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. 2024 Mar;9(3):102903.
doi: 10.1016/j.esmoop.2024.102903. Epub 2024 Mar 6.

Analytical validation of HER2DX genomic test for early-stage HER2-positive breast cancer

M Marín-Aguilera  1 , P Jares  2 , E Sanfeliu  3 , G Villacampa  4 , E Hernández-Lllán  5 , A I Martínez-Puchol  5 , S Shankar  6 , B González-Farré  3 , A G Waks  6 , F Brasó-Maristany  7 , F Pardo  8 , D K Manning  6 , J A Abery  9 , J Curaba  9 , L Moon  9 , O Gordon  9 , P Galván  7 , P Wachirakantapong  6 , O Castillo  8 , C M Nee  6 , P Blasco  8 , T H Senevirathne  6 , V Sirenko  8 , O Martínez-Sáez  10 , A Aguirre  8 , I E Krop  11 , Z Li  12 , P Spellman  13 , O Metzger Filho  14 , K Polyak  12 , P Michaels  6 , J A Puig-Butillé  5 , A Vivancos  15 , J Matito  16 , W Buckingham  1 , C M Perou  17 , P Villagrasa-González  1 , A Prat  18 , J S Parker  17 , L Paré  19
Affiliations

Analytical validation of HER2DX genomic test for early-stage HER2-positive breast cancer

M Marín-Aguilera et al. ESMO Open. 2024 Mar.

Abstract

Background: HER2DX, a multianalyte genomic test, has been clinically validated to predict breast cancer recurrence risk (relapse risk score), the probability of achieving pathological complete response post-neoadjuvant therapy (pCR likelihood score), and individual ERBB2 messenger RNA (mRNA) expression levels in patients with early-stage human epidermal growth factor receptor 2 (HER2)-positive breast cancer. This study delves into the comprehensive analysis of HER2DX's analytical performance.

Materials and methods: Precision and reproducibility of HER2DX risk, pCR, and ERBB2 mRNA scores were assessed within and between laboratories using formalin-fixed paraffin-embedded (FFPE) tumor tissues and purified RNA. Robustness was appraised by analyzing the impact of tumor cell content and protocol variations including different instruments, reagent lots, and different RNA extraction kits. Variability was evaluated across intratumor biopsies and genomic platforms [RNA sequencing (RNAseq) versus nCounter], and according to protocol variations.

Results: Precision analysis of 10 FFPE tumor samples yielded a maximal standard error of 0.94 across HER2DX scores (1-99 scale). High reproducibility of HER2DX scores across 29 FFPE tumors and 20 RNAs between laboratories was evident (correlation coefficients >0.98). The probability of identifying score differences >5 units was ≤5.2%. No significant variability emerged based on platform instruments, reagent lots, RNA extraction kits, or TagSet thaw/freeze cycles. Moreover, HER2DX displayed robustness at low tumor cell content (10%). Intratumor variability across 212 biopsies (106 tumors) was <4.0%. Concordance between HER2DX scores from 30 RNAs on RNAseq and nCounter platforms exceeded 90.0% (Cohen's κ coefficients >0.80).

Conclusions: The HER2DX assay is highly reproducible and robust for the quantification of recurrence risk, pCR likelihood, and ERBB2 mRNA expression in early-stage HER2-positive breast cancer.

Keywords: HER2DX; RNA; analytical validation; breast cancer; gene expression.

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Conflict of interest statement

Disclosure MMA is an employee at Reveal Genomics; GV has received a speaker’s fee from MSD, Pfizer, GSK, and Pierre Fabrer, has held an advisory role with AstraZeneca, and received consultant fees from Reveal Genomics; FBM has a HER2DX patent application EP21383165; PG is an employee at Reveal Genomics; OMS has declared travel expenses and consulting fees from Roche and Reveal, and speaker fees from Eisai, Daiichi, and Novartis; KP serves on the Scientific Advisory Board of Novartis, Ideaya Biosciences, and Scorpion Therapeutics, holds equity options in Scorpion Therapeutics and Ideaya Biosciences, and receives sponsored research funding from Novartis through DFCI; and has patents on S100A7 antibody and BET inhibitor resistance held by DFCI; JM is an employee at Reveal Genomics; WB is an employee at Reveal Genomics; CMP is an equity stockholder and consultant of BioClassifier LLC, and for Reveal Genomics, is also listed as an inventor on patent applications for the Breast PAM50 assay; PVG is one of the stockholders of Reveal Genomics; AP reports advisory and consulting fees from Roche, Pfizer, Novartis, Amgen, BMS, Puma, Oncolytics Biotech, MSD, Guardant Health, Peptomyc, and Lilly, lecture fees from Roche, Pfizer, Novartis, Amgen, BMS, and Daiichi Sankyo, institutional financial interests from Novartis, Roche, and Pfizer, stockholder and consultant of Reveal Genomics, SL; AP is also listed as an inventor on patent applications for the HER2DX assay; JSP is an equity stockholder and consultant for Reveal Genomics and is also listed as an inventor on patent applications for the Breast PAM50 assay; LP is listed as an inventor on HER2DX patent PCT/EP2021/070788 and is an employee at Reveal Genomics. All other authors have declared no conflicts of interest.

Figures

Figure 1
Figure 1
Precision of HER2DX intra-laboratory. (A) Schematic outline of experimental design; (B) scatter plot revealing both intra- and inter-run variability across the three distinct HER2DX scores (i.e. relapse risk, pCR likelihood, and ERBB2 mRNA); (C) correlation matrix comparing runs from arm 1 and 2 with respect to the normalized gene expression of the 27 genes analyzed in the test. Correlation coefficients are shown. Ctl, control; mRNA, messenger RNA; pCR, pathological complete response.
Figure 2
Figure 2
Testing the accuracy and reproducibility of HER2DX between laboratories from FFPE. (A) Overview of the experiment design when initiating from FFPE blocks; (B) correlation plots illustrating the concordance among the three scores (i.e. relapse risk, pCR likelihood, and ERBB2 mRNA) obtained in two distinct laboratories, both starting from FFPE blocks; (C) Bland–Altman plots including Pearson correlation coefficients offering a comprehensive analysis of the discrepancies between the three HER2DX scores obtained in both laboratories starting from FFPE blocks; (D) correlation plot between the three scores in the simulation data with 1 × 106 estimated scores. Ctl, control; FFPE, formalin-fixed paraffin-embedded; mRNA, messenger RNA; pCR, pathological complete response.
Figure 3
Figure 3
Testing the accuracy and reproducibility of HER2DX between laboratories from RNA. (A) Overview of the experiment design when initiating from RNAs; (B) correlation plots illustrating the concordance among the three scores (i.e. relapse risk, pCR likelihood, and ERBB2 mRNA) obtained in two distinct laboratories, both starting from FFPE blocks from RNAs; (C) correlation plot between the three scores in the simulation data with 1×106 estimated scores. Cor., correlation; Ctl, control; FFPE, formalin-fixed paraffin-embedded; mRNA, messenger RNA; pCR, pathological complete response.
Figure 4
Figure 4
Robustness of HER2DX: evaluation of non-tumor component and different starting RNA quantities. (A) Overview of the spiking experiment designed; (B) boxplot representing the percentiles for the three HER2DX scores (i.e. relapse risk score, pCR likelihood score, and ERBB2 score) after diluting tumor-derived RNA in stroma-derived RNA; (C) overview of the experiment design to evaluate the effect of starting from different RNA quantities; (D) boxplot representing the percentiles for the three HER2DX scores (relapse risk score, pCR likelihood score, and ERBB2 score) after starting from different quantities of RNA. Ctl, control; FFPE, formalin-fixed paraffin-embedded; mRNA, messenger RNA; pCR, pathological complete response.
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