HER2 Immunohistochemistry Score Research Articles

Performance of HER2 DAKO HercepTest and Ventana 4B5 immunohistochemical assays on detecting HER2 gene-amplification in uterine serous carcinomas

We compared the performance of two commonly-used HER2 immunohistochemistry (IHC) assays in uterine serous carcinomas (USC), correlating with HER2 gene amplification by fluorescence in-situ hybridization (FISH). Sixty-five USCs were stained by both HercepTest™ and PATHWAY 4B5 assays. FISH was performed by HER2 IQFISH pharmDx. Consensus HER2 IHC scoring was performed, and HER2 testing results were evaluated using USC-specific criteria. Complete concordance between HercepTest and 4B5 assays was achieved in 44/65 tumors (68%). The overall HER2 IHC/FISH concordance was 94% (45/48) by HercepTest and 91% (42/46) by 4B5. All HER2 IHC 3+ cases with HercepTest (n = 6) and 4B5 (n = 4) were gene-amplified, corresponding to specificities of 100%. For cases with IHC 2+, 41% (7/17) by HercepTest and 42% (8/19) by 4B5 had HER2 gene amplification. The sensitivity for HercepTest and 4B5 were 38% and 25%, respectively, at a cut-off of IHC 3+ (P = 0.50), and were 81% and 75%, respectively, at a cut-off of IHC 2+ (P > 0.99). Among HER2 IHC 0–1+ cases, 3/42 cases by HercepTest and 4/42 cases by 4B5 showed amplified FISH results, corresponding to overall false negative rates of 19% for HercepTest and 25% for 4B5. By using USC-specific IHC scoring criteria, both HercepTest and 4B5 assays showed high specificities (100%) for HER2 gene amplification in IHC 3+ cases, high IHC/FISH concordance, and comparable sensitivity for detecting HER2 gene amplification. The notable false negative rates using IHC 2+ as a cut-off for reflexing FISH analysis may warrant consideration for performing FISH in IHC 1+ cases until more data become available.

Abstract PO1-15-05: Gene expression signature as a predictor of pathological complete response to neoadjuvant docetaxel, carboplatin, trastuzumab, and pertuzumab treatment in locally advanced HER2-positive breast cancer

Abstract Background: Neoadjuvant pertuzumab and trastuzumab in combination with chemotherapy can achieve pathological complete response (pCR, defined as ypT0/is ypN0) in approximately 40–60% of patients with human epidermal growth factor receptor 2-positive (HER2+) breast cancer (BC). Patients who achieve pCR have significantly improved survival. Therefore, the prediction of pCR is crucial for optimizing neoadjuvant therapy. Methods: To identify a gene expression signature that could predict pCR in patients with HER2+ BC, we used the nCounter Breast Cancer 360TM V2 panel to quantify the expression of 758 genes in 104 HER2+ BC samples from patients who received neoadjuvant docetaxel, carboplatin, trastuzumab, and pertuzumab (TCHP) chemotherapy and subsequent curative breast surgery. Data normalization, differentially expressed gene (DEG) analysis, and gene set enrichment analysis (GSEA) were performed using the NanoTube Bioconductor package. Using the elastic net logistic regression model with optimal hyperparameters determined by 5-fold cross-validation (5-CV), we developed a chemosensitivity score based on the expression of significant DEGs (genes with absolute log2-fold-change ≥0.5 and q-value < 0.1). We tested its predictive value for pCR using multivariable logistic regression analysis. Results: The median age at diagnosis was 49.5 years. The clinical stage was II in 53 (51%) patients and III in 51 (49%) patients at diagnosis. Ninety (86.5%) patients had a HER2 immunohistochemistry (IHC) score of 3+, while the remaining 14 (13.5%) patients had HER2 IHC 2+ but HER2 amplification detected by in situ hybridization. Pathological evaluation of surgical specimens revealed that 55 (52.9%) patients achieved pCR. Menopausal status (p = 0.01), PAM50 intrinsic subtype (p = 0.002), hormone receptor (HR) status (p < 0.001), and HER2 IHC score (p = 0.019) were significantly correlated with the pCR rate. DEG analysis using the PAM50 intrinsic subtype, HR status, and HER2 IHC score as covariates revealed 6 downregulated DEGs and 33 upregulated DEGs in patients who achieved pCR compared to patients who did not (Table 1). The 39 DEGs were significantly enriched in nine preselected MSigDB hallmark gene sets representing immunological processes (p < 0.001). The optimal elastic net logistic regression model containing 39 DEGs achieved the mean 5-CV area under the receiver operating characteristic curve of 0.81. The chemosensitivity score calculated using the optimal model parameters was significantly correlated with pCR after adjustment for the PAM50 intrinsic subtype, HR status, and HER2 IHC score (p < 0.001). GSEA revealed eight hallmark gene sets that were significantly up- or downregulated in samples with pCR, including epithelial-mesenchymal transition (normalized enrichment score [NES] = -2.16; q < 0.001), late estrogen response (NES = -2.01; q = 0.004), early estrogen response (NES = -1.972; q = 0.005), and inflammatory response (NES = 1.53; q = 0.06). Conclusion: Our findings demonstrate that gene expression patterns can predict the response to neoadjuvant TCHP chemotherapy in HER2+ BC. The functional characterization of 39 DEGs suggests that the tumor microenvironment, including adjacent immune cells, plays a significant role in modulating the response to neoadjuvant chemotherapy in HER2+ BC. Table 1. Significant DEGs Citation Format: Junghoon Shin, Ji-Yeon Kim, Eun Yoon Cho, Seok Won Kim, Jeong Eon Lee, Hyunwoo Lee, Yeon Hee Park, Jin Seok Ahn, Young-Hyuck Im. Gene expression signature as a predictor of pathological complete response to neoadjuvant docetaxel, carboplatin, trastuzumab, and pertuzumab treatment in locally advanced HER2-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-15-05.

Abstract PO4-26-09: The Advantage of Artificial-Intelligence in HER2 IHC 0 and 1+ Scoring in Breast Cancer

Abstract Background: Trastuzumab deruxtecan has substantially changed the treatment of HER2-low breast cancer, emphasizing the need for accurate differentiation between HER2 immunohistochemistry (IHC) scores of 0 and 1+. However, the current accuracy of HER2 IHC 0 and 1+ scoring in real-world is inadequate. Artificial intelligence (AI) has emerged as a potential solution to improve interpretation accuracy. We developed an AI algorithm based on whole slide images (WSI) to quantitatively assess HER2 expression and its role in interpreting HER2 IHC scores of 0 and 1+. Methods: Our three-phase AI analysis framework involved segmenting tumor areas (excluding ductal carcinoma in situ), detecting and classifying tumor cells based on membrane staining patterns, and grading HER2 IHC scores according to the 2018 ASCO/CAP HER2 guideline. The AI tool was trained using 6012 patches annotated by experienced pathologists. Performance evaluation was conducted using a test dataset comprising 265 slides. A total of 141 HER2 IHC slides (27 IHC 0 and 114 IHC 1+) from patients diagnosed with invasive breast cancer at Fudan University Shanghai Cancer Center in 2021 were included. Two pretrained expert pathologists independently rescored the HER2 slides, followed by analysis using the AI tool. All inconsistent cases were also reviewed by a third senior pathologist. Interobserver agreement between the pathologists and concordance between the pathologists and the AI interpretation results were assessed. We also explored potential ranges where HER2 interpretation by pathologists exhibited inconsistency or inaccuracy. Results: The HER2 AI algorithm showed high performance in interpreting all levels of HER2 expression, with an overall kappa value of 0.85. For HER2 IHC 0 and 1+ cases, the AI model demonstrated high sensitivities (0.839 and 0.914) and specificities (0.983 and 0.890) (Table 1). After the rescoring process, 51 cases were reclassified as IHC 0 and 90 cases as IHC 1+. The overall agreement rate between the historical and re-scoring results was 73.05% (103/141). The agreement rate between the AI and re-scoring results was 91.49% (129/141) (Table 2), indicating comparable interpretation capabilities between the AI model and well-trained pathologists. The interobserver agreement between the two pathologists was 83.69% (118/141), with agreement rates of 88.23% (45/51) for IHC 0 and 81.11% (73/90) for IHC 1+. Analyzing the inconsistent cases (n=12) between IHC 0 and IHC 1+, we examined the percentage of weak and incomplete expression provided by the AI. In 9 out of 12 cases, the AI provided the same results as the third senior pathologist, suggesting that AI could assist in interpretation within this range. We identified a range of 2.83% to 24.98% as a "grey-zone," where even well-trained pathologists provided inconsistent results. Conclusion: Our study demonstrates the excellent performance of an AI-based tool for scoring HER2 IHC 0 and 1+. The AI model exhibits comparable capabilities to well-trained pathologists in interpreting HER2 expression. Additionally, we identified a "grey-zone" among pathologists, highlighting the limitations of manual subjective interpretation. AI assistance within this zone may help mitigate subjective variations. Table 1. Correlation between pathologist read and AI read of HER2 immunohistochemistry. Table 2. Correlation between pathologist rescoring and AI read of HER2 immunohistochemistry. Citation Format: Ming Li, Hong Lv, Yizhi Zhao, Chenglu Zhu, Hansheng Li, Mingzhen Lin, Wen-Tao Yang. The Advantage of Artificial-Intelligence in HER2 IHC 0 and 1+ Scoring in Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-26-09.

Abstract PO2-14-12: Accurate quantification of slide-level HER2 scores in breast cancer using a machine-learning model, AIM-HER2 Breast Cancer

Abstract Background: HER2 expression level is a key factor in determining the optimal treatment course for breast cancer patients. Roughly 15% of breast cancers are HER2(+), and determination of HER2 status is routinely assessed by immunohistochemistry (IHC). Accurate assessment of the HER2 IHC score (0, 1+, 2+, 3+) by pathologists is therefore critical, especially in light of novel therapeutic approaches demonstrating efficacy in the HER2-low setting (IHC scores 1+, and 2+/FISH-)1,2. To assist pathologists with the consistent provision of reproducible and accurate scores across the entire HER2 scoring range, we developed a machine-learning model (“AIM-HER2”) to generate accurate, slide-level HER2 scores aligned with ASCO-CAP guidelines in clinical breast cancer HER2 IHC specimens. Methods: AIM-HER2 was developed using whole-slide images (WSI; N=4261) from clinical and commercial sources. WSI were split into training (N=2694, 63%) and optimization (N=1567, 37%) sets. An additive multiple instance learning (aMIL) model3 was trained to predict HER2 scores directly from WSI and create interpretable heatmaps that depict HER2 predictions in tissue images. Image artifacts and in situ carcinomas were identified using previously trained artifact and tissue segmentation models and were excluded, leaving only regions of invasive carcinoma to be analyzed. AIM-HER2 performance was assessed on additional slides obtained from five academic or commercial sources (N=804 total, 770 evaluable) on which HER2 IHC was performed. Board-certified pathologists (N=52) with relevant experience provided manual HER2 scores based on ASCO-CAP guidelines. Nested pairwise non-inferiority analysis4 was used to compare model performance to that of pathologists (N=3 pathologists per slide). In the nested pairwise framework, agreement among pathologists was compared to agreement between AIM-HER2 and pathologists via linear kappa, so that summary metrics account for inter-pathologist variability. Results: High concordance was observed between AIM-HER2-predicted and pathologist-labeled slide-level HER2 scores, both overall and for each scoring level. Similar results were observed when assessing AIM-HER2 performance on multiple slide scanners and after IHC with multiple HER2 IHC antibody clones. Results are summarized in Table 1. Conclusions: We developed AIM-HER2, a novel aMIL-based approach for predicting slide-level HER2 IHC scores. AIM-HER2 has similar levels of agreement with pathologists as pathologists have with each other for determining HER2 score. This result is upheld when slides imaged using multiple scanning platforms and stained using multiple HER2 antibody clones. The performance of AIM-HER2 on multiple scanners and after multiple assays supports broad applicability of this algorithm in clinical laboratories, including for the identification of HER2-low cases. Work is ongoing to perform similar analyses in an independent, real-world dataset.

Abstract PO4-26-07: HER2-low prevalence in early breast cancer (BC) across reference laboratories in Brazil: potential impact of immunohistochemistry (IHC) antibody assay sensitivity

Abstract Background: Current definition of HER2-positive and HER2-low BC follows ASCO/CAP guidelines using IHC and/or in situ hybridization (ISH)-based techniques. Although there is no recommendation for a specific antibody assay, the companion diagnostic test for trastuzumab deruxtecan in the USA is Ventana PATHWAY anti-HER2 monoclonal antibody (4B5) run on Benchmark Ultra instrument. In this study, we assessed the prevalence of HER2-low BC in samples stained with Dako anti-HER2 polyclonal antibody (A0485) and analyzed with Autostainer Link 48 Agilent/Dako, commonly used in different large-volume laboratories in Brazil. Methods: Retrospective study of all early-stage BC samples without exposure to neoadjuvant therapy tested for HR plus HER2 status in Oncoclínicas Precision Medicine (Locus Lab) from 2021 to 2023 and analyzed following ASCO/CAP guidelines. Tumors with HER2 IHC score 0 were classified as HER2-zero, whereas tumors with HER2 score 1+ and those with HER2 score 2+ with ISH-negative were classified as HER2-low. To assess the impact of preanalytical factors on HER2-low positivity, we stratified samples as in-house (part of Oncoclínicas network) or external (other institutions’ FFPE preparation conditions not controlled). Results were also validated in independent IRA Lab using the same Dako anti-HER2 polyclonal antibody (A0485) on Autostainer Link 48 Agilent/Dako. Results: Out of 1,638 eligible samples from Locus Lab, 84% were classified as HR-positive/HER2 negative, 12% HER2-positive, and 4% triple-negative. In 1,386 HR-positive/HER2 negative samples, 83% were HER2-zero, and 17% (CI95% 15%-19%) were HER2-low (1+ in 79% and 2+ with ISH negative in 21%). In 70 triple-negative samples, 62% were HER2-zero and 38% (CI95% 27%-51%) were HER2-low (1+ in 44% and 2+ with ISH negative in 56%). HER2-low positivity was 17% in the subset of in-house samples (N=1.090) and 16% in external referrals (P=0.41). In the independent cohort, out of 441 samples from IRA Lab, 80% were HR-positive/HER2 negative, 12% HER2-positive and 8% triple negative. In 352 HR positive/HER2 negative samples, 80% were HER2-zero and 20% (CI95% 16%-25%) were HER2-low. In 36 triple-negative samples, 72% were HER2-zero and 28% (CI95% 14%-45%) were HER2-low. Conclusions: Different from another Brazilian cohort where the prevalence of HER2-low was 50% with monoclonal anti-HER2 antibodies (Reinert et al., SABCS 2020), our data suggests that the reduced proportion of samples with HER2-low status may be related to antibody clone sensitivity and not linked to preanalytical factors or local ancestry/cancer biology. Indeed, a recent Italian cohort also had lower than expected prevalence of HER2-low in surgical specimens using anti-HER2 Dako A0485 assay on Omnis instrument (Rossi et al., Breast Cancer Res Treat 2023). Prior studies have found differential analytical sensitivity of antibodies for HER2-low status, while HER2 score 3+ detection is equivalent (Ruschoff et al., Virchows Archiv 2022). We recommend prospective evaluation of the HER2-low status with companion-diagnostic Ventana 4B5 assay and alternative antibodies and instruments. Citation Format: Leonard M da Silva, Fernanda Orpinelli Rego, Matheus Costa e Silva, Tomas Reinert, Fernanda Koyama, Daniel Bueno da Cunha, Vagner Duarte, Emílio Pereira, Bruno Ferrari, Rodrigo Dienstmann. HER2-low prevalence in early breast cancer (BC) across reference laboratories in Brazil: potential impact of immunohistochemistry (IHC) antibody assay sensitivity [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-26-07.

Abstract PO4-14-08: A gene expression-based classifier for HER2-low breast cancer

Abstract Background: The definition of HER2-low breast cancer from clinical trials of antibody-conjugated drugs (ADCs) relies on immunohistochemistry scoring (IHC). However, in daily-practice the accuracy of IHC is hindered by inter-observer variability in assessing HER2 non-overexpressing status. Here, we aimed to identify breast cancer primary tumors with low HER2 expression by leveraging gene expression profiling. Materials and methods: A discovery approach was applied to gene expression profile of internal INT1 (n= 125) and INT2 (n= 84) datasets. We identified specific differently expressed genes (DEGs) according to HER2 IHC categories 0, 1+, 2+ and 3+. Principal Component Analysis (PCA) was used to generate a HER2-low signature whose performance was confirmed in the independent INT3 (n= 95), and TCGA and GSE20194 publicly available datasets. The association between the HER2-low signature and HER2 IHC categories was evaluated by non-parametric Kruskal-Wallis (KW) test with post hoc pair wise comparisons (i.e. contrasts); the HER2-low signature discriminatory capability was assessed by estimating the area under the receiver operating characteristic (ROC) curve (AUC) with its corresponding 95% Confidence Interval (CI). Gene Ontology and KEGG analyses were performed to enrich the DEGs for functional information. Results: A 20-gene HER2-low signature, consisting of both up-regulated (n=11) and down-regulated (n=9) genes, was computed based on the DEGs according to HER2 IHC categories as follows: 11 genes specific for the 1+ category, 8 genes for the 2+ category, and 1 gene for the HER2-low category. HER2-low signature genes were significantly enriched with lipid and steroid metabolism pathways, peptidase regulation, and humoral immune response. The HER2-low signature levels demonstrated a significant association with HER2 IHC categories (KW p-value < 0.001) and were distributed in a bell-shaped pattern across IHC categories (low values 0 and 3+; high values 1+ and 2+), effectively distinguishing HER2-low from 0 (contrast p-value < 0.001) and 3+ (contrast p-value < 0.001). Notably, the signature levels were significantly higher in tumors scored with 1+ as compared to 0 (contrast p =0.002). The HER2-low signature association with IHC categories and discriminatory capability was confirmed in the independent INT3 and TCGA datasets with higher values in HER2-low compared to both 0 and 3+. The HER2-low signature achieved an AUC value of 0.72 (95%CI 0.62-0.83) in differentiating HER2 0/3+ from HER2 1+/2+ categories, which is worth noting in light of the individual ERBB2 mRNA AUC value of 0.48 (95%CI 0.34-0.62). Conclusions: The 20-gene HER2-low signature was generated by maximizing the differences in gene expression between tumors with different HER2 status according to IHC. In contrast to the previously published single ERBB2 gene expression assessment, our data presents compelling evidence for effectively distinguishing HER2-low tumors, including those scored as 1+ from HER2-0 tumors. Our signature holds potential in selecting novel candidates for ADC therapy. Citation Format: Serena Di Cosimo, Sara Pizzamiglio, Chiara Maura Ciniselli, Valeria Duroni, Vera Cappelletti, Loris de Cecco, Maria Carmen De Santis, Rosaria Orlandi, Marilena Iorio, Marco Silvestri, Giancarlo Pruneri, Paolo Verderio. A gene expression-based classifier for HER2-low breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-14-08.

Abstract PO3-15-12: Expression and co-expression patterns of TROP2 and HER2 in breast cancer: implications for bispecific antibody-drug conjugate therapy

Abstract Introduction: Antibody-drug conjugates (ADCs) targeting trophoblast cell surface antigen 2 (TROP2), such as sacituzumab-govitecan and datopotamab-deruxtecan, have recently been developed. A preclinical study has reported the efficacy of anti-human epidermal growth factor receptor 2 (HER2) and TROP2 bispecific ADCs in treating HER2 and TROP2 co-expressing tumors, including HER2-low tumors in various solid tumors (Chengzhang Shang et al: AACR 2023). In this study, we aimed to evaluate the expression of TROP2 by immunohistochemistry (IHC) at different sites and investigate the changing status and co-expression of HER2 and TROP2 among breast cancer patients. Methods: We collected 244 archival paired samples of primary tumors and metastatic sites from 111 breast cancer patients treated in our hospital from 2000 to 2018. Estrogen receptor (ER), progesterone receptor (PgR), and HER2 status were determined from previous pathology reports. HER2 low was defined as HER2 IHC score of 1+ or 2+ with negative fluorescent in situ hybridization (ISH). In addition, we determined the expression of TROP2 using IHC assays and categorized the results based on the histochemical score (H-score). Results: Among the samples, ER+HER2- was 139, ER+HER2+ was nine, ER-HER2+ was eight, and triple-negative (TN) was 88. HER2 3+ or 2+ with ISH positive was 17, HER2 low was 112, HER2 negative was 115. The median TROP2 H-score was 10, with a mean of 37.5 (range: 0-265). Overall, 27% of the samples had H-score of 0, 73% had H-score of 1<, 47% had H-score of 10<, and 12% had H-score of 100< . No statistically significant association was observed between TROP2 expression and breast cancer subtypes (ER+HER2-; H-score of 0 24%, H-score of 1< 76%, H-score of 10< 44%, H-score of 100< 14%, TN; H-score of 0 30%, H-score of 1< 70%, H-score of 10< 54%, H-score of 100< 11%). On the other hand, samples from the metastatic site showed a higher rate of TROP-2 expression H-score of 1< compared to the primary site (primary site; H-score of 0 36%, H-score of 1< 64%, metastatic site; H-score of 0 18%, H-score of 1< 82%; p< 0.01). The rates of TROP2-positive expression defined as H-score of 1<, varied among the metastatic sites, with 100% in liver, 92% in brain, 89% in lymph nodes, 64% in lung, and 60% in bone. Regarding the co-expression of HER2 and TROP2, among HER2-positive samples, 35% had TROP2 H-score of 0, 24% had TROP2 H-score of 1<, and 41% had TROP2 H-score of 10<, while among HER2 low samples, 21% had TROP2 H-score of 0, 27% had TROP2 H-score of 1<, 39% had TROP2 H-score of 10<, and TROP2 H-score of 100< was found in 13% of patients. Based on the definition of co-expression such as TROP2 H-score of 1< and HER2 1+ or more, overall, 40% of the samples had co-expression of HER2 and TROP2, and the metastatic site had a higher rate of co-expression than the primary tumor (49%, 32% respectively, p=0.002). Conclusions: This study demonstrates that TROP-2 expression by IHC in breast cancer varies between primary and metastatic sites in breast cancer. Metastatic sites and specific organs show higher rate of TROP2 expression. Additionally, 40% of the samples had the co-expression of HER2 and TROP2, suggesting a potential target for future bispecific ADCs therapy. Citation Format: Mai Onishi, Tatsunori Shimoi, Yuki Kojima, Shu Yazaki, Taro Yamanaka, Rui Kitadai, Asuka Kawachi, Hitomi Okuma, Mai Hoshino, Ayumi Saito, Munehiro Ito, Aiko Maejima, Tadaaki Nishikawa, Kazuki Sudo, Emi Noguchi, Yasuhiro Fujiwara, Masayuki Yoshida, Kan Yonemori. Expression and co-expression patterns of TROP2 and HER2 in breast cancer: implications for bispecific antibody-drug conjugate therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-15-12.

Abstract PO4-26-08: Prevalence of HER2-low and IHC >0 to < 1+ in breast cancer and its concordance between historical and rescored results: a multi-center, retrospective study in China

Abstract Background: With Trastuzumab deruxtecan (T-DXd) changing the anti-HER2 treatment paradigm, HER2-low (defined as IHC2+/ISH- and IHC1+) breast cancer patients have emerged as a new targetable population. The lower threshold for HER2 expression that can benefit from HER2-directed antibody-drug conjugates (ADCs) is still being investigated, such as HER2 immunohistochemistry (IHC) >0 to < 1+ (defined as IHC 0 with incomplete and faint staining in ≤10% of tumor cells) in the DESTINY-Breast06 trial. Accurate determination of HER2 scores has become a critical topic in clinical discussions, given its clinical relevance to HER2-directed treatment strategies. Hence, we conduct this study (HER2-PATH, NCT05203458) to evaluate the distribution of HER2 status including HER2 IHC >0 to < 1+ in Chinese patients with breast cancer. Concordance between rescored and historical results was also analyzed. Methods: A retrospective study was conducted in breast cancer patients who underwent surgery at 10 sites in China between July 2021 and July 2022. Archived HER2 IHC slides from these patients were subjected to rescoring by a review committee comprising two readers and one adjudicator. The two readers independently evaluated each slide blinded to the historical scores. If their results matched, the recorded outcome was considered final. In cases of disagreement, the adjudicator made the final judgment. All slides were stained using Ventana 4B5 and scored following the ASCO/CAP 2018 guidelines, including the addition of the IHC >0< 1+ as defined in the DESTINY-Breast06 trial. The prevalence of the rescored HER2 status was calculated, and the concordance between the historical and rescored HER2 status was assessed using the Cohen's Kappa coefficient. Results: A total of 2868 patients were included in the analysis. The rescored results categorized 682 (23.8%) patients as HER2 IHC 0, 871 (30.4%) patients as IHC 1+, 800 (27.9%) patients as IHC 2+, and 515 (18.0%) patients as IHC 3+. The rates of HER2-positive, HER2-low, and HER2 IHC 0 (including HER2 null and HER2 IHC >0 to < 1+) were 21.8%, 54.3% and 23.9%, respectively. Notably, the prevalence of HER2-low was numerically higher in the HR-positive subgroup compared to the HR-negative subgroup (60.2% vs 30.6%). Furthermore, the prevalence of HER2 IHC >0 to < 1+ and HER2 null was 10.6% and 13.2% among all patients, respectively. Among the HR-positive subgroup, the prevalence of HER2 IHC >0 to < 1+ was 10.9%, while the rate was 9.1% among the HR-negative subgroup. Overall, there was an 83.1% concordance between the historical and rescored results for HER2 IHC scores. The concordance rate for IHC 1+ was numerically lower (74.5%) compared to IHC 0 (85.2%), IHC 2+ (81.4%), and IHC 3+ (98.6%). However, considering that 12.0% of IHC 1+ patients turned into IHC 2+ with limited impact on HER2-low diagnosis, the concordance rate for HER2-low remained as 91.6%. Conclusion: HER2-low prevalence in Chinese breast cancer patients was found to be consistent with global data, while additional 10.6% of patients were identified as HER2 IHC >0 to < 1+, which is currently being investigated in a randomized controlled trial comparing T-DXd with SoC. To our knowledge, this is the first study to report the prevalence of HER2-low, including HER2 IHC >0 to < 1+, in the Chinese breast cancer population based on rescored results. The concordance of HER2-low between the historical and rescored results was 91.6%, indicating that most cases could be reproducibly classified. Table: Rescored HER2 Expression Level Citation Format: Hong Lv, Junqiu Yue, Qingfu Zhang, Fangping Xu, Jianming Li, Lingfei Kong, Peng Gao, Guanjun Zhang, Haifeng Yang, Xiu Nie, Wen-Tao Yang. Prevalence of HER2-low and IHC >0 to < 1+ in breast cancer and its concordance between historical and rescored results: a multi-center, retrospective study in China [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-26-08.

Concordance between ER, PR, Ki67, and HER2-low expression in breast cancer by MammaTyper RT-qPCR and immunohistochemistry: implications for the practising pathologist.

There are limited data on the role of multigene tests and their correlation with immunohistochemistry (IHC), especially on core biopsy. MammaTyper is a quantitative conformite Europeeanne (CE) marked, National Institute for Health and Care excellence (NICE) approved, in in vitro diagnostic quantitative real-time polymerase chain reaction (RT-qPCR) test for assessment of mRNA expression of four biomarkers (ESR1, PGR, ERBB2, MKI67). We evaluated the concordance of MammaTyper with oestrogen receptor (ER), progesterone receptor(PR), HER2, and Ki67 by IHC on 133 core needle biopsies of breast cancer. HER2 was positive if IHC 3+ or 2+ and fluorescence in situ hybridization (FISH)-amplified. Global and hotspot Ki67 expression was analysed using a cutoff of ≥20% assessed manually and by digital image analysis. Agreements were expressed as overall percent agreement (OPA), positive percent agreement (PPA), negative percent agreement (NPA), and Cohen's kappa. RT-qPCR results of ESR1 were highly concordant with IHC with OPA of 94.7% using 1% cutoff and 91.7% when the low ER-positive category was included. The PPA and NPA between RT-qPCR and IHC for PR was 91.5% and 88.0%, respectively, when using the 1% cutoff. For ERBB2/HER2, the OPA was 95% and the PPA was 84.6%. 40 of 72 HER2 IHC score 0 tumours were classified as ERBB2 low. Best concordance between MKI67 by MammaTyper and Ki67 IHC was achieved using hotspot digital image analysis (OPA: 87.2%, PPA: 90.6%, NPA: 80%). RT-qPCR-based assessment of the mRNA expression of ESR1, PGR, ERBB2, and MKI67 showed high concordance with IHC, suggesting that the MammaTyper test on core needle biopsies represents a reliable, efficient, and reproducible alternative for breast cancer classification and refining HER2 low categorisation.

Trastuzumab deruxtecan in patients with metastatic non-small-cell lung cancer (DESTINY-Lung01): primary results of the HER2-overexpressing cohorts from a single-arm, phase 2 trial

DESTINY-Lung01 is a multicentre, open-label, phase 2 study evaluating the antitumour activity and safety of trastuzumab deruxtecan, a HER2-directed antibody-drug conjugate, in patients with HER2-overexpressing or HER2 (ERBB2)-mutant unresectable or metastatic non-small-cell lung cancer (NSCLC). The results of the HER2-mutant cohort (cohort 2) have been reported elsewhere. Herein, we report the primary analysis of cohorts 1 and 1A, which aimed to evaluate the activity and safety of trastuzumab deruxtecan 5·4 mg/kg and 6·4 mg/kg in patients with HER2-overexpressing NSCLC. Patients aged 18 years or older with unresectable or metastatic (or both unresectable and metastatic) non-squamous NSCLC who had relapsed following or were refractory to standard treatment or for whom no standard treatment was available, with an HER2 immunohistochemistry score of 3+ or 2+ (without known HER2 mutations) and an Eastern Cooperative Oncology Group performance status score of 0 or 1, were enrolled at 20 specialist hospitals in France, Japan, the Netherlands, Spain, and the USA. Patients were assigned to cohorts sequentially, first to cohort 1, to receive trastuzumab deruxtecan 6·4 mg/kg (cohort 1), then to cohort 1A, to receive trastuzumab deruxtecan 5·4 mg/kg, both administered intravenously once every 3 weeks. The primary endpoint was confirmed objective response rate by independent central review and was assessed in the full analysis set, which included all patients who signed an informed consent form and were enrolled in the study. Safety was assessed in all enrolled patients who received at least one dose of trastuzumab deruxtecan. This trial is registered with ClinicalTrials.gov, NCT03505710, and is ongoing (closed to recruitment). Between Aug 27, 2018, and Jan 28, 2020, 49 patients were enrolled in cohort 1 (median age 63·0 years [IQR 58·0-68·0], 30 [61%] male, 19 [39%] female, and 31 [63%] White), and from June 16 to Dec 9, 2020, 41 patients were enrolled in cohort 1A (median age 62·0 years [IQR 56·0-66·0], 22 [54%] male, 19 [46%] female, and 31 [76%] White). As of data cutoff (Dec 3, 2021), the median treatment duration was 4·1 months (IQR 1·4-7·1) in cohort 1 and 5·5 months (1·4-8·7) in cohort 1A, and median follow-up was 12·0 months (5·4-22·4) in cohort 1 and 10·6 months (4·5-13·5) in cohort 1A. Confirmed objective response rate by independent central review was 26·5% (95% CI 15·0-41·1; 13 of 49, all partial responses) in cohort 1 and 34·1% (20·1-50·6; 14 of 41; two complete responses and 12 partial responses) in cohort 1A. The most common treatment-emergent adverse events of grade 3 or worse were neutropenia (12 [24%] of 49 in cohort 1, none in cohort 1A), pneumonia (six [12%] and two [5%], respectively), fatigue (six [12%] and three [7%], respectively), and disease progression (six [12%] and four [10%], respectively). Drug-related treatment-emergent adverse events of grade 3 or worse occurred in 26 (53%) of 41 patients in cohort 1 and nine (22%) of 49 patients in cohort 1A. Drug-related serious adverse events were reported in ten (20%) patients and three (7%) patients, respectively. Deaths due to treatment-emergent adverse events occurred in ten (20%) patients in cohort 1 (disease progression in six (12%) patients and bronchospasm, hydrocephalus, respiratory failure, and pneumonitis in one [2%] patient each), and in seven (17%) patients in cohort 1A (due to disease progression in four (10%) patients and dyspnoea, malignant neoplasm, and sepsis in one (2%) patient each). One death due to a treatment-emergent adverse event was determined to be due to study treatment by the investigator, which was in cohort 1 (pneumonitis). Independent adjudication of interstitial lung disease or pneumonitis found that drug-related interstitial lung disease or pneumonitis occurred in ten (20%) patients in cohort 1 (two [4%] grade 1, five [10%] grade 2, and three [6%] grade 5) and two (5%) patients in cohort 1A (one [2%] grade 2 and one [2%] grade 5). An additional patient in cohort 1A had grade 4 pneumonitis after the data cutoff, which was subsequently adjudicated as drug-related grade 5 interstitial lung disease or pneumonitis. Given the low antitumour activity of existing treatment options in this patient population, trastuzumab deruxtecan might have the potential to fill a large unmet need in HER2-overexpressing NSCLC. Our findings support further investigation of trastuzumab deruxtecan in patients with HER2-overexpressing NSCLC. Daiichi Sankyo and AstraZeneca.

Abstract 7391: Beyond traditional staining: A virtual-IHC digital assay for HER2 score prediction from routine H&E images

Abstract Background: In breast cancer diagnostics, the accurate assessment of HER2 status is essential for treatment stratification. Conventionally, HER2 immunohistochemistry (IHC) scoring necessitates specialized staining protocols, which are resource- and time-intensive. This study presents a virtual IHC (vIHC) digital assay to predict HER2 scores directly from hematoxylin and eosin (H&E) stained images, which are routinely acquired for all specimens. This methodology aims to accelerate standard practices by reducing the need for additional IHC staining, thereby decreasing laboratory workload, costs, and turnaround time. It also presents an opportunity to screen a wider array of samples promptly, potentially identifying candidates for HER2-targeted therapy that might otherwise be delayed or overlooked. Methods: Serially sectioned breast core needle biopsy samples were stained with H&E and HER2-IHC following Leica-Bond staining protocols. Stained slides were imaged using the Hologic GeniusTM Digital Diagnostics System with Research Use Only software for Whole Slide Imaging. From this initial image dataset, 240 regions of interest (ROIs) were manually selected in order to create a dataset that uniformly covers: (i) all HER2-IHC scores (0-3), (ii) a wide range of HER2 stain intensities, and (iii) expression patterns. The IHC scores were assigned to each selected ROI by a pathology expert, following the scoring recommendations of ASCO/CAP American Society of Clinical Oncology and College of American Pathologists, with the caveat that ductal carcinoma in situ was also given a score for the purpose of training the model. These ROIs were then extracted from the images of the serially sectioned H&E-stained slides using Reveal Bio’s proprietary co-registration digital assay. A predictive digital assay was developed at Reveal Biosciences to classify the 240 H&E-stained breast tissue ROIs into four HER2 IHC score categories: 0, 1, 2, and 3. For model evaluation, cross-validation was performed 10 times by randomly splitting the dataset of H&E ROIs into 80% for training MIL and 20% for testing the model, followed by averaging the performance metrics across the 10 dataset splits. The performance metrics included classification accuracy, precision, recall, and F1 scores for each HER2 score category. Results: The vIHC digital assay for predicting the HER2 scores directly from H&E stained breast tissue images achieved the average classification accuracy of 93% across the four HER2 score categories on the test set of ROIs. This performance demonstrates that the model is capable of successfully distinguishing between the HER2 score categories directly from H&E images without the need for IHC staining. The results suggest that the vIHC digital assay could be a reliable and cost-effective tool for preliminary breast cancer screening, providing a valuable adjunct to traditional histopathological analysis. Citation Format: Misagh Naderi, Nikola Mitic, Nikola Spasic, Djordje Cikic, Kristen Ruf, Nikolaus Wagner, Igor Mihajlovic, Sinisa Todorovic, Ray Jenoski, Sid Mayer, Michael Quick, Claire Weston. Beyond traditional staining: A virtual-IHC digital assay for HER2 score prediction from routine H&E images [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7391.

Immunohistochemical assessment of HER2 low breast cancer: interobserver reproducibility and correlation with digital image analysis.

The recent findings from the DESTINY-Breast04 trial highlighted the clinical importance of distinguishing between HER2 immunohistochemistry (IHC) scores 0 and 1 + in metastatic breast cancer (BC). However, pathologist interpretation of HER2 IHC scoring is subjective, and standardized methodology is needed. We evaluated the consistency of HER2 IHC scoring among pathologists and the accuracy of digital image analysis (DIA) in interpreting HER2 IHC staining in cases of HER2-low BC. Fifty whole-slide biopsies of BC with HER2 IHC staining were evaluated, comprising 25 cases originally reported as IHC score 0 and 25 as 1 +. These slides were digitally scanned. Six pathologists with breast expertise independently reviewed and scored the scanned images, and DIA was applied. Agreement among pathologists and concordance between pathologist scores and DIA results were statistically analyzed using Kendall coefficient of concordance (W) tests. Substantial agreement among at least five of the six pathologists was found for 18 of the score 0 cases (72%) and 15 of the score 1 + cases (60%), indicating excellent interobserver agreement (W = 0.828). DIA scores were highly concordant with pathologist scores in 96% of cases (47/49), indicating excellent concordance (W = 0.959). Although breast subspecialty pathologists were relatively consistent in evaluating BC with HER2 IHC scores of 0 and 1 +, DIA may be a reliable supplementary tool to enhance the standardization and quantification of HER2 IHC assessment, especially in challenging cases where results may be ambiguous (i.e., scores 0-1 +). These findings hold promise for improving the accuracy and consistency of HER2 testing.

A gene expression-based classifier for HER2-low breast cancer

In clinical trials evaluating antibody-conjugated drugs (ADCs), HER2-low breast cancer is defined through protein immunohistochemistry scoring (IHC) 1+ or 2+ without gene amplification. However, in daily practice, the accuracy of IHC is compromised by inter-observer variability. Herein, we aimed to identify HER2-low breast cancer primary tumors by leveraging gene expression profiling. A discovery approach was applied to gene expression profile of institutional INT1 (n = 125) and INT2 (n = 84) datasets. We identified differentially expressed genes (DEGs) in each specific HER2 IHC category 0, 1+, 2+ and 3+. Principal Component Analysis was used to generate a HER2-low signature whose performance was evaluated in the independent INT3 (n = 95), and in the publicly available TCGA and GSE81538 datasets. The association between the HER2-low signature and HER2 IHC categories was evaluated by Kruskal–Wallis test with post hoc pair-wise comparisons. The HER2-low signature discriminatory capability was assessed by estimating the area under the receiver operating characteristic curve (AUC). Gene Ontology and KEGG analyses were performed to evaluate the HER2-low signature genes functional enrichment. A HER2-low signature was computed based on HER2 IHC category-specific DEGs. The twenty genes included in the signature were significantly enriched with lipid and steroid metabolism pathways, peptidase regulation, and humoral immune response. The HER2-low signature values showed a bell-shaped distribution across IHC categories (low values in 0 and 3+; high values in 1+ and 2+), effectively distinguishing HER2-low from 0 (p < 0.001) to 3+ (p < 0.001). Notably, the signature values were higher in tumors scored with 1+ as compared to 0. The HER2-low signature association with IHC categories and its bell-shaped distribution was confirmed in the independent INT3, TCGA and GSE81538 datasets. In the combined INT1 and INT3 datasets, the HER2-low signature achieved an AUC value of 0.74 (95% confidence interval, CI 0.67–0.81) in distinguishing HER2-low vs. the other categories, outperforming the individual ERBB2 mRNA AUC value of 0.52 (95% CI 0.43–0.60). These results represent a proof-of-concept for an observer-independent gene-expression-based classifier of HER2-low status. The herein identified 20-gene signature shows promise in distinguishing between HER2 0 and HER2-low expressing tumors, including those scored as 1+ at IHC, and in developing a selection approach for ADCs candidates.

Predictive markers for pathological complete response (pCR) after neo-adjuvant chemotherapy in HER2-positive breast carcinoma.

Patients with HER2-positive invasive breast cancer that is node-positive and/or larger than 3 cm are generally treated with neoadjuvant chemotherapy (NAC). We aimed to identify predictive markers for pathological complete response (pCR) after NAC in HER2-positive breast carcinoma. Hematoxylin/eosin-stained slides of 43 HER2-positive breast carcinoma biopsies were histopathologically reviewed. Immunohistochemistry (IHC) was performed on pre-NAC biopsies, comprising HER2, estrogen receptor (ER), progesterone receptor (PR), Ki-67, epidermal growth factor receptor (EGFR), mucin-4 (MUC4), p53 and p63. Dual-probe HER2 in situ hybridization (ISH) was performed to study the mean HER2 and CEP17 copy numbers. ISH and IHC data were retrospectively collected for a validation cohort, comprising 33 patients. Younger age at diagnosis, 3+ HER2 IHC scores, high mean HER2 copy numbers and high mean HER2/CEP17 ratios were significantly associated with an increased chance of achieving a pCR, and the latter two associations were confirmed in the validation cohort. No other immunohistochemical or histopathological markers were associated with pCR. This retrospective study of two community-based NAC-treated HER2-positive breast cancer patient cohorts identified high mean HER2 copy numbers as a strong predictor for pCR. Further studies on larger cohorts are required to determine a precise cut-point for this predictive marker.

HER2/ERBB2 copy number analysis by targeted next-generation sequencing in breast cancer.

A combination of immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) is the current standard of care for HER2 evaluation in breast cancer. Here, we investigate the potential clinical utility of next-generation sequencing (NGS)-derived HER2/ERBB2 copy number (CN) data for predicting HER2 status as defined by American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines. In total, 294 locally recurrent and metastatic breast cancers previously tested by targeted hybrid capture-based NGS and by HER2 IHC/FISH were included. Analyses focused on the ERBB2 median log2 ratios and start-end genomic coordinates from NGS, average HER2 CN and HER2/CEP17 ratios from FISH, and the HER2 IHC scores. We also determined a more stringent log2 ratio cutoff to predict HER2-positive status with 100% specificity. Sixty-four (22%) cases were HER2 positive and 230 (78%) were HER2 negative by ASCO/CAP guidelines. The ERBB2 median log2 ratios from NGS strongly correlated with HER2 status by IHC/FISH (area under receiver operator characteristic curve = 0.951). ERBB2 log2 ratio more than 1.7 was 100% specific for HER2-positive results by IHC/FISH. Start and end genomic coordinates for regions of gain near ERBB2 by NGS also predicted HER2 status. Copy number data from our NGS panel strongly correlate with HER2 status. Using a stringent cutoff, ERBB2 log2 ratio accurately predicts HER2 positivity with high specificity. The NGS CN assessment may have utility in determining HER2 status in certain clinical settings.

Preoperative systemic inflammation response index: Clinicopathologic predictor of pathological complete response in HER2-positive breast cancer patients receiving neoadjuvant systemic therapy.

Multiple pretreatment systemic inflammatory markers (SIMs) have been reported as predictors of pathological complete response (pCR) after neoadjuvant systemic therapy (NST) in patients with breast cancer (BC). However, the most significant SIM remains to be conclusively identified, and variations among different molecular subtypes remain unknown. The objective of the study was to identify the most significant SIM in patients with human epidermal growth factor receptor 2 (HER2) positive BC, to construct a pCR-predictive nomogram combining it with other clinicopathologic factors, and to evaluate its prognostic value on survival. We retrospectively reviewed the findings for 240 patients with stage I-III HER2-positive BC who underwent NST and subsequent surgery at Kaohsiung and Taichung Veterans General Hospital from 2011 to 2021. Clinicopathologic factors were analyzed by stepwise logistic regression with backward selection. The data were used to construct a nomogram plot for determining the pCR probability. Kaplan-Meier curves and log-rank test were used to evaluate disease-free survival (DFS) and overall survival (OS). Among the pretreatment SIMs, only the systemic inflammation response index (SIRI) was significantly related to pCR, with an optimal cutoff value of 1.27 × 10 9 /L. Stepwise logistic analyses indicated that clinical N stage, HER2 immunohistochemistry score, hormone receptor status, targeted therapy regimen, and SIRI were independent predictors of pCR, with an area under the curve of 0.722. The Hosmer-Lemeshow test and calibration curve revealed that the predictive ability was a good fit to actual observations. A nomogram was constructed based on the logistic model. The external validation of the model also revealed satisfactory discrimination and calibration. Kaplan-Meier analysis showed that patients with SIRI <1.27 had longer DFS and OS. Pretreatment SIRI <1.27 is predictive of pCR, DFS, and OS in HER2-positive BC. Our nomogram could efficiently predict pCR and facilitate clinical decision-making before neoadjuvant treatment.

Diagnostic Accuracy of Immunohistochemistry for HER2-Positive Breast Cancer.

Currently, human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells are diagnosed under the American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) 2018 guidelines. The guideline combined the results of in situ hybridization (DISH) and immunohistochemistry (IHC) techniques. The IHC technique is easy, cheap, and suitable for developing country. Therefore, in this study, we validated the use of IHC alone compared to the results of HER2 amplification under ASCO/CAP 2018 guidelines in diagnosed HER2 positive breast cancer cells. A total of 510 breast cancer tissue samples from Rajavithi Hospital in Bangkok, Thailand, from January 1st, 2022, to May 31st, 2023, were analyzed by IHC, followed by dual ISH (DISH). We selected 58 samples of IHC equivocal (score 2+) and 98 samples of IHC positive (score 3+) to analyze the diagnostic values by comparing them to the results of HER2 amplification. The HER2 IHC score was found to agree with HER2 amplification with a sensitivity of 87.96%, a specificity of 93.75%, a positive predictive value of 96.94%, a negative predictive value of 77.59%, a positive likelihood ratio of 14.07, a negative likelihood ratio of 0.13, and an accuracy of 89.74%. The promising outcomes suggest that a positive IHC test result (score 3+) could potentially stand alone for patients with breast cancer undergoing anti-HER2 treatment, even without DISH confirmation.

Abstract A036: A subset of lung adenocarcinomas defined by high-level ERBB2 amplification is vulnerable to HER2-targeted therapy

Abstract Background: Most lung adenocarcinomas (LUAD) are driven by activation of receptor tyrosine kinases (RTK). HER2 (encoded by ERBB2) is an RTK capable of ligand-independent dimerization and signaling when overexpressed. ERBB2 amplification (ERBB2amp) is a common cause of HER2 overexpression. While activating ERBB2 mutations (e.g., ERBB2 exon 20 YVMA insertions) are targetable drivers of LUAD, the role of ERBB2amp in lung cancer (LC) is unclear and there is no FDA-approved therapy for this indication in LC. Here, we report the clinicopathological and molecular characteristics of LUAD with high-level ERBB2amp and investigate the efficacy of HER2-targeted agents in LC models with ERBB2amp. We also demonstrate the clinical benefit of trastuzumab-deruxtecan (T-DXd) in a patient with ERBB2amp LUAD. Design: An institutional database of hybrid capture DNA NGS panel targeting 290-447 genes (v1-v3.1) was interrogated for LUAD with high-level ERBB2amp (defined as at least 6 calculated gene copies by NGS). HER2 immunohistochemistry (IHC) was performed on 29 available cases and scored according to ASCO/CAP scoring criteria. HER2 IHC scores of 2+ and 3+ were considered overexpression. Two LC cell lines, H2170 and CALU3 were identified as ERBB2amp and their responses to HER2 inhibitors were characterized with Western blotting, viability, and apoptosis assays. CALU3 and H2170 xenograft models were also established and treated with HER2 inhibitors or T-DXd. Results: ERBB2amp was identified in 31/3,684 (0.8%) LUAD patients. HER2 overexpression was present in 24 out of 29 evaluable cases (79.3%). Eighteen cases (58%) showed high-level ERBB2amp with no concurrent drivers and were more likely to be male (P = 0.001) and have a history of tobacco use (P = 0.001), compared to patients with high-level ERBB2amp with a concurrent driver mutation (activating ERBB2 SNV/indels, activating EGFR and MET mutations, RET fusion). H2170 and CALU3 cells displayed overexpression of HER2 and phospho-HER2, and growth of these cells showed sub-nanomolar sensitivity to trastuzumab and T-DXd, and low nanomolar sensitivity to afatinib. T-DXd induced apoptosis in both cell lines (assessed by activation of caspase 3/7 and elevation of BIM and cleaved PARP levels). While trastuzumab and afatinib significantly slowed down growth of H2170 and CALU3 xenografts, only T-DXd achieved tumor shrinkage equivalent to partial (H2170) or complete response (CALU3) in all animals. We describe the case of a LUAD patient with a RET rearrangement who acquired high-level ERBB2amp after progression on selpercatinib and was treated with T-DXd, with evidence of disease stability and clinical benefit. Conclusion: High-level ERBB2 amplification detected by NGS reliably predicts HER2 overexpression in lung adenocarcinoma. LUAD with ERBB2 amplification and no concurrent oncogenic drivers is a rare event that occurs predominantly in male smokers. This data can inform future trials investigating the utility of HER2-targeting agents in this population. Citation Format: Igor Odintsov, Maisam Makarem, Sara E. Bachert, Tom Zhang, Romel Somwar, Pasi A. Jänne, Lynette M. Sholl. A subset of lung adenocarcinomas defined by high-level ERBB2 amplification is vulnerable to HER2-targeted therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A036.

Standardized pathology report for HER2 testing in compliance with 2023 ASCO/CAP updates and 2023 ESMO consensus statements on HER2-low breast cancer.

Since the release of the DESTINY-Breast04 (DB-04) trial findings in June 2022, the field of pathology has seen a renaissance of HER2 as a predictive biomarker in breast cancer. The trial focused on patients with metastatic breast cancer who were classified as "HER2-low," i.e., those with immunohistochemistry (IHC) HER2 1 + or 2 + and negative in situ hybridization (ISH) results. The study revealed that treating these patients with trastuzumab deruxtecan (T-DXd) instead of the oncologist's chosen chemotherapy led to outstanding improvements in survival. This has challenged the existing binary HER2 pathological classification system, which categorized tumors as either positive (overexpression/amplification) or negative, as per the ASCO/CAP 2018 guideline reaffirmed by ASCO/CAP 2023 guideline update. Given that DB-04 excluded patients with HER2 IHC score 0 status, the results of the ongoing DB-06 trial may shed further light on the potential benefits of T-DXd therapy for these patients. Roughly half of all breast cancers are estimated to belong to the HER2-low category, which does not represent a distinct or specific subtype of cancer. Instead, it encompasses a diverse group of tumors that exhibit clinical, morphological, immunohistochemical, and molecular variations. However, HER2-low offers a distinctive biomarker status that identifies a specific therapeutic regimen (i.e.,T-DXd) linked to a favorable prognosis in breast cancer. This unique association emphasizes the importance of accurately identifying these tumors. Differentiating between a HER2 IHC score 0 and score 1 + has not been clinically significant until now. To ensure accurate classification and avoid misdiagnosis, it is necessary to adopt standardized procedures, guidelines, and specialized training for pathologists in interpreting HER2 expression in the lower spectrum. Additionally, the utilization of artificial intelligence holds promise in supporting this endeavor. Here, we address the current state of the art and unresolved issues in assessing HER2-low status, with a particular emphasis on the score 0. We explore the dilemma surrounding the exclusion of HER2-zero patients from potentially beneficial therapy based on traditional HER2 testing. Additionally, we examine the clinical context, considering that DB-04 primarily involved heavily pretreated late-stage metastatic breast cancers. We also delve into emerging evidence suggesting that extrapolating HER2-low status from the original diagnosis may lead to misleading results. Finally, we provide recommendations for conducting high-quality testing and propose a standardized pathology report in compliance with 2023 ASCO/CAP updates and 2023 ESMO consensus statements on HER2-low breast cancer.

Effect of HER2-low expression on neoadjuvant efficacy in operable breast cancer.

To investigate the effects of HER2-low expression (HER2-low) and HER2-zero expression (HER2-0) on the pathological complete response (pCR) rate and survival of patients following neoadjuvant chemotherapy. Eighty-six patients were followed up. Patients were divided into HER2-0 (immunohistochemistry (IHC) score of 0 (IHC0)) and HER2-low (IHC1+ or IHC2+/in situ hybridization non-amplified (ISH-)) groups according to the IHC detection of puncture tissues. After neoadjuvant chemotherapy, the clinical characteristics, pCR rate and DFS were compared between the two groups. There were 24 (27.9%) cases with HER2-0 and 62 (72.1%) cases with HER2-low. Hormone receptor-positive (HR+) patients accounted for 77.4% of the HER2-low group, which was higher than 70.8% in the HER2-0 group, and there was no significant difference between the two groups (p = 0.524). There were statistical differences in the pT and pN stages between HER2-low and HER2-0 subgroups in the triple-negative breast cancer (TNBC) group after neoadjuvant chemotherapy. The HER2-low subgroup had an earlier T stage (p = 0.009), and the ratio of N0 to N1 in the HER2-low and HER2-0 subgroups was 92.9% and 71.4%, respectively (p = 0.037). The Ki-67 index and median PR value were significantly lower in the HER2-low group after neoadjuvant chemotherapy (p = 0.002, p = 0.018). The HER2 IHC score was altered in the HER2-low group, and the HER-2 (2+) score changed significantly (p = 0.002). Seventy-eight patients with complete immunohistochemical data were analyzed. The discordance rate of the IHC score of HER2 after neoadjuvant chemotherapy was 38.5%, and eight patients with HER2-low showed HER2-0 status, with a discordance rate of 10.3%. After neoadjuvant chemotherapy, The pCR rate was significantly lower in the HER2-low group compared with that in the HER2-0 group (4.8% vs. 8.3%; p = 0.914), but the recurrence and metastasis rates were lower in the HER2-low group (9.7% vs. 20.8%; p = 0.165). There were no differences in DFS between the two groups at 6, 12, 24, and 36months (p = 0.076; p = 0.518; p = 0.245; p = 0.406). The subgroup analysis demonstrated no significant difference in DFS between HER2-low and HER2-0 subgroups in the HR + and TNBC groups (p = 0.141, p = 0.637). This retrospective study indicates that HER2-low has no significant effect on neoadjuvant efficacy in operable breast cancer. There were no statistical differences in clinical characteristics, pCR rate, and DFS between the HER2-low and the HER2-0 groups. There was no evidence that a HER2-low status constitutes a unique biological subtype, suggesting that more clinical data might be needed to verify these observations.