# ERBB2 Comprehensive Profiling and Prognostication in Stage III Colon Cancer: Findings From PETACC8 and IDEA-France Cohorts

Camilla Pilati, Audrey Soulabaille, Claire Gallois, Hélène Blons, Anne Cayre, Marine Sroussi, Delphine Le Corre, Sophie Mouillet-Richard, Claire Mulot, Karine Le Malicot, et al.

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## To cite this version:

Camilla Pilati, Audrey Soulabaille, Claire Gallois, Hélène Blons, Anne Cayre, et al., ERBB2 Comprehensive Profiling and Prognostication in Stage III Colon Cancer: Findings From PETACC8 and IDEA-France Cohorts. Gastroenterology, 2024, 10.1053/j.gastro.2024.10.046. hal-04955612

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Submitted on 21 Feb 2025

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# ERBB2 Comprehensive Profiling and Prognostication in Stage III Colon Cancer: Findings From PETACC8 and IDEA-France Cohorts

Camilla Pilati,<fnref n="1" /> Audrey Soulabaille,<fnref n="1" /> Claire Gallois,<fnref n="1" />,² Hélène Blons,<fnref n="1" />,² Anne Cayre,³

Marine Sroussi,<fnref n="1" />,⁴ Delphine Le Corre,<fnref n="1" /> Sophie Mouillet-Richard,<fnref n="1" /> Claire Mulot,<fnref n="1" />

Karine Le Malicot,⁵ Aurélien De Reynies,<fnref n="1" /> Jean-Baptiste Bachet,<fnref n="1" />,⁶,<fnref n="7" /> Christophe Borg,⁸

Frédéric Di Fiore,⁹ Rosine Guimbaud,¹⁰ Jaafar Bennouna,¹¹ Thierry André,¹² Julien Taieb,<fnref n="1" />,²,<fnref n="7" />

Frédérique Penault-Llorca,³ and Pierre Laurent-Puig<fnref n="1" />,²

<figure><img src="image_3.png" /></figure>

**BACKGROUND & AIMS:** ERBB2 pathway activation, through amplification or activating mutations, represents a new target for colon cancer (CC) treatment. Molecular methods were compared with the gold standard for assessing ERBB2 status, and the prognostic value of ERBB2 amplification, mutations, and expression was determined using data from 2 phase 3 trials involving nearly 3000 patients with stage III CC.

**METHODS:** In the PETACC8 trial, immunohistochemistry and fluorescence in situ hybridization, DNA, and RNA analysis were

performed on 1813, 1719, and 1733 samples, respectively. In the IDEA-France trial, DNA and RNA sequencing was performed on 1129 and 1263 samples. The breast cancer SCAN-B cohort (N = 3409) served as an external reference. A new molecular ERBB2-amplified status was defined using ERBB2 next-generation sequencing score, RNA sequencing expression, and clustering based on ERBB2 neighboring gene expression. Concordance between diagnostic techniques and the association between time to recurrence (TTR) and ERBB2-status were

<footnote n="1">¹Centre de Recherche des Cordeliers, Sorbonne Université, Institut National de la Santé et de la Recherche Médicale, Université Paris Cité, Personalized Medicine, Pharmacogenomics and Therapeutic Optimization, Paris, France; ²Institut du Cancer Paris, Cancer Research for Personalized Medicine, Assistance Publique-Hôpitaux de Paris Centre, Hôpital Européen Georges Pompidou, Paris, France; ³Department of Pathology, Centre Jean Perrin, UNICANCER, Institut National de la Santé et de la Recherche Médicale, UMR 1240, Imagerie Moléculaire et Stratégies Théranostiques, University Clermont Auvergne, Clermont-Ferrand, France; ⁴Institut Chimie Biologie Innovation, Laboratoire de BioChimie, École Supérieure de Physique et de Chimie Industriel, UMR 8231, Centre National de la Recherche Scientifique, Paris Sciences et Lettres University, Paris, France; ⁵Fédération Francophone de Cancérologie Digestive, Epidémiologie et Recherche Clinique en Cancérologie Digestive, Institut National de la Santé et de la Recherche Médicale, Lipides Nutrition Cancer, UMR 1231, University of Burgundy and Franche Comté, Dijon, France; ⁶Department of Hepato-Gastroenterology, Groupe Hospitalier Pitié Salpêtrière, Paris, France;</footnote>

<footnote n="7">⁷Association des Gastroentérologues Oncologues, Paris, France; ⁸Department of Medical Oncology, University Hospital of Besançon, Besançon, France; ⁹Hepatogastroenterology Department, Centre Hospitalier Universitaire Rouen, University of Rouen Normandy, Institut National de la Santé et de la Recherche Médicale 1245, Institut de Recherche en Oncologie Group, Normandie University, Rouen, France; ¹⁰Digestive Medical Oncology, Centre Hospitalier Universitaire de Toulouse, Toulouse, France; ¹¹Thoracic Oncology Unit, University Hospital of Nantes and Institut National de la Santé et de la Recherche Médicale, Regional Center for Research in Cancerology and Immunology, Nantes, France; and ¹²Sorbonne Université and Hôpital Saint Antoine, Institut National de la Santé et de la Recherche Médicale 938, Site de Recherche Intégrée sur le Cancer Curamus, Paris, France</footnote>
**RESULTS:** The prevalence of the molecular ERBB2-amplified group was 1.85% in PETACC8 and 1.5% in IDEA-France, with a concordance of 0.81 (95% CI, 0.70-0.92) with the gold standard immunohistochemistry and fluorescence in situ hybridization method in PETACC8. A nonlinear relationship was observed between TTR and ERBB2 expression, with extreme groups showing a less favorable prognosis (*P* < .0001) in both colon and breast cancers. Patients with molecular ERBB2-amplified status or mutations had the poorest prognosis, followed by low-expression and intermediate-expression groups (3-year TTR of 67.0%, 71.2%, and 77.9%, respectively). In multivariate analysis, the low-expression group had a significantly shorter TTR (hazard ratio, 1.28; 95% CI, 1.07-1.52). **CONCLUSIONS:** The molecular definition of ERBB2 status could represent a cost-effective alternative in stage III CC. ERBB2 alterations and low RNA expression significantly reduced TTR, highlighting the complex role of ERBB2.

**Keywords:** HER2 Amplification; ERBB2 Expression; IHC/FISH; RNA-seq; NGS Score; Stage III CC Prognosis.

Colon cancer (CC) is a major public health concern, ranking third in cancer incidence and second in cancer-related deaths.¹ Stage III localized CC, constituting approximately 35% of diagnoses, presents heterogeneous prognoses, with 5-year overall survival rates ranging from 45% to 65%.²

The *ERBB2* gene, also known as HER2, encodes a tyrosine kinase receptor belonging to the epidermal growth factor receptor (EGFR) family. The amplification of *ERBB2* and the subsequent overexpression of the HER2 protein are observed in 15%-30% of breast cancers and 10%-30% of gastric cancers. This overexpression is a poor prognostic marker that is effectively targeted by anti-HER2 therapy.³ ERBB2 amplification occurs in 5% of metastatic colorectal cancer cases with *RAS* wild type and 2%-3% of cases regardless of *RAS* status, yet its prognostic and predictive significance remains uncertain.⁴⁻⁷ Somatic activating mutations in ERBB2, which occasionally occur alongside amplification, lack clear prognostic data, but suggest potential therapeutic relevance.⁴⁻⁶,⁸⁻¹² Results of phase 2 studies testing dual anti-HER2 inhibition in ERBB2-amplified metastatic colorectal cancer showed promise in heavily pretreated patients,¹⁰,¹¹,¹³ emphasizing the need for further research into their predictive and prognostic value, particularly in stage III CC, where adjuvant strategies require improvement.

Currently, the gold standard for assessing *ERBB2* amplification status in CC involves immunohistochemistry (IHC), followed by fluorescence in situ hybridization (FISH) in ambiguous cases. Although widely established and used in the clinical setting, these techniques rely on pathologists' expertise. Meanwhile, "omic" methods, particularly those leveraging next-generation sequencing (NGS), have become increasingly popular for detailed examination of the tumor's molecular profile at both DNA and RNA levels. This study aims to identify surrogate markers for *ERBB2* amplification through RNA and DNA analyses, comparing them with established methods. Using data from the prospective

## WHAT YOU NEED TO KNOW

### BACKGROUND AND CONTEXT

Activation of the ERBB2/HER2 pathway in colon cancer presents a critical opportunity for targeted therapies, necessitating accurate methods for assessing ERBB2 status.

### NEW FINDINGS

This study introduces a novel molecular definition of ERBB2 amplification and highlights the nonlinear relationship between survival and ERBB2 expression in both colon and breast cancers.

### LIMITATIONS

Clinical trials investigating anti-HER2 therapies as neoadjuvant treatments in stage III colon cancer are currently underway, which may limit the immediate applicability of these findings in clinical practice.

### CLINICAL RESEARCH RELEVANCE

Understanding ERBB2 status is crucial for effectively stratifying patients, as those with ERBB2 amplification or low RNA expression tend to have poorer prognoses and exhibit differing responses to anti-epidermal growth factor receptor treatments. This knowledge can guide treatment decisions and inform future clinical trials.

### BASIC RESEARCH RELEVANCE

This study enhances the foundational understanding of ERBB2's role in colorectal cancer biology, providing insights that may lead to innovative therapeutic strategies.

PETACC8 and IDEA-France studies, we analyzed concordance among techniques (IHC, FISH, NGS, and RNA sequencing [RNA-seq]) in assessing ERBB2 genomic amplification and its impact on RNA and protein expressions, ultimately determining its prognostic value in stage III CC.

## Methods

### *Patient Population*

The PETACC8 trial, a phase 3 randomized study, enrolled 2559 patients with stage III CC comparing the FOLFOX4 (5-fluorouracil, leucovorin, and oxaliplatin) regimen with FOLFOX4 plus cetuximab in the adjuvant setting.¹⁴ Among them, 2043 patients consented to participate in translational research. Of these, 1813 (88.7%) had samples for immunohistochemistry and FISH analyses. Tumor DNA and RNA were available for 1719 patients (84.1%) and 1733 patients (84.8%).

IDEA-France, a part of the IDEA international collaboration, is a phase 3 randomized trial involving 2000 patients with stage III

Abbreviations used in this paper: CC, colon cancer; CMS, consensus molecular subtype; EGFR, epidermal growth factor receptor; FFPE, formalin-fixed, paraffin-embedded; FISH, fluorescence in situ hybridization; FOLFOX4, oxaliplatin, leucovorin, and 5-fluorouracil; HR, hazard ratio; IHC, immunohistochemistry; NGS, next-generation sequencing; RNA-seq, RNA sequencing; TTR, time to recurrence.
CC, comparing 3–6 months of oxaliplatin-based adjuvant treatment (modified FOLFOX6 or CAPOX [capecitabine plus oxaliplatin]).¹⁵ Among these patients, 1612 consented to participate in translational research. Tumor DNA and RNA were available for 1129 patients (70.0%) and 1263 patients (78.3%). ERBB2 status was not assessed by IHC and FISH in the IDEA-France population.

The SCAN-B cohort (GSE96058 dataset; N = 3409; Illumina HiSeq 2000) was used as an external cohort.¹⁶

## Molecular Analysis

**ERBB2 immunohistochemistry and fluorescence in situ hybridization status.** ERBB2 IHC and FISH were conducted on tissue microarrays. Tissue microarrays were generated with a 2-mm punch size from macrodissected formalin-fixed, paraffin-embedded (FFPE) blocks from the primary tumor after deparaffinization and rehydration. HER2/ERBB2 IHC staining was performed on fresh sections, cut within 24 hours, using the anti-HER2/neu monoclonal antibody (Clone: 4B5 Ventana predilution; Roche Diagnostics, Rotkreuz, Switzerland) on tissue microarray. ERBB2 FISH analysis used the Zytolight SPEC ERBB2/CEN 17 Dual Color Probe kit. Locus-specific ERBB2 probe (610-kb ZyGreen directly labeled fluorescent DNA probe) and CEP17 probe (ZyOrange, targeting sequences mapping in 17p11.1-q11.1 specific for the alpha satellite centromeric region D17Z1 of chromosome 17) were employed as per manufacturer's recommendations (ZytoVision GmbH, Bremerhaven, Germany). Nonoverlapping nuclei of tumor cells were assessed for HER2 probe and CEP17 probe signals. The FISH result was considered positive with a ratio HER2/CEP17 ≥2 in ≥50% of tumor cells. Classification followed the HERACLES scoring system,¹⁷ categorizing IHC staining into the following 4 groups: 0, 1+ (both "negative"), 2+ ("equivocal"), and 3+ ("positive"). ERBB2 amplification status was determined accordingly: IHC 0 or 1+ as "negative," IHC 2+ FISH negative as "negative," IHC 2+ FISH positive as "positive," and IHC 3+ as "positive."

**3' RNA sequencing.** In both cohorts, tumor RNA was extracted from macrodissected FFPE blocks from primary tumors using the Maxwell RSC RNA FFPE Kit (Promega). PolyA-RNA sequencing (RNA-seq) library preparation protocols were conducted using 400 ng of template RNA and the QuantSeq 3' mRNA-Seq Kit FWD for Illumina (Lexogen GmbH) following manufacturer's instructions. Libraries were sequenced on NovaSeq6000 (Illumina). FASTQ RNA-seq files were mapped using STAR aligner 2.7.9a, and raw read counts were obtained using the "Rsubread" package in R software (R Project for Statistical Computing). Before mapping, the genome index was built on GRCh38.p13 human genome. Samples with more than 10,000 genes detected were kept for further analysis. Batch correction was performed using the Combat_seq function from the "sva" package in R software. Mitochondrial and housekeeping genes were excluded from the raw count matrix. For each sample, the corresponding vector of raw counts was scaled: counts were divided by the total number of counts of the vector and multiplied by the median of the total number of counts across samples. Finally, the scaled data were log2-transformed.

**Multigene DNA sequencing.** Tumor DNA was extracted from macrodissected FFPE blocks from primary tumors. NGS was conducted with a dedicated panel of 92 amplicons (Ion AmpliSeq Colon-Lung Cancer Research Panel version 2; Life Technologies, Carlsbad, CA) covering >500 hotspot mutations in 22 genes, including ERBB2, BRAF, KRAS, NRAS, and TP53.

Multiplex polymerase chain reaction libraries were prepared using 30 ng of DNA whenever possible and 3 µL of DNA for samples with DNA concentration <10 ng/µL by AmpliSeq technology (Ion AmpliSeq library kit, version 2, Ion library equalizer kit; Life Technologies). Clonal amplification and sequencing were done on the Ion Chef System (Ion PI Hi-Q Chef, Ion PI Chip Kit, version 3) and Ion Torrent Proton sequencer (Life Technologies). Data were analyzed by the Torrent Suite, versions 4.4.3 and 5.0.4 (Life Technologies) using optimized parameters: minimal depth of 300× and detection thresholds of 2% and 1% for hotspots. Variant call files from the variant caller were loaded on a galaxy platform and annotated using the Safir2report tool.¹⁸

NGS, IHC, and FISH analyses for this study were performed and interpreted before 2015¹⁹ and RNA-seq analysis were performed in 2019–2023.

## Statistical Analyses

Bioinformatic analyses were performed with R software, version 4.3.1.

ERBB2 NGS score was calculated to estimate ERBB2 copy number according to the following formula: (number of ERBB2 reads – number of ERBB2 reads expected per patient) / number of ERBB2 reads expected per patient. The number of ERBB2 reads expected per patient was determined by the total reads per patient multiplied by the mean ratio of ERBB2 reads to total reads in the series (minus extremes).

The optimal thresholds for "ERBB2 NGS score" and "ERBB2 RNA-seq expression" were determined using Youden's J-index method with the "cutpointr" package in R software. Differential gene expression analysis between IHC/FISH ERBB2-amplified and nonamplified ERBB2 samples was conducted based on RNA-seq data from PETACC-8 with the DESeq2 package. Nonhierarchical clustering with complete linkage method was performed on RNA-seq data from ERBB2 and 4 neighboring genes from PETACC8 and IDEA-France cohorts separately.

Associations between categorical variables were analyzed using Fisher exact test or χ² test as appropriate. Associations between categorical and continuous variables were assessed using Wilcoxon/Mann-Whitney test. Concordance between different diagnostic methods for ERBB2 amplification status was evaluated using Cohen's κ coefficient. Univariate Cox models analyzed time to recurrence (TTR) as a function of continuous ERBB2 RNA-seq expression, with a restricted cubic spline used for modeling. Univariate and multivariate Cox models were employed to analyze associations between clinicobiological variables and TTR. Kaplan-Meier method estimated TTR curves, which were compared using 2-sided log-rank test in the "survival" package in R software. Patients alive and relapse-free were censored at last follow-up and deceased patients without relapse were censored at date of death. Significant variables in univariate analyses (*P* < .05) were included in multivariate analyses. Analyses were conducted with a 2-sided statistical significance level of 5%.

## Data Availability

All clinical and biological variables, as well as the RNA-seq counts used in this article, are available on request for academic researchers in the field of cancer research at the following e-mail addresses: daniel.gonzalez@u-bourgogne.fr and pierre.laurent-puig@parisdescartes.fr.
## Results

### ERBB2 Status in PETACC8 and IDEA-France Cohorts

**ERBB2 status in PETACC8 cohort.** Among PETACC8 patients, ERBB2 protein expression was evaluated using IHC in 1813 patients (88.7%) and the copy number of *ERBB2* gene was determined using FISH in 1691 patients (82.8%). According to the HERACLES criteria, we identified IHC/FISH *ERBB2* amplification in 34 cases (1.9%) and 1779 were considered as ERBB2 nonamplified.

Using Youden's J-Index method, we calculated the optimal thresholds for *ERBB2* NGS score (n = 1719) and *ERBB2* RNA-seq expression (n = 1733) to maximize the sensitivity and specificity for identifying IHC/FISH *ERBB2*-amplified tumors. A high *ERBB2* NGS score (cutoff at 1.47) was observed in 33 patients (1.9%) and high *ERBB2* RNA-seq expression (cutoff at 9.34) was observed in 95 patients (5.5%). Somatic activating mutations of *ERBB2* were detected in 15 patients (0.9%) (Table 1 and Supplementary Table 1). Concordance rates between these diagnostic methods, measured by Cohen's κ coefficient, were 0.78 (95% CI, 0.66-0.90) for IHC/FISH vs NGS score and 0.40 (95% CI, 0.23-0.54) for IHC/FISH vs RNA-seq expression. Interestingly, we observed that high *ERBB2* RNA-seq expression was only mildly specific to amplified tumors, as some nonamplified samples also exhibited increased *ERBB2* RNA-seq expression. This suggests the existence of additional mechanisms besides amplification regulating *ERBB2* transcriptomic activity (Supplementary Figure 1).

**ERBB2 status in IDEA-France cohort.** Applying the same thresholds in the IDEA-France cohort allowed the identification of *ERBB2* NGS high score in 18 patients (1.6%) and *ERBB2* RNA-seq high expression in 55 patients (4.4%). In this cohort, somatic activating mutations of the *ERBB2* gene were found in 17 patients (1.5%) (Table 1).

**Identification of surrogate biomarkers of ERBB2 amplification.** We used RNA sequencing and differential gene expression analysis to compare IHC/FISH *ERBB2*-amplified and nonamplified tumors (Figure 1). As expected, the *ERBB2* gene exhibited the most pronounced increase in expression in IHC/FISH-amplified tumors compared with nonamplified tumors, showing the most significant *P* value. We observed that nearly all of the genes overexpressed in IHC/FISH-amplified tumors, compared with nonamplified tumors, were located on chromosome 17, specifically in the close vicinity of the *ERBB2* gene (17q12). Indeed, *ERBB2* gene amplification rarely occurs in isolation; most of the time, neighboring genes are also amplified, leading to their overexpression. Taking advantage of this co-amplification phenomenon, we investigated the expression of the following 4 *ERBB2* neighboring genes: *STARD3*, *PGAP3*, *MIEN1*, and *GRB7*. To establish a connection between regional amplification and RNA overexpression of the genes close to *ERBB2*, we performed nonhierarchical clustering and identified 2 clusters. In the PETACC8 cohort, cluster A included 28 of the 1733 tumors analyzed (1.6%), which exhibited overexpression of *ERBB2* neighboring genes (including *ERBB2* itself) (Figure 2). Among the 95 samples with high RNA-seq expression, 27 belonged to cluster A (28.4%). In contrast, 68 belonged to cluster B (71.6%), confirming that RNA-seq overexpression is only associated with regional amplification mechanisms in some cases. The concordance between cluster A tumors and the IHC/FISH *ERBB2*-amplified tumors in the PETACC8 cohort, as measured by Cohen's κ coefficient, was 0.74 (95% CI, 0.60-0.88). In the IDEA-France cohort, 15 patients (1.2%) belonged to cluster A. Among the 55 samples with high RNA-seq expression, 15 belonged to cluster A (27.3%) and 40 belonged to cluster B (72.7%). No patient with *ERBB2* NGS low score and *ERBB2* RNA-seq low expression belonged to cluster A (Supplementary Figure 2). To confirm

<table><caption>Table 1. *ERBB2* Status in PETACC8, IDEA-France, and Pooled Population</caption><thead><tr><th>Characteristic</th><th>PETACC8<br>(N = 2043)</th><th>IDEA-France<br>(N = 1612)</th><th>Pooled population<br>(N = 3655)</th></tr></thead><tbody><tr><td><strong>ERBB2 IHC/FISH</strong></td><td></td><td></td><td></td></tr><tr><td>Positive, n (%)</td><td>34 (1.9)</td><td>—</td><td>34 (1.9)</td></tr><tr><td>Negative, n (%)</td><td>1779 (98.1)</td><td>—</td><td>1779 (98.1)</td></tr><tr><td>Undetermined, n</td><td>230</td><td>1612</td><td>1842</td></tr><tr><td><strong>ERBB2 NGS score</strong></td><td></td><td></td><td></td></tr><tr><td>NGS high score, n (%)</td><td>33 (1.9)</td><td>18 (1.6)</td><td>51 (1.8)</td></tr><tr><td>NGS low score, n (%)</td><td>1686 (98.1)</td><td>1111 (98.4)</td><td>2797 (98.2)</td></tr><tr><td>Undetermined, n</td><td>324</td><td>483</td><td>807</td></tr><tr><td><strong>ERBB2 RNA expression</strong></td><td></td><td></td><td></td></tr><tr><td>RNA high expression, n (%)</td><td>95 (5.5)</td><td>55 (4.4)</td><td>150 (5.0)</td></tr><tr><td>RNA low expression, n (%)</td><td>1638 (94.5)</td><td>1208 (95.6)</td><td>2846 (95.0)</td></tr><tr><td>Undetermined, n</td><td>310</td><td>349</td><td>659</td></tr><tr><td><strong>ERBB2 activating mutation</strong></td><td></td><td></td><td></td></tr><tr><td>Mutated, n (%)</td><td>15 (0.9)</td><td>17 (1.5)</td><td>32 (1.1)</td></tr><tr><td>Nonmutated, n (%)</td><td>1681 (99.1)</td><td>1114 (98.5)</td><td>2795 (98.9)</td></tr><tr><td>Undetermined, n</td><td>347</td><td>481</td><td>828</td></tr></tbody></table>
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Volcano plot of IHC/FISH ERBB2 differential gene expression analysis in PETACC8 cohort. Dashed lines correspond to *P* value cutoff of 10⁻⁵ and log₂ fold-change of -1 and 1 on x- and y-axis, respectively. Genes in red are located on chromosome 17.</figcaption></figure>

these results, we performed the same analysis on a cohort of 3409 breast cancers (SCAN-B cohort).¹⁶ The concordance between cluster A tumors and the HER-amplified tumors in the SCAN-B cohort, as measured by Cohen's κ coefficient, was 0.785 (95% CI, 0.75-0.82) (Supplementary Figure 3).

**Comparative analysis between ERBB2 diagnostic methods.** In the PETACC8 cohort, 1463 patients had complete data for all 5 methods (ie, IHC, FISH, RNA *ERBB2*

expression, NGS *ERBB2* score, and clustering) used to characterize ERBB2 status. Of these, 141 tumor samples (9.6%) showed positivity in at least 1 of the techniques: 17 (12.1%) were concordant across all 5 techniques, 6 (4.3%) were concordant across 4 techniques, 4 (2.8%) were concordant across 3 techniques, 9 (6.4%) were concordant across 2 techniques, and 105 (74.5%) were positive with only 1 of the techniques. Among the latter group, 50 showed

<figure><img src="image_5.png" /><figcaption>**Figure 2.** Nonhierarchical clustering according to expression of ERBB2 and 4 neighboring genes. (A) Schematic representation of neighboring genes at the ERBB2 locus in the 17q12 region. (B, C) Heatmaps for PETACC8 (B) and IDEA-France (C) transcriptomic data. In the heatmap, each row represents a gene and each column represents a sample. The color and intensity of the boxes are used to represent changes in gene expression. Red represents up-regulated genes and blue represents down-regulated genes. Above the heatmap, the bars represent the distribution of samples according to ERBB2 mutational status, ERBB2 RNA-seq, ERBB2 NGS score, and ERBB2 status in IHC/FISH (only in PETACC8), respectively. The results of clustering are shown on top of each heatmap.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 3.** Comparative analysis between ERBB2 diagnostic methods in PETACC-8 cohort. (A) Venn diagram comparing the 5 diagnostic methods in PETACC8 (among the 141 patients with no missing data for IHC, FISH, ERBB2 NGS score, ERBB2 RNA-seq expression and clustering, and showing positivity in at least 1 of the techniques). (B) ERBB2 RNA-seq expression as a function of ERBB2 NGS score according to FISH status, IHC score, and cluster membership in the 145 samples from PETACC8, with at least 1 positive test and no missing data for RNA-seq and NGS tests. Samples without a label belong to cluster B. Dashed lines correspond to thresholds for ERBB2 RNA-seq expression and ERBB2 NGS score on x- and y-axis, respectively.</figcaption></figure>

only *ERBB2* RNA-seq high expression, and 40 showed only IHC 2+ staining (Figure 3A). Similar to previous studies,¹⁷ all cases (19 of 19) with IHC 3+ showed amplification by FISH, and only a minority of patients (9 of 52 [17%]) with equivocal IHC (2+) were FISH-positive. Conversely, 17 patients classified as amplified by FISH tested negative on IHC, indicating likely partial amplification of the *ERBB2* gene or a false-positive FISH result.

**Toward a new molecular definition of ERBB2 amplification by the combination of RNA and DNA testing.** Based on the previous results, we defined a new molecular classification of ERBB2 status based on a combination of RNA and DNA testing and refer to it as "molecular ERBB2-amplified" in the following paragraphs. Samples belonging to this new molecular ERBB2-amplified group exhibit either RNA overexpression of *ERBB2* and its neighboring genes (cluster A) or high *ERBB2* NGS score and high RNA-seq expression of *ERBB2* (eg, samples with *ERBB2* focal amplification). In the PETACC8 cohort, the concordance measured by Cohen's κ coefficient between molecular ERBB2 amplification and IHC/FISH test is 0.81 (95% CI, 0.70-0.92). The prevalence of molecular ERBB2-amplified status is 1.85% in the PETACC8 cohort (n = 32 of 1726) and 1.5% in the IDEA cohort (n = 19 of 1253).

Considering the 3 potential mechanisms of ERBB2 pathway activation, that is, the amplification of the *ERBB2* gene or the presence of an activating mutation in the catalytic domain of ERBB2, we combined the molecular ERBB2 status with the ERBB2 mutational status. In both cohorts, 81 patients belonged to the "molecular ERBB2-amplified or ERBB2 mutated" group.

**Clinicobiological characteristics associated with ERBB2 amplification or ERBB2 mutation in PETACC8 cohort.** Tumors with *ERBB2* amplification determined by IHC/FISH were more frequently located in the distal part of the colon (73.5% vs 60.1%; *P* = .014), were more often *TP53*-mutated (83.3% vs 60.4%; *P* = .011), and none of them were deficient mismatch repair (0% vs 10.1%; *P* = .043). A similar trend was observed in molecular ERBB2-amplified tumors, which were more frequently located

distally (78.1% vs 60.0%; *P* = .002), had an even greater prevalence of *TP53* mutations (92.6% vs 59.9%; *P* < .001), and were all proficient mismatch repair (0% vs 10.2%; *P* = .068). In addition, they were less frequently RAS-mutated (22.2% vs 49.0%; *P* = .006) (Supplementary Table 2). Tumors with *ERBB2* somatic activating mutations were more frequently deficient mismatch repair than nonmutated *ERBB2* tumors (35.7% vs 10.3%; *P* = .011). They were more commonly classified in the consensus molecular subtype (CMS) as CMS1 (57.1% vs 20.6%; *P* = .009).²⁰ There was no significant difference between mutated and nonmutated tumors in the remaining tested clinical and molecular variables (Supplementary Table 2). In addition, there was no association between *ERBB2* RNA-seq expression and *ERBB2*-activating mutations (*P* = .36) (Supplementary Figure 4).

## Survival Analysis According to ERBB2 Status in PETACC8 and IDEA-France Pooled Cohorts

Although several studies have been conducted during the last decade, the prognostic role of ERBB2 alterations in CC continues to be debated. To better understand the complex role of ERBB2 in the prognosis of stage III CC, we investigated survival based on *ERBB2* transcriptomic expression level as a continuous variable. Interestingly, *ERBB2* RNA expression and TTR were significantly associated in the univariate Cox model in the PETACC8 cohort (hazard ratio [HR], 0.88; 95% CI, 0.79-0.98; *P* = .021), indicating a protective effect of *ERBB2* expression level on TTR. However, previous reports suggested a pejorative prognostic value of *ERBB2* amplification.⁵ Therefore, we performed a restricted cubic spline regression, revealing a nonlinear relationship between *ERBB2* RNA-seq expression level and survival in PETACC8 and IDEA-France cohorts (Figure 4A). Interestingly, our findings suggested that patients with either low or high *ERBB2* transcript levels in their tumors have a shorter TTR than those with intermediate levels. This pattern was consistent across both cohorts. We observed a very similar trend in the SCAN-B cohort of breast cancers, thus confirming the results identified in our
<figure><img src="image_7.png" /><figcaption>**Figure 4.** Survival analysis according to ERBB2 expression status. (A) Restricted cubic spline curves of the risk of relapse according to ERBB2 RNA-seq expression value in PETACC8 and IDEA-France cohorts. (B) Kaplan-Meier TTR curves according to ERBB2 RNA-seq group expression in PETACC8 and IDEA-France pooled population.</figcaption></figure>

2 CC cohorts and extending our findings to other ERBB2-dependent tumor types (Supplementary Figure 5).

We divided the PETACC8 and IDEA-France pooled population into 3 categories based on ERBB2 RNA-seq expression levels, centered around a pivotal value of 8.51, correlating with the lowest HR for tumor recurrence. These groups were categorized as follows: "low expression" with ERBB2 RNA-seq levels <7.68 (n = 717), "intermediate expression" ranged from 7.68 to 9.34 (n = 2129, reference group), and "high expression" was ≥9.34 (n = 150), which was the previously set threshold. Results of Kaplan-Meier analysis showed a significant difference in TTR among these groups (*P* < .0001). Patients with low ERBB2

expression had the poorest prognosis, with a 3-year TTR of 71.2%, compared with 73.3% and 77.9% for those with high and intermediate ERBB2 expression, respectively (Figure 4B and Supplementary Figure 6). Compared with the intermediate ERBB2 expression group, tumors with low ERBB2 expression were more often located in the proximal colon (45.9% vs 37.4%; *P* < .001), had higher rates of RAS mutations (53.3% vs 48.3%; *P* = .028) and BRAF mutations (15.9% vs 10.1%; *P* < .001), were more frequently classified as pT4 stage (24.3% vs 19.5%; *P* = .007), had higher histopathology grading (19.4% vs 12.5%; *P* < .001), and were more likely to fall into CMS1 and CMS4 categories (20.5% vs 15.8% and 40.9% vs 23.0%, respectively; *P* < .001)
(Supplementary Table 3). Low *ERBB2* expression was prognostic in patients with CMS2 and CMS4 (HR, 1.45; 95% CI, 1.08–1.94; *P* = .013; HR, 1.31; 95% CI, 1.03–1.68; *P* = .028) (Supplementary Figure 7). Among patients with low *ERBB2* expression tumors, those treated with FOLFOX plus cetuximab had shorter TTR than those receiving FOLFOX alone (Supplementary Figure 8A). This observation persisted across *RAS* wild-type or *RAS*-mutated tumors (Supplementary Figure 9). Conversely, in high or intermediate *ERBB2* expression groups, no significant outcome difference was observed between treatment arms (Supplementary Figure 8B). When treated with FOLFOX alone, lower (quartile 1) and higher (quartile 4) *ERBB2* expression quartile groups have similar TTR; however, when treated with FOLFOX + cetuximab, the 2 groups displayed opposite responses (Supplementary Figure 8C).

We found that high *ERBB2* RNA expression without amplification did not have prognostic significance in the overall population (Figure 5A). Conversely, TTR was significantly shorter for patients activating ERBB2 either by harboring molecular ERBB2-amplified status or ERBB2 mutations, compared with the group with intermediate *ERBB2* expression (HR, 1.5; 95% CI, 1.06–2.3; *P* = .025; 3-

year TTR: 67.0% vs 78.0%) (Figure 5B). In the multivariate analysis using a Cox proportional hazard model, the low *ERBB2* expression group was confirmed as a significant independent factor of bad prognosis (HR, 1.28; 95% CI, 1.07–1.52; *P* = .006) (Figure 6). Conversely, the molecular ERBB2-amplified or ERBB2-mutated group was not more significant when adjusting for the other prognostic factors in the multivariate model.

Discussion

Selecting patients with cancer with rare genetic events is difficult and costly and raises the question of a parsimonious use of tissues. Furthermore, the development of new technologies allowing the characterization of numerous genetic alterations, leading to the simultaneous detection of multiple markers, raises the question of developing alternative strategies to the classical detection of theragnostic markers. Assessing ERBB2 amplification status in CC traditionally involves IHC followed by FISH when ambiguity arises. Although these techniques have long served as the gold standard in clinical practice, they are difficult to standardize and may lead to false-negative results.²¹,²² For

A

<table><thead><tr><th>Variable</th><th>N</th><th>Hazard ratio</th><th>P-Value</th></tr></thead><tbody><tr><td><strong>ERBB2 groups</strong></td><td></td><td></td><td></td></tr><tr><td>Intermediate ERBB2 RNA expression</td><td>2106</td><td></td><td></td></tr><tr><td>Molecular-ERBB2 positive or ERBB2 Mutated</td><td>81</td><td></td><td></td></tr><tr><td>Non-Amplified, High ERBB2 RNA expression</td><td>82</td><td></td><td></td></tr><tr><td>Low ERBB2 RNA expression</td><td>706</td><td></td><td></td></tr></tbody></table><table><thead><tr><th></th><th>Reference</th><th></th></tr></thead><tbody><tr><td>1.55 (1.06, 2.26)</td><td>.025</td></tr><tr><td>1.09 (0.72, 1.66)</td><td>.685</td></tr><tr><td>1.43 (1.22, 1.66)</td><td>&lt;.001</td></tr></tbody></table>

B

<figure><img src="image_8.png" /><figcaption>**Figure 5.** Survival analysis according to ERBB2 expression and mutational status. (A) Forest plot for TTR according to ERBB2 status in PETACC8 and IDEA-France pooled population. (B) Kaplan-Meier curves for TTR according to ERBB2 genomic status (amplification or mutation) in PETACC8 and IDEA-France pooled population.</figcaption></figure>
<table><caption>**Figure 6.** Forest plot based on the results of multivariate analysis of the factors associated with TTR in PETACC8 and IDEA-France pooled population.</caption><thead><tr><th>Variable</th><th>N</th><th>Hazard ratio</th><th>P-Value</th></tr></thead><tbody><tr><td><strong>ERBB2 groups</strong></td><td></td><td></td><td></td></tr><tr><td>Intermediate ERBB2 RNA expression</td><td>1784</td><td></td><td>Reference</td></tr><tr><td>Molecular-ERBB2 positive or ERBB2 Mutated</td><td>70</td><td></td><td>1.31 (0.84, 2.04)</td><td>.228</td></tr><tr><td>Non-Amplified, High ERBB2 RNA expression</td><td>76</td><td></td><td>1.13 (0.73, 1.73)</td><td>.584</td></tr><tr><td>Low ERBB2 RNA expression</td><td>583</td><td></td><td>1.28 (1.07, 1.52)</td><td>.006</td></tr><tr><td><strong>pT</strong></td><td></td><td></td><td></td></tr><tr><td>pT1-3</td><td>1993</td><td></td><td>Reference</td></tr><tr><td>pT4</td><td>520</td><td></td><td>1.93 (1.64, 2.28)</td><td>&lt;.001</td></tr><tr><td><strong>pN</strong></td><td></td><td></td><td></td></tr><tr><td>pN1</td><td>1683</td><td></td><td>Reference</td></tr><tr><td>pN2</td><td>830</td><td></td><td>2.62 (2.25, 3.05)</td><td>&lt;.001</td></tr><tr><td><strong>MMR status</strong></td><td></td><td></td><td></td></tr><tr><td>pMMR</td><td>2278</td><td></td><td>Reference</td></tr><tr><td>dMMR</td><td>235</td><td></td><td>0.94 (0.63, 1.40)</td><td>.763</td></tr><tr><td><strong>BRAF status</strong></td><td></td><td></td><td></td></tr><tr><td>NM</td><td>2225</td><td></td><td>Reference</td></tr><tr><td>M</td><td>288</td><td></td><td>1.35 (1.02, 1.79)</td><td>.036</td></tr><tr><td><strong>RAS status</strong></td><td></td><td></td><td></td></tr><tr><td>NM</td><td>1295</td><td></td><td>Reference</td></tr><tr><td>M</td><td>1218</td><td></td><td>1.32 (1.12, 1.56)</td><td>&lt;.001</td></tr><tr><td><strong>CMS group</strong></td><td></td><td></td><td></td></tr><tr><td>CMS1</td><td>434</td><td></td><td>Reference</td></tr><tr><td>CMS2</td><td>1046</td><td></td><td>1.13 (0.86, 1.48)</td><td>.374</td></tr><tr><td>CMS3</td><td>377</td><td></td><td>1.31 (0.97, 1.77)</td><td>.077</td></tr><tr><td>CMS4</td><td>656</td><td></td><td>1.39 (1.06, 1.81)</td><td>.016</td></tr></tbody></table>

example, the pre-analytical phase (eg, time and type of fixative and old blank slides) may influence IHC sensitivity and, to a lesser extent, FISH. To mitigate these risks, we ensured that IHC and FISH were performed on fresh sections, cut within 24 hours of testing, to maintain optimal quality. Nevertheless, traditional methods are being challenged by the advent of new DNA and RNA sequencing technologies, which have emerged as powerful tools for characterizing CC's molecular landscape, simultaneously analyzing various molecular features in a standardized and efficient quantitative approach.

In our study, NGS and RNA-seq revealed ERBB2 amplification in approximately 1.7% of CC cases, which aligns with previous studies using IHC/FISH.⁴ Although the NGS score demonstrated excellent concordance with IHC/FISH in our study and multiple other studies,²³⁻²⁵ 7 patients (0.43%) showed an ERBB2 amplification by NGS without positive IHC/FISH. Data from the MOUNTAINEER trial²⁶ demonstrated that among 10 patients negative by central HER2 IHC/FISH, the only patient who responded to HER2-targeted therapy was found to be ERBB2-amplified by NGS. This suggests that NGS may identify additional patients who could benefit from targeted therapy, even when traditional IHC/FISH methods indicate a negative result. However, ERBB2 RNA and protein overexpression, identified by RNA-seq and IHC without DNA amplification, may be attributed to transcriptional regulations, such as the activation of transcription factors influencing ERBB2 expression. In that subgroup of patients, sensitivity to HER2 inhibitors remains to be explored in both digestive and breast cancers.

To explore these variations, we expanded our analysis beyond ERBB2 RNA expression, examining the RNA expression of neighboring genes that may be co-amplified, but are not transcriptionally co-regulated. This broader approach allows for extrapolating ERBB2 amplification status from whole transcriptome data, applicable to patients analyzed via RNA-seq or transcriptome microarrays. Furthermore, this method can potentially identify other amplified genes in CC, such as KRAS, EGFR, or MET.

Currently, anti-HER2 treatments are being developed in randomized trials for ERBB2-amplified, RAS wild-type metastatic CC, with promising efficacy and acceptable tolerability reported in phase 2 trials using compounds such as tucatinib and trastuzumab-deruxtecan. Given the tumor shrinkage observed with these new drugs, they are now being tested in neoadjuvant proof-of-concept platforms, such as the NEO French platform in collaboration with the FOxTROT UK platform (NEO-HER2 trial) and the Unicorn Italian platform. Patients prioritized for screening would likely be those with high-risk stage III disease who could benefit from an intensified preoperative treatment approach. In this context, having comprehensive data from a large patient population on the outcomes of different HER2 profiles in stage III CC, before the introduction of anti-HER2 therapies, will be essential for developing accurate statistical models and hypotheses for future clinical trials evaluating these treatments.

Regarding prognostic impact, the low incidence of this alteration hampers the assessment of the potential prognostic effect of ERBB2 amplification in CC. Up-to-date studies present divergent results, although there is a trend toward an association between ERBB2 amplification and
less favorable overall or progression-free survival. We showed here that these inconsistencies could also be explained by the fact that, in addition to *ERBB2* over-expression, tumors with low *ERBB2* expression are also associated with a guarded prognosis. Furthermore, we demonstrated that patients with low *ERBB2*-expressing tu-mors are more frequently located in the proximal colon and, when treated with FOLFOX plus cetuximab—an EGFR-blocking antibody—have poorer survival than patients treated with FOLFOX alone. Together, these results could partly explain the lower impact of cetuximab treatment in proximal CC compared with distal CC.²⁷ Although HER2 amplification has been suggested previously as a negative predictor of response to cetuximab,²⁸ our findings sug-gested that this may not hold true for RNA expression (Supplementary Figure 8C). Indeed, patients with high *ERBB2* RNA expression appear to derive benefits from anti-EGFR treatment. Similar results were reported recently by Battaglin et al²⁹ in a cohort of metastatic colorectal cancer with a nonlinear association between ERBB2 expression and outcome, particularly in association with cetuximab treat-ment. By blocking EGFR activation, cetuximab may prevent the formation of heterodimers with ERBB2. This action could be particularly beneficial in patients with high ERBB2 expression, as it might negatively modulate the mitogen-activated protein kinase signaling pathway, thereby reducing cell proliferation. Overall, these findings suggest that although patients with HER2 amplification may benefit from targeted anti-HER2 therapies, those with elevated ERBB2 RNA expression could potentially respond favorably to cetuximab, underscoring the theragnostic value of molecular profiling in optimizing treatment strategies for colorectal cancer.

The results presented in this study provide valuable in-
sights into the prevalence and prognostic significance of *ERBB2*
amplification, molecular alterations, and expression in CC. Our
study suggests a Janus aspect of the expression of ERBB2. The
nonlinear relation between the *ERBB2* expression and TTR was
evidenced for the first time in stage III CC and breast cancer.

These findings refine the prognostic implications of such
alterations, introducing the potential role of assessing
ERBB2 status in stage III colorectal cancer. Despite *ERBB2*
amplification being a relatively rare event, the considerable
efficacy and tolerable safety profile demonstrated by anti-
ERBB2 drugs in patients with chemorefractory disease un-
derscore the importance of early ERBB2 screening to assist
clinicians in selecting optimal treatment options and
directing patients toward relevant clinical trials.

**Supplementary Material**

Note: To access the supplementary material accompanying
this article, visit the online version of *Gastroenterology* at
www.gastrojournal.org, and at http://doi.org/10.1053/
j.gastro.2024.10.046.

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Received July 12, 2024. Accepted October 22, 2024.

**Correspondence**

Address correspondence to: Camilla Pilati, PhD, Centre de Recherche des Cordeliers, 15 rue de l'école de médecine, Paris 75006, France. e-mail: camilla.pilati@inserm.fr.

**Acknowledgments**

Camilla Pilati and Audrey Soulabaille contributed equally to this work.

**CrediT Authorship Contributions**

Camilla Pilati, PhD (Conceptualization: Lead; Formal analysis: Lead;

Methodology: Equal; Project administration: Equal; Supervision: Equal;

Validation: Equal; Visualization: Lead; Writing – original draft: Lead)

Audrey Soulabaille, MD (Conceptualization: Equal; Formal analysis: Lead;

Methodology: Equal; Visualization: Equal; Writing – original draft: Equal)

Claire Gallois, MD, PhD (Data curation: Equal; Formal analysis: Supporting;

Resources: Equal)

Hélène Blons, PharmD, PhD (Data curation: Supporting; Resources: Equal)

Anne Cayre, PhD (Investigation: Equal; Resources: Supporting)

Marine Sroussi, MD (Data curation: Supporting; Formal analysis: Equal)

Delphine Le Corre, MS (Data curation: Supporting; Investigation: Equal)

Sophie Mouillet-Richard, PhD (Conceptualization: Equal; Funding acquisition: Supporting; Supervision: Supporting)

Claire Mulot, MS (Data curation: Supporting; Investigation: Supporting)
Karine Le Malicot, PhD (Data curation: Supporting; Formal analysis: Supporting)

Aurélien De Reynies, PhD (Formal analysis: Supporting)

Jean-Baptiste Bachet, MD, PhD (Resources: Equal)

Christophe Borg, MD, PhD (Resources: Equal)

Frédéric Di Fiore, MD, PhD (Resources: Equal)

Rosine Guimbaud, MD, PhD (Resources: Equal)
Jaafar Bennouna, MD, PhD (Resources: Equal)

Thierry André, MD, PhD (Resources: Equal)

Julien Taieb, MD, PhD (Resources: Lead)
Frédérique Penault-Llorca, MD, PhD (Data curation: Supporting; Formal analysis: Equal; Investigation: Equal; Resources: Supporting)

Pierre Laurent-Puig, MD, PhD (Conceptualization: Lead; Formal analysis: Equal; Funding acquisition: Lead; Methodology: Equal; Project administration: Equal; Supervision: Lead; Validation: Equal; Writing – original draft: Lead)

**Conflicts of interest**

The authors disclose no conflicts.

**Funding**

This work was supported by Aide et Recherche en Cancérologie Digestive Fondation and Association pour la Recherche sur le Cancer.
<table><caption>Supplementary Table 1. ERBB2-Activating Mutations Observed in PETACC8 and IDEA-France Cohorts</caption><thead><tr><th rowspan="2">Variable</th><th colspan="2">PETACC8 (n = 15)</th><th colspan="2">IDEA-France (n = 19<sup>a</sup>)</th></tr><tr><th>n</th><th>Case with amplification</th><th>n</th><th>Case with amplification</th></tr></thead><tbody><tr><td>Mutation type</td><td></td><td></td><td></td><td></td></tr><tr><td>Missense</td><td></td><td></td><td></td><td></td></tr><tr><td>p.Leu755Ser</td><td>3</td><td>1<sup>b</sup></td><td>8</td><td>1<sup>c</sup></td></tr><tr><td>p.Val842Ile</td><td>5</td><td>—</td><td>3</td><td>—</td></tr><tr><td>p.Val777Leu</td><td>3</td><td>—</td><td>1</td><td>—</td></tr><tr><td>p.Asp769Tyr</td><td>1</td><td>—</td><td>2</td><td>—</td></tr><tr><td>p.Val859Ile</td><td>1</td><td>—</td><td>—</td><td>—</td></tr><tr><td>p.Gly882Ala</td><td>1</td><td>—</td><td>—</td><td>—</td></tr><tr><td>p.Ala771Val</td><td>—</td><td>—</td><td>1</td><td>—</td></tr><tr><td>p.Asn857His</td><td>—</td><td>—</td><td>1</td><td>—</td></tr><tr><td>p.Asp873Gly</td><td>—</td><td>—</td><td>1</td><td>—</td></tr><tr><td>p.Leu755Pro</td><td>—</td><td>—</td><td>1</td><td>—</td></tr><tr><td>Deletion</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>p.Glu770_splice</td><td>1</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Insertion/deletion</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>p.Ala775_Gly776insValAla</td><td>—</td><td>—</td><td>1</td><td>—</td></tr></tbody></table>

<footnote><sup>a</sup>19 mutations in 17 tumor samples (2 patients with ERBB2 double mutation).</footnote>

<footnote><sup>b</sup>ERBB2 status: IHC/FISH-positive, NGS high score, RNA-seq high expression, molecular ERBB2-amplified.</footnote>

<footnote><sup>c</sup>ERBB2 status: NGS high score, RNA-seq high expression, molecular ERBB2-amplified.</footnote>
<table><caption>**Supplementary Table 2.** Clinicobiological Characteristics Associated With Immunohistochemistry and Fluorescence in Situ Hybridization Status, Molecular ERBB2 Status, and ERBB2 Mutation Status in PETACC8 Cohort</caption><thead><tr><th rowspan="2">Characteristic</th><th colspan="3">IHC/FISH status</th><th colspan="3">Molecular ERBB2 status</th><th colspan="3">ERBB2 mutation status</th></tr><tr><th>Positive<br>(n = 34)</th><th>Negative<br>(n = 1779)</th><th>P value<sup>a</sup></th><th>Amplified<br>(n = 32)</th><th>Negative<br>(n = 1694)</th><th>P value<sup>a</sup></th><th>M (n = 15)</th><th>NM<br>(n = 1681)</th><th>P value<sup>a</sup></th></tr></thead><tbody><tr><td>Age, n (%)</td><td></td><td></td><td>.3</td><td></td><td></td><td>.3</td><td></td><td></td><td>>.9</td></tr><tr><td>&nbsp;&nbsp;&lt;70 y</td><td>32 (94.1)</td><td>1542 (86.7)</td><td></td><td>30 (93.8)</td><td>1461 (86.2)</td><td></td><td>13 (86.7)</td><td>1453 (86.4)</td><td></td></tr><tr><td>&nbsp;&nbsp;≥70 y</td><td>2 (5.9)</td><td>237 (13.3)</td><td></td><td>2 (6.3)</td><td>233 (13.8)</td><td></td><td>2 (13.3)</td><td>228 (13.6)</td><td></td></tr><tr><td>Gender, n (%)</td><td></td><td></td><td>.2</td><td></td><td></td><td>.037</td><td></td><td></td><td>.2</td></tr><tr><td>&nbsp;&nbsp;Female</td><td>11 (32.4)</td><td>764 (42.9)</td><td></td><td>8 (25.0)</td><td>735 (43.4)</td><td></td><td>9 (60.0)</td><td>725 (43.1)</td><td></td></tr><tr><td>&nbsp;&nbsp;Male</td><td>23 (67.6)</td><td>1015 (57.1)</td><td></td><td>24 (75.0)</td><td>959 (56.6)</td><td></td><td>6 (40.0)</td><td>956 (56.9)</td><td></td></tr><tr><td>WHO</td><td></td><td></td><td>.034</td><td></td><td></td><td>.3</td><td></td><td></td><td>.2</td></tr><tr><td>&nbsp;&nbsp;0, n (%)</td><td>23 (67.6)</td><td>1403 (81.9)</td><td></td><td>23 (74.2)</td><td>1335 (81.8)</td><td></td><td>10 (66.7)</td><td>1320 (81.4)</td><td></td></tr><tr><td>&nbsp;&nbsp;≥1, n (%)</td><td>11 (32.4)</td><td>311 (18.1)</td><td></td><td>8 (25.8)</td><td>298 (18.2)</td><td></td><td>5 (33.3)</td><td>301 (18.6)</td><td></td></tr><tr><td>&nbsp;&nbsp;Unknown, n</td><td>0</td><td>65</td><td></td><td>1</td><td>61</td><td></td><td>0</td><td>60</td><td></td></tr><tr><td>Tumor site</td><td></td><td></td><td>.014</td><td></td><td></td><td>.002</td><td></td><td></td><td>.3</td></tr><tr><td>&nbsp;&nbsp;Proximal, n (%)</td><td>7 (20.6)</td><td>687 (38.8)</td><td></td><td>5 (15.6)</td><td>656 (38.9)</td><td></td><td>9 (60.0)</td><td>656 (39.2)</td><td></td></tr><tr><td>&nbsp;&nbsp;Distal, n (%)</td><td>25 (73.5)</td><td>1065 (60.1)</td><td></td><td>25 (78.1)</td><td>1012 (60.0)</td><td></td><td>6 (40.0)</td><td>999 (59.7)</td><td></td></tr><tr><td>&nbsp;&nbsp;Dual, n (%)</td><td>2 (5.9)</td><td>19 (1.1)</td><td></td><td>2 (6.3)</td><td>18 (1.1)</td><td></td><td>0 (0.0)</td><td>19 (1.1)</td><td></td></tr><tr><td>&nbsp;&nbsp;Unknown, n</td><td>0</td><td>8</td><td></td><td>0</td><td>8</td><td></td><td>0</td><td>7</td><td></td></tr><tr><td>Bowel obstruction and/or<br>perforation, n (%)</td><td></td><td></td><td>.3</td><td></td><td></td><td>.085</td><td></td><td></td><td>>.9</td></tr><tr><td>&nbsp;&nbsp;Yes</td><td>9 (26.5)</td><td>336 (18.9)</td><td></td><td>10 (31.3)</td><td>324 (19.1)</td><td></td><td>3 (20.0)</td><td>315 (18.7)</td><td></td></tr><tr><td>&nbsp;&nbsp;No</td><td>25 (73.5)</td><td>1443 (81.1)</td><td></td><td>22 (68.8)</td><td>1370 (80.9)</td><td></td><td>12 (80.0)</td><td>1366 (81.3)</td><td></td></tr><tr><td>pT</td><td></td><td></td><td>.10</td><td></td><td></td><td>.066</td><td></td><td></td><td>>.9</td></tr><tr><td>&nbsp;&nbsp;pT1-3, n (%)</td><td>23 (67.6)</td><td>1407 (79.1)</td><td></td><td>21 (65.6)</td><td>1338 (79.0)</td><td></td><td>12 (80.0)</td><td>1328 (79.0)</td><td></td></tr><tr><td>&nbsp;&nbsp;pT4, n (%)</td><td>11 (32.4)</td><td>371 (20.9)</td><td></td><td>11 (34.4)</td><td>355 (21.0)</td><td></td><td>3 (20.0)</td><td>353 (21.0)</td><td></td></tr><tr><td>&nbsp;&nbsp;Unknown, n</td><td>0</td><td>1</td><td></td><td>0</td><td>1</td><td></td><td></td><td></td><td></td></tr><tr><td>pN</td><td></td><td></td><td>.3</td><td></td><td></td><td>.4</td><td></td><td></td><td>.2</td></tr><tr><td>&nbsp;&nbsp;pN1, n (%)</td><td>18 (52.9)</td><td>1110 (62.4)</td><td></td><td>18 (56.3)</td><td>1064 (62.8)</td><td></td><td>7 (46.7)</td><td>1048 (62.3)</td><td></td></tr><tr><td>&nbsp;&nbsp;pN2, n (%)</td><td>16 (47.1)</td><td>669 (37.6)</td><td></td><td>14 (43.8)</td><td>630 (37.2)</td><td></td><td>8 (53.3)</td><td>633 (37.7)</td><td></td></tr><tr><td>Histopathology grading</td><td></td><td></td><td>.5</td><td></td><td></td><td>.2</td><td></td><td></td><td>.7</td></tr><tr><td>&nbsp;&nbsp;G1-2, n (%)</td><td>26 (76.5)</td><td>1428 (81.3)</td><td></td><td>23 (71.9)</td><td>1362 (81.4)</td><td></td><td>11 (78.6)</td><td>1337 (80.6)</td><td></td></tr><tr><td>&nbsp;&nbsp;G3-4, n (%)</td><td>8 (23.5)</td><td>328 (18.7)</td><td></td><td>9 (28.1)</td><td>312 (18.6)</td><td></td><td>3 (21.4)</td><td>322 (19.4)</td><td></td></tr><tr><td>&nbsp;&nbsp;Unknown, n</td><td>0</td><td>23</td><td></td><td>0</td><td>20</td><td></td><td>1</td><td>22</td><td></td></tr><tr><td>RAS</td><td></td><td></td><td>.10</td><td></td><td></td><td>.006</td><td></td><td></td><td>.6</td></tr><tr><td>&nbsp;&nbsp;M, n (%)</td><td>10 (33.3)</td><td>797 (48.6)</td><td></td><td>6 (22.2)</td><td>767 (49.0)</td><td></td><td>8 (53.3)</td><td>774 (46.0)</td><td></td></tr><tr><td>&nbsp;&nbsp;NM, n (%)</td><td>20 (66.7)</td><td>843 (51.4)</td><td></td><td>21 (77.8)</td><td>799 (51.0)</td><td></td><td>7 (46.7)</td><td>907 (54.0)</td><td></td></tr><tr><td>&nbsp;&nbsp;Unknown, n</td><td>4</td><td>139</td><td></td><td>5</td><td>128</td><td></td><td>0</td><td>0</td><td></td></tr></tbody></table>
<table><caption>Supplementary Table 2. Continued</caption><thead><tr><th rowspan="2">Characteristic</th><th colspan="3">IHC/FISH status</th><th colspan="3">Molecular ERBB2 status</th><th colspan="3">ERBB2 mutation status</th></tr><tr><th>Positive<br>(n = 34)</th><th>Negative<br>(n = 1779)</th><th>P value<sup>a</sup></th><th>Amplified<br>(n = 32)</th><th>Negative<br>(n = 1694)</th><th>P value<sup>a</sup></th><th>M (n = 15)</th><th>NM<br>(n = 1681)</th><th>P value<sup>a</sup></th></tr></thead><tbody><tr><td><strong>BRAF</strong></td><td></td><td></td><td>.2</td><td></td><td></td><td>.2</td><td></td><td></td><td>>.9</td></tr><tr><td>M, n (%)</td><td>1 (2.9)</td><td>195 (11.5)</td><td></td><td>1 (3.1)</td><td>183 (11.3)</td><td></td><td>1 (6.7)</td><td>197 (11.7)</td><td></td></tr><tr><td>NM, n (%)</td><td>33 (97.1)</td><td>1504 (88.5)</td><td></td><td>31 (96.9)</td><td>1438 (88.7)</td><td></td><td>14 (93.3)</td><td>1484 (88.3)</td><td></td></tr><tr><td>Unknown, n</td><td>0</td><td>80</td><td></td><td>0</td><td>73</td><td></td><td>0</td><td>0</td><td></td></tr><tr><td><strong>MMR</strong></td><td></td><td></td><td>.043</td><td></td><td></td><td>.068</td><td></td><td></td><td>.011</td></tr><tr><td>MSI/dMMR, n (%)</td><td>0 (0.0)</td><td>177 (10.1)</td><td></td><td>0 (0.0)</td><td>170 (10.2)</td><td></td><td>5 (35.7)</td><td>161 (10.3)</td><td></td></tr><tr><td>MSS/pMMR, n (%)</td><td>34 (100.0)</td><td>1580 (89.9)</td><td></td><td>32 (100.0)</td><td>1491 (89.8)</td><td></td><td>9 (64.3)</td><td>1403 (89.7)</td><td></td></tr><tr><td>Unknown, n</td><td>0</td><td>22</td><td></td><td>0</td><td>33</td><td></td><td>1</td><td>117</td><td></td></tr><tr><td><strong>CMS</strong></td><td></td><td></td><td>.3</td><td></td><td></td><td>.3</td><td></td><td></td><td>.009</td></tr><tr><td>CMS1, n (%)</td><td>3 (9.4)</td><td>339 (20.3)</td><td></td><td>4 (12.5)</td><td>340 (20.2)</td><td></td><td>8 (57.1)</td><td>309 (20.6)</td><td></td></tr><tr><td>CMS2, n (%)</td><td>20 (62.5)</td><td>764 (45.8)</td><td></td><td>19 (59.4)</td><td>767 (45.7)</td><td></td><td>3 (21.4)</td><td>689 (46.0)</td><td></td></tr><tr><td>CMS3, n (%)</td><td>2 (6.3)</td><td>187 (11.2)</td><td></td><td>1 (3.1)</td><td>192 (11.4)</td><td></td><td>2 (14.3)</td><td>162 (10.8)</td><td></td></tr><tr><td>CMS4, n (%)</td><td>7 (21.9)</td><td>379 (22.7)</td><td></td><td>8 (25.0)</td><td>381 (22.7)</td><td></td><td>1 (7.1)</td><td>338 (22.6)</td><td></td></tr><tr><td>Unknown, n</td><td>2</td><td>110</td><td></td><td>0</td><td>14</td><td></td><td>1</td><td>183</td><td></td></tr><tr><td><strong>TP53</strong></td><td></td><td></td><td>.011</td><td></td><td></td><td>&lt;.001</td><td></td><td></td><td>.3</td></tr><tr><td>M, n (%)</td><td>25 (83.3)</td><td>952 (60.4)</td><td></td><td>25 (92.6)</td><td>903 (59.9)</td><td></td><td>7 (46.7)</td><td>1023 (60.9)</td><td></td></tr><tr><td>NM, n (%)</td><td>5 (16.7)</td><td>625 (39.6)</td><td></td><td>2 (7.4)</td><td>604 (40.1)</td><td></td><td>8 (53.3)</td><td>658 (39.1)</td><td></td></tr><tr><td>Unknown, n</td><td>4</td><td>202</td><td></td><td>5</td><td>187</td><td></td><td>0</td><td>0</td><td></td></tr></tbody></table>

dMMR, deficient mismatch repair; M, mutated; MMR, mismatch repair; MSI, microsatellite instability; MSS, microsatellite stability; NM, nonmutated; pMMR, proficient mismatch repair; WHO, World Health Organization.

<footnote><sup>a</sup>Fisher exact test; Pearson χ² test.</footnote>
<table><caption>**Supplementary Table 3.** Clinicobiological Characteristics Associated With ERBB2 RNA Sequencing Expression Group in Pooled Population</caption><thead><tr><th rowspan="2">Characteristic</th><th colspan="2">ERBB2 RNA-seq expression group</th><th rowspan="2">P value<sup>a</sup></th></tr><tr><th>Intermediate (n = 2128)</th><th>Low (n = 717)</th></tr></thead><tbody><tr><td>Age</td><td></td><td></td><td>.12</td></tr><tr><td>&lt;70 y, n (%)</td><td>1 717 (81.0)</td><td>561 (78.4)</td><td></td></tr><tr><td>≥70 y, n (%)</td><td>402 (19.0)</td><td>155 (21.6)</td><td></td></tr><tr><td>Unknown, n</td><td>9</td><td>1</td><td></td></tr><tr><td>Gender</td><td></td><td></td><td>.2</td></tr><tr><td>Female, n (%)</td><td>933 (44.0)</td><td>296 (41.3)</td><td></td></tr><tr><td>Male, n (%)</td><td>1 186 (56.0)</td><td>420 (58.7)</td><td></td></tr><tr><td>Unknown, n</td><td>9</td><td>1</td><td></td></tr><tr><td>WHO</td><td></td><td></td><td>.8</td></tr><tr><td>0, n (%)</td><td>1 628 (78.5)</td><td>547 (78.0)</td><td></td></tr><tr><td>≥1, n (%)</td><td>447 (21.5)</td><td>154 (22.0)</td><td></td></tr><tr><td>Unknown, n</td><td>53</td><td>16</td><td></td></tr><tr><td>Tumor site</td><td></td><td></td><td>&lt;.001</td></tr><tr><td>Proximal, n (%)</td><td>781 (37.4)</td><td>323 (45.9)</td><td></td></tr><tr><td>Distal, n (%)</td><td>1 289 (61.8)</td><td>372 (52.9)</td><td></td></tr><tr><td>Dual, n (%)</td><td>16 (0.8)</td><td>8 (1.1)</td><td></td></tr><tr><td>Unknown, n</td><td>42</td><td>14</td><td></td></tr><tr><td>Bowel obstruction and/or perforation</td><td></td><td></td><td>.065</td></tr><tr><td>Yes, n (%)</td><td>392 (18.5)</td><td>155 (21.6)</td><td></td></tr><tr><td>No, n (%)</td><td>1 727 (81.5)</td><td>561 (78.4)</td><td></td></tr><tr><td>Unknown, n</td><td>9</td><td>1</td><td></td></tr><tr><td>pT</td><td></td><td></td><td>.007</td></tr><tr><td>pT1-3, n (%)</td><td>1 704 (80.5)</td><td>542 (75.7)</td><td></td></tr><tr><td>pT4, n (%)</td><td>414 (19.5)</td><td>174 (24.3)</td><td></td></tr><tr><td>Unknown, n</td><td>10</td><td>1</td><td></td></tr><tr><td>pN</td><td></td><td></td><td>.051</td></tr><tr><td>pN1, n (%)</td><td>1 448 (68.3)</td><td>461 (64.4)</td><td></td></tr><tr><td>pN2, n (%)</td><td>671 (31.7)</td><td>255 (35.6)</td><td></td></tr><tr><td>Unknown, n</td><td>9</td><td>1</td><td></td></tr><tr><td>Histopathology grading</td><td></td><td></td><td>&lt;.001</td></tr><tr><td>G1-2, n (%)</td><td>1 815 (87.5)</td><td>562 (80.6)</td><td></td></tr><tr><td>G3-4, n (%)</td><td>259 (12.5)</td><td>135 (19.4)</td><td></td></tr><tr><td>Unknown, n</td><td>54</td><td>20</td><td></td></tr><tr><td>RAS</td><td></td><td></td><td>.028</td></tr><tr><td>M, n (%)</td><td>910 (48.3)</td><td>337 (53.3)</td><td></td></tr><tr><td>NM, n (%)</td><td>975 (51.7)</td><td>295 (46.7)</td><td></td></tr><tr><td>Unknown, n</td><td>243</td><td>85</td><td></td></tr><tr><td>BRAF</td><td></td><td></td><td>&lt;.001</td></tr><tr><td>M, n (%)</td><td>194 (10.1)</td><td>103 (15.9)</td><td></td></tr><tr><td>NM, n (%)</td><td>1 723 (89.9)</td><td>545 (84.1)</td><td></td></tr><tr><td>Unknown, n</td><td>211</td><td>69</td><td></td></tr><tr><td>MMR</td><td></td><td></td><td>.6</td></tr><tr><td>MSI/dMMR, n (%)</td><td>194 (9.5)</td><td>60 (8.8)</td><td></td></tr><tr><td>MSS/pMMR, n (%)</td><td>1 847 (90.5)</td><td>620 (91.2)</td><td></td></tr><tr><td>Unknown, n</td><td>87</td><td>37</td><td></td></tr><tr><td>CMS</td><td></td><td></td><td>&lt;.001</td></tr><tr><td>CMS1, n (%)</td><td>336 (15.8)</td><td>146 (20.5)</td><td></td></tr><tr><td>CMS2, n (%)</td><td>958 (45.2)</td><td>190 (26.7)</td><td></td></tr><tr><td>CMS3, n (%)</td><td>338 (15.9)</td><td>85 (11.9)</td><td></td></tr><tr><td>CMS4, n (%)</td><td>488 (23.0)</td><td>291 (40.9)</td><td></td></tr><tr><td>Unknown, n</td><td>8</td><td>5</td><td></td></tr></tbody></table>

dMMR, deficient mismatch repair; M, mutated; MMR, mismatch repair; MSI, microsatellite instability; MSS, microsatellite stability; NM, nonmutated; pMMR, proficient mismatch repair; WHO, World Health Organization.

<footnote>aPearson χ² test.</footnote>