Cancer Inst. 2004;96:926–935. doi: 10.1093/jnci/djh166. PubMed DOI

Olsson H., Jansson A., Holmlund B., Gunnarsson C. Methods for Evaluating HER2 Status in Breast Cancer: Comparison of IHC, FISH, and Real-Time PCR Analysis of Formalin-Fixed Paraffin-Embedded Tissue. Pathol. Lab. Med. Int. 2013;2013:31–37. doi: 10.2147/PLMI.S44976. DOI

Piccart-Gebhart M.J., Procter M., Leyland-Jones B., Goldhirsch A., Untch M., Smith I., Gianni L., Baselga J., Bell R., Jackisch C., et al. Trastuzumab after Adjuvant Chemotherapy in HER2-Positive Breast Cancer. N. Engl. J. Med. 2005;353:1659–1672. doi: 10.1056/NEJMoa052306. PubMed DOI

Sawaki M., Taira N., Uemura Y., Saito T., Baba S., Kobayashi K., Kawashima H., Tsuneizumi M., Sagawa N., Bando H., et al. Randomized Controlled Trial of Trastuzumab With or Without Chemotherapy for HER2-Positive Early Breast Cancer in Older Patients. J. Clin. Oncol. 2020;38:3743–3752. doi: 10.1200/JCO.20.00184. PubMed DOI

Sauter G., Lee J., Bartlett J.M.S., Slamon D.J., Press M.F. Guidelines for Human Epidermal Growth Factor Receptor 2 Testing: Biologic and Methodologic Considerations. J. Clin. Oncol. 2009;27:1323–1333. doi: 10.1200/JCO.2007.14.8197. PubMed DOI

Zoppoli G., Garuti A., Cirmena G., Di Cantogno L.V., Botta C., Gallo M., Ferraioli D., Carminati E., Baccini P., Curto M., et al. Her2 Assessment Using Quantitative Reverse Transcriptase Polymerase Chain Reaction Reliably Identifies Her2 Overexpression without Amplification in Breast Cancer Cases. J. Transl. Med. 2017;15:91. doi: 10.1186/s12967-017-1195-7. PubMed DOI PMC

Wang Z., Portier B.P., Gruver A.M., Bui S., Wang H., Su N., Vo H.-T., Ma X.-J., Luo Y., Budd G.T., 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;15:210–219. doi: 10.1016/j.jmoldx.2012.10.003. PubMed DOI

Nakanishi Y., Mizutani G., Sano M., Oinuma T., Nemoto N. Comparison of HER2 mRNA Amplification with Immunohistochemistry in Human Breast Cancer Using Laser Assisted Microdissection Technique. Acta Histochem. Cytochem. 2004;37:73–79. doi: 10.1267/ahc.37.73. DOI

Richard F., De Schepper M., Geukens T., Vos H., Bassez A., Nguyen H.L., Maetens M., Wang L., Nevelsteen I., Wildiers H., et al. 14P Comparison of Single-Cell ERBB2 mRNA Expression Levels with HER2 Status by Immunohistochemistry Reveals Heterogeneity of the HER2-Low Status. ESMO Open. 2023;8:101238. doi: 10.1016/j.esmoop.2023.101238. DOI

Lyng M.B., Lænkholm A.-V., Pallisgaard N., Ditzel H.J. Identification of Genes for Normalization of Real-Time RT-PCR Data in Breast Carcinomas. BMC Cancer. 2008;8:20. doi: 10.1186/1471-2407-8-20. PubMed DOI PMC

Ross J.S., Fakih M., Ali S.M., Elvin J.A., Schrock A.B., Suh J., Vergilio J.-A., Ramkissoon S., Severson E., Daniel S., et al. Targeting HER2 in Colorectal Cancer: The Landscape of Amplification and Short Variant Mutations in ERBB2 and ERBB3. Cancer. 2018;124:1358–1373. doi: 10.1002/cncr.31125. PubMed DOI PMC

Noske A., Loibl S., Darb-Esfahani S., Roller M., Kronenwett R., Müller B.M., Steffen J., von Toerne C., Wirtz R., Baumann I., et al. Comparison of Different Approaches for Assessment of HER2 Expression on Protein and mRNA Level: Prediction of Chemotherapy Response in the Neoadjuvant GeparTrio Trial (NCT00544765) Breast Cancer Res. Treat. 2011;126:109–117. doi: 10.1007/s10549-010-1316-y. PubMed DOI

Borges P.C.C., Spencer H.B., Barbosa C., Costa V., Furtado A., Leal M.C., Lopes C., Ferreira D., Carvalho A.L., Dos-Santos-Silva I., et al. XPERT® Breast Cancer STRAT4 as an Alternative Method of Identifying Breast Cancer Phenotype in Cape Verde (Preliminary Results) Ecancermedicalscience. 2023;17:1530. doi: 10.3332/ecancer.2023.1530. PubMed DOI PMC

Koudelakova V., Berkovcova J., Trojanec R., Vrbkova J., Radova L., Ehrmann J., Kolar Z., Melichar B., Hajduch M. Evaluation of HER2 Gene Status in Breast Cancer Samples with Indeterminate Fluorescence in Situ Hybridization by Quantitative Real-Time PCR. J. Mol. Diagn. 2015;17:446–455. doi: 10.1016/j.jmoldx.2015.03.007. PubMed DOI

Skálová H., Dundr P., Povýšil C., Velenská Z., Petruželka L., Tvrdík D. Study of the Effect of Neoadjuvant Chemotherapy on the Status of Her2/Neu. Folia Biol. 2011;57:191–199. doi: 10.14712/fb2011057050191. PubMed DOI

Bièche I., Onody P., Laurendeau I., Olivi M., Vidaud D., Lidereau R., Vidaud M. Real-Time Reverse Transcription-PCR Assay for Future Management of ERBB2-Based Clinical Applications. Clin. Chem. 1999;45:1148–1156. doi: 10.1093/clinchem/45.8.1148. PubMed DOI

Gjerdrum L.M., Sorensen B.S., Kjeldsen E., Sorensen F.B., Nexo E., Hamilton-Dutoit S. Real-Time Quantitative PCR of Microdissected Paraffin-Embedded Breast Carcinoma: An Alternative Method for HER-2/Neu Analysis. J. Mol. Diagn. 2004;6:42–51. doi: 10.1016/S1525-1578(10)60490-4. PubMed DOI PMC

Gaudio M., Jacobs F., Benvenuti C., Saltalamacchia G., De Sanctis R., Santoro A., Zambelli A. 53P HER2 Low by Immunohistochemistry (IHC) and Gene Expression by qRT-PCR Using OncotypeDX in ER+ Early Breast Cancer. ESMO Open. 2023;8:101277. doi: 10.1016/j.esmoop.2023.101277. DOI

Do H., Dobrovic A. Sequence Artifacts in DNA from Formalin-Fixed Tissues: Causes and Strategies for Minimization. Clin. Chem. 2015;61:64–71. doi: 10.1373/clinchem.2014.223040. PubMed DOI

Qi P., Bai Q.-M., Yao Q.-L., Yang W.-T., Zhou X.-Y. Performance of Automated Dissection on Formalin-Fixed Paraffin-Embedded Tissue Sections for the 21-Gene Recurrence Score Assay. Technol. Cancer Res. Treat. 2020;19:1533033820960760. doi: 10.1177/1533033820960760. PubMed DOI PMC

First Strand cDNA Synthesis (Standard Protocol) [(accessed on 15 November 2024)]. Available online: https://www.neb.com/en/products/e6560-protoscript-ii-first-strand-cdna-synthesis-kit.

Gorden E.M., Sturk-Andreaggi K., Marshall C. Repair of DNA Damage Caused by Cytosine Deamination in Mitochondrial DNA of Forensic Case Samples. Forensic. Sci. Int. Genet. 2018;34:257–264. doi: 10.1016/j.fsigen.2018.02.015. PubMed DOI

Tomlins S.A., Hovelson D.H., Suga J.M., Anderson D.M., Koh H.A., Dees E.C., McNulty B., Burkard M.E., Guarino M., Khatri J., et al. Real-World Performance of a Comprehensive Genomic Profiling Test Optimized for Small Tumor Samples. JCO Precis. Oncol. 2021;5:1312–1324. doi: 10.1200/PO.20.00472. PubMed DOI PMC

Schlemmer B.O., Sorensen B.S., Overgaard J., Olsen K.E., Gjerdrum L.M., Nexo E. Quantitative PCR--New Diagnostic Tool for Quantifying Specific mRNA and DNA Molecules: HER2/Neu DNA Quantification with LightCycler Real-Time PCR in Comparison with Immunohistochemistry and Fluorescence in Situ Hybridization. Scand. J. Clin. Lab. Investig. 2004;64:511–522. doi: 10.1080/00365510410002922. PubMed DOI

Monfort-Lanzas P., Rusu E.C., Parrakova L., Karg C.A., Kernbichler D.-E., Rieder D., Lackner P., Hackl H., Gostner J.M. ExonSurfer: A Web-Tool to Design Primers at Exon–Exon Junctions. [(accessed on 15 September 2024)];BMC Genom. 2024 25:594. doi: 10.1186/s12864-024-10456-2. Available online: https://exonsurfer.i-med.ac.at. PubMed DOI PMC

Luna® Universal qPCR Master Mix Protocol. [(accessed on 15 November 2024)]. Available online: https://www.neb.com/en/protocols/2016/11/08/luna-universal-qpcr-master-mix-protocol-m3003.

Wolff A.C., Hammond M.E.H., Hicks D.G., Dowsett M., McShane L.M., Allison K.H., Allred D.C., Bartlett J.M.S., Bilous M., Fitzgibbons P., et al. Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Update. J. Clin. Oncol. 2013;31:3997–4013. doi: 10.1200/JCO.2013.50.9984. PubMed DOI