HER2 Immunohistochemistry Research Articles

Best practices for achieving consensus in HER2-low expression in breast cancer: current perspectives from practising pathologists.

Human epidermal growth factor receptor 2 (HER2) expression is an important biomarker in breast cancer (BC). Most BC cases categorised as HER2-negative (HER2-) express low levels of HER2 [immunohistochemistry (IHC) 1+ or IHC 2+/in-situ hybridisation not amplified (ISH-)] and represent a clinically relevant therapeutic category that is amenable to targeted therapy using a recently approved HER2-directed antibody-drug conjugate. A group of practising pathologists, with expertise in breast pathology and BC biomarker testing, outline best practices and guidance for achieving consensus in HER2 IHC scoring for BC. The authors describe current knowledge and challenges of IHC testing and scoring of HER2-low expressing BC and provide best practices and guidance for accurate identification of BCs expressing low levels of HER2. These expert pathologists propose an algorithm for assessing HER2 expression with validated IHC assays and incorporate the 2023 American Society of Clinical Oncology and College of American Pathologist guideline update. The authors also provide guidance on when to seek consensus for HER2 IHC scoring, how to incorporate HER2-low into IHC reporting and present examples of HER2 IHC staining, including challenging cases. Awareness of BC cases that are negative for HER protein overexpression/gene amplification and the related clinical relevance for targeted therapy highlight the importance of accurate HER2 IHC scoring for optimal treatment selection.

HER2-negative or low expression as an unfavorable prognostic factor in patients with stage I/II uterine carcinosarcoma.

Uterine carcinosarcoma (UCS) is uncommon high-grade endometrial cancer with limited treatment options. We evaluated the prognostic significance of human epidermal growth factor receptor 2 (HER2) expression and HER2 gene amplification within large cohorts of UCS, and clarify clinicopathologic characteristics of HER2-low UCS. We examined HER2 protein expression in 148 patients of UCS using in vivo diagnostic HER2 immunohistochemistry (IHC) kits and HER2 gene amplification using fluorescence in situ hybridization (FISH) in 72 patients. HER2 IHC score was evaluated according to the latest American Society of Clinical Oncology/College of American Pathologists criteria for gastric cancer, which was negative in 41 patients, low expression of 1+ was observed in 57 patients, and HER2 high expression was observed in 50 patients (2+ in 38 and 3+ in 12 patients). There was no significant statistical difference in clinicopathological characteristics based on HER2 protein expression status. HER2 negative and low expression compared to high expression revealed poor overall survival in stage I/ II. The concordance between IHC and FISH results were relatively low compared to other cancer types (HER2 IHC score 1+, 2+, and 3+ were 5%, 15%, and 50%), and combining these results was not efficient as a prognostic factor in UCS. In contrast, the HER2 IHC score alone was a prognostic factor in stage I/II UCS. HER2 low group did not show specific clinicopathologic features. Since the HER2 IHC score low in advanced UCS is a predictive factor, stratification of UCS using HER2 IHC score for HER2 IHC score low group and developing adjuvant therapy may be proposed in the near future.

Quantitative Measurement of HER2 Expression in Non–Small Cell Lung Cancer With a High-Sensitivity Assay

Recently, low human epidermal growth factor receptor 2 (HER2) protein expression has been proposed as a predictive biomarker for response to the antibody-drug conjugate trastuzumab deruxtecan (T-DXd) in metastatic breast cancer. HER2 expression in non–small cell lung cancer (NSCLC) patients has never been carefully measured, and little is known about the frequency of cases with unamplified but detectable levels of the protein. Although some HER2-targeted therapies have been studied in NSCLC patients, they have been restricted to those with genomic ERBB2 gene alterations, which only represent relatively rare cases of NSCLC. Still, emerging investigations of T-DXd in NSCLC have shown promise in patients with unamplified HER2. Taken together, we hypothesize that there may be many cases of NSCLC with levels of HER2 protein expression comparable with levels seen in breast cancer that benefit from T-DXd.Here, we used a previously validated, analytic, quantitative immunofluorescence (QIF) assay that is more sensitive than legacy clinical HER2 immunohistochemistry assays. We measured HER2 protein levels in NSCLC cases to determine the proportion of cases with detectable HER2 expression. Using cell line calibration microarrays alongside our QIF method enabled us to convert HER2 signal into units of attomoles per mm2. We found that over 63% of the 741 analyzed NSCLC cases exhibited HER2 expression above the limit of detection, with more than 17% of them exceeding the lower limit of quantification. Although the threshold for response to T-DXd in breast cancer is still unknown, many cases of NSCLC have expression in a range comparable to breast cancer cases with immunohistochemistry scores of 1+ or 2+. Our assay could potentially select NSCLC cases with a detectable target (ie, HER2) that might benefit from HER2 antibody-drug conjugates, irrespective of ERBB2 genomic alterations.

Human epidermal growth factor receptor 2 (HER2) status in breast cancer: practice points and challenges.

Human epidermal growth factor receptor 2 (HER2)-enriched breast cancer benefits significantly from anti-HER2 targeted therapies. This highlights the critical need for precise HER2 immunohistochemistry (IHC) interpretation serving as a triage tool for selecting patients for anti-HER2 regimens. Recently, the emerging eligibility of patients with HER2-low breast cancers for a novel HER2-targeted antibody-drug conjugate (T-DXd) adds challenges to HER2 IHC scoring interpretation, notably in the 0-1+ range, which shows high interobserver and interlaboratory staining platform variability. In this review, we navigate evolving challenges and suggest practical recommendations for HER2 IHC interpretation.

Impact of HER2 low status on genomic signatures in triple negative breast cancer (TNBC).

1092 Background: HER2 status has become more important with new treatment regimens subcategorizing HER2 to into low (1+or 2+ FISH-) or absent HER2 expression. Through comprehensive genomic profiling (CGP), we aim to identify distinctive molecular features associated with various immunohistochemistry (IHC) categories of HER2 low tumors. Methods: Retrospective review of pathology specimens submitted to Foundation Medicine were done and 514 TNBC (ER-/PR-/HER20,1+,2+FISH-) and 191 3+/HER2 amplified clinically advanced BC underwent hybrid capture-based CGP to evaluate genomic alterations (GA) in 324 genes, MSI status and tumor mutational burden (TMB, mutations/Mb). PD-L1 expression was determined by IHC (Dako 22C3 TPS). Chi-square test and Mann Whitney U test were used in the statistical comparisons of the 2 groups. Statistical significance was defined as p < 0.05. Results: 53.5% were HER2 IHC 0+, 31.5% were HER2 IHC 1+ and 15.0% were IHC 2+/FISH negative. The HER2 3+/amplified BC were 59% ER+ and 41% ER-. Notable differences in GA frequencies included a significant increase in PIK3CA GA in the HER2 2+ vs the HER 0+ and 1+ groups (p=.0003). A lower frequency in BRCA1 GA in the HER2 2+ (5.2%) vs the HER2 0+ (9.8%) was significant when compared to the 1.0% in the HER2 3+/amplified cases (p<.0001). BRCA2 GA frequencies did not show statistically significant differences among the TNBC HER2 IHC score groups. TP53 and RB1 had the highest frequency in HER2 0+ samples, with decreasing prevalence in the HER2 1+, 2+ and 3+/amplified cases (p<.0001 for both). There was also a trend towards higher frequency of CDH1 GA as the HER2 IHC score increased from 0 to 2+ among TNBC. There were no significant differences in immunotherapy biomarkers (MSI, TMB and PD-L1) based on HER2 status among the TNBC groups. TMB >= 10 mutations/Mb was significantly higher in the HER2 3+/amplified group (p=.004). Conclusions: We found that TNBC HER2 2+ positive FISH show key molecular differences from their HER2 low (1+) or absent (0+) counterparts. The most substantial difference was an increase in GA in PIK3CA. While this is targetable in the hormone positive setting, its potential role as a target in the hormone negative setting is uncertain. Also noted but did not reach significance was a decrease in GA within BRCA1/2 and a positive linear relationship with CDH1 and HER2 IHC expression. These GA differences may indicate potential treatment options for different HER2 subgroups and warrant further investigation. Limitations of our study include the retrospective nature and the lack of clinical data. [Table: see text]

Off-label use of fam-trastuzumab deruxtecan-nxki with early activity in a cohort of patients with desmoplastic small round cell tumor.

11584 Background: Desmoplastic small round cell tumor (DSRCT) is a rare and aggressive sarcoma occurring predominantly in pediatric, adolescent and young adult biologic males, and is molecularly characterized by the pathognomonic EWSR1-WT1 fusion. Despite aggressive multi-modality therapy outcomes for those with DSRCT remain poor, and more effective therapies are urgently required. Prior pre-clinical data and transcriptomic analyses of DSRCT patients has suggested the relevance of the ERBB2 pathway in this disease, prompting the off-label use of fam-trastuzumab deruxtecan-nxki (T-DXd) in the treatment of patients of DSRCT patient with relapsed and refractory disease. Methods: Patients with relapsed/refractory DSRCT received T-DXd 5.4 mg/kg intravenously in 21-day cycles. Whole body PET and CT of the chest, abdomen and pelvis were used for monitoring. Response was correlated with HER2 immunohistochemistry (IHC) status and transcriptomic analysis by RNA sequencing (RNAseq) when available. Results: Seven patients received the agent, and at the time of submission 3 patients had at least one follow up imaging test after receiving between 6 and 9 cycles of T-DXd. Treatment was overall well tolerated with the only grade 3 toxicity of transaminitis (in patients with known liver metastases), which self-resolved. All other toxicities including constipation, nausea, and fatigue were grade 2 or less. All 3 patients have experienced clinical benefit including one patient with a RECIST 1.1 partial response and two patients with stable disease. Additionally, favorable responses were noted by PET imaging. Four additional patients with relapsed/refractory DSRCT have initiated off-label treatment with T-DXd and are pending imaging responses. 7/7 and 4/7 patients have available IHC and ERBB2 RNA expression data available respectively. All ERBB2 RNA expression data is elevated when compared with the median across all evaluated pediatric tumors in our institutional series. Conclusions: The use of anti-HER2 therapy in a limited and unselected group of heavily pre-treated DSRCT patients is demonstrating an early signal of activity with minimal toxicity and suggests that formal assessment as monotherapy or in other relevant combinations in clinical trials is warranted. Further correlation with IHC and RNAseq expression data may help optimize correlatives and/or generate biomarkers for future clinical trials.

A meta-analysis of the difference in response to trastuzumab-deruxtecan (T-DXd) based on HER2 immunohistochemistry (IHC) in HER2 low metastatic breast cancer (mBC).

e13164 Background: HER2 low BC have an IHC staining of 1+ or 2+ with negative fluorescence in situ hybridization (ISH). Although T-DXd is approved for this, difference in activity between 1+ and 2+ is uncertain. Methods: A systematic search with controlled vocabulary encompassing BC and T-DXd was conducted in PubMed, Embase, Scopus, and Cochrane on November 11 2023 with no search restrictions. 2318 records were identified and duplicates were removed using Mendeley. The remaining 1189 records were imported into Rayyan, and the titles were screened independently by 2 reviewers. 47/75 full texts were relevant, of which 4 [3 clinical trials, 1 retrospective study] were included for analysis. Only studies that provided outcome data for HER2 1+ and 2+ ISH- were included. RevMan was used to analyze the Risk Ratio (RR) and Odds Ratio (OR) with 95% Confidence Interval (CI), derived from Random Effects (RE) model with Mantel-Haenszel (MH) method. Pooled proportions (PP) were determined using OpenMeta. Results: There were a total of 264 patients in 1+ and 204 in 2+ISH-. Median age was 57 years. All studies had pre-treated mBC. T-DXd dose was at least 5.4 mg/kg every 3 weeks. Visual inspection of our funnel plots revealed no bias. PP (3/4 studies) for patients achieving an Objective Response Rate (ORR) [Complete Response (CR)+Partial Response (PR)] was 42.9% (31.6-54.2%) in 1+ and 38% (26.3-49.7%) in 2+. The median Progression free survival (2/4 studies) was 8.6 (1+) and 7.95 (2+) months respectively. RE RR (3/4 studies) for Progressive Disease (PD) showed no difference between 1+ and 2+ [0.93 (0.80, 1.10), p=0.4, I²=0%]. Similarly, RE OR (3/4 studies) for Disease Control Rate (DCR) [CR+PR+Stable disease (SD)] [1.20 (0.78, 1.85), p=0.41, I²=0%], ORR [1.17 (0.58, 2.34), p=0.66, I²=0%], and PR [1.05 (0.52, 2.11), p=0.89, I²=0%] showed no significant difference between 1+ and 2+ (Table). Conclusions: Our results indicate no difference in response to T-DXd between 1+ and 2+, solidifying its role as a treatment option in HER2 low mBC. Limited number and heterogeneity between studies are limitations of our study, which ought to be addressed in future trials through clear subgroup stratification between 1+ and 2+. [Table: see text]

Influence of neoadjuvant regimen on the prognosis of locally advanced gastric and gastroesophageal junction cancer with HER2 overexpression.

e16082 Background: The relative high recurrence rate in locally advanced gastric or gastroesophageal junction (G/GEJ) cancer with Her2 overexpression after surgery, along with the absence of a standard neoadjuvant treatment protocol, necessitates exploration of novel approaches for this patient population. Methods: In this study, we conducted a retrospective analysis of clinical characteristics, postoperative pathological results, and survival data for patients who underwent neoadjuvant treatment and subsequent surgery at our center from September 2020 to December 31, 2022. The inclusion criteria were as follows: 1) pathologically confirmed adenocarcinoma of the G/GEJ; 2) diagnosed as cT3-4 or N+ by enhanced CT and endoscopic ultrasound; 3) Her2 immunohistochemistry 3+ or 2+ with Fish positive in biopsy specimens; 4) discussed in a multidisciplinary team for the need for neoadjuvant treatment; 5) received 2-4 cycles of neoadjuvant therapy; 6) underwent radical D2 surgery; 7) had complete follow-up data. Results: Seventy-three patients met the inclusion criteria, with 19 receiving chemotherapy + anti-PD-1, 10 receiving chemotherapy + anti-Her2, and 44 receiving chemotherapy alone. Their pCR rates were 31.6%, 10%, and 2.3%, respectively (P = 0.003); MPR rates were 42.1%, 30%, and 11.4% (P = 0.022). As of the last follow-up on December 31, 2023, the median DFS for the chemotherapy group was 18.1 months (95% confidence interval 14.9-21.3); median DFS for the other two groups had not been reached. DFS analysis revealed differences in median DFS among different neoadjuvant treatment regimens (P = 0.025); further pairwise comparisons indicated that this difference mainly stemmed from the chemotherapy + anti-Her2 group compared to the chemotherapy group. Conclusions: In locally advanced gastric and gastroesophageal junction cancer with Her2 overexpression, neoadjuvant chemotherapy combined with anti-Her2 treatment improves the pCR rate and prolongs DFS compared to neoadjuvant chemotherapy alone.

Biologic and prognostic significance of HER2-low and ER-low expressions in de novo metastatic breast cancer.

e13170 Background: It is unclear whether ER-low-positive (ER-low) and HER2-low-positive (HER2-low) biomarkers, as new biological subtypes, have different biological and clinicopathological characteristics in de novo metastatic breast cancer (dnMBC). Per ASCO/CAP guidelines, tumors were defined as HER2-low-positive (HER2-low) if they had an HER2 immunohistochemical (IHC) score of 1+ or 2+ with negative in situ hybridization assay and HER2-0 if they had an HER2 IHC score of 0; ER-low refers to the tumor IHC staining of 1%-10%, if the staining is less than 1%, it is defined as ER-0. This study aimed to investigate whether ER-low and HER2-low expressions is associated with clinicopathologic characteristics and prognosis among patients with de novo metastatic breast cancer. The study was approved by the Ethics Committee of Hebei Cangzhou Hospital of Integrated Traditional Chinese and Western Medicine (2017-AF29-058). Methods: Using hospital-based database, we identified patients with dnMBC diagnosed between 2010 and 2017, retrospectively collecting demographic data, tumor characteristics, treatment types, histopathological report and survival data. According to ASCO/CAP guideline recommendations, patient-specific histopathological results were classified, and IHC-based breast cancer markers, mainly including ER-low and HER2-low, were evaluated and analyzed. Median survival time (MST) was estimated by Kaplan-Meier method and log rank test. Univariable and multivariable analyses were performed using the Cox proportional hazard model to identify statistically significant prognostic factors. All statistical analyses were performed using Stata 18.0. Results: A total of 518 patients (median age of 53 years) were included in this study, including 194 patients with HER2-low, 201 patients with HER2-low, 36 patients with ER-low, and 179 patients with ER-0. Expression of HER2-low was significantly more common among ER-positive tumors (excluding the ER-low) compared with HER2-0 tumors (71.1% vs. 62.2%; P < .001). The MST in the HER2-low cohort was longer than that in the HER2-0 cohort (40 vs. 30 months; HR,0.65; 95% CI, 0.52-0.82, p=.0001); while the MST of ER-low cohort was slightly longer than that of the ER-0 cohort (27 vs. 20 months; HR,0.75; 95% CI, 0.51-1.11, p=.231), but there was no statistical difference. Notably, in the metastasis pattern cohort, HER2-low showed a significantly higher MST compared with HER2-0 (P<.001), with a MST of up to 68 months for bone-only metastases. There was no significant difference in MST between ER-low and ER-0 regardless of whether patients received endocrine therapy (P=.135). Conclusions: In the present study, for dnMBC patients, ER-low expression has similar biological and clinical outcome characteristics as ER-0. However, unlike HER2-0, HER2-low expression may predict a survival benefit in bone-only metastases.

Identification of HER2 ultra-low based on an artificial intelligence (AI)-powered HER2 subcellular quantification from HER2 immunohistochemistry images.

1115 Background: HER2-targeted antibody-drug conjugates (ADCs) can effectively target tumor cells even in HER2-low breast cancers (1+ and 2+/no amp). We developed an AI-based whole-slide image (WSI) analyzer for immunohistochemistry (IHC)-stained slides, exploring a cutoff differentiating between true HER2 negative versus HER2 ultra-low. Methods: An AI-based IHC WSI analyzer was developed using 6,188 WSIs stained by 19 types of IHC including HER2 and from various cancers, annotated by 153 pathologists. The model predicts cells as tumor cells or non-tumor cells and generates HER2 expression continuous score (HER2ecs, 0-100%), quantifying IHC staining intensity of membrane, nucleus, cytoplasm, and membrane+cytoplasm separately for each cell. A total of 401 HER2 IHC WSIs were acquired from Samsung Medical Center (n=275), Kyung Hee University Hospital (n=78), and Commercial Biobank (n=48), scored as 0 (n=347) and 1+ (n=54) by pathologists. Results: In total, 67,169,114 tumor cells and 119,113,886 non-tumor cells from 401 WSIs were successfully evaluated. Comparing HER2 score 0 versus 1+ by pathologists, mean HER2ecs of nucleus (1.7% vs 4.1%), cytoplasm (6.5% vs 12.9%), and membrane (3.6% vs 8.2%) were comparable. In HER2 score 0 WSIs, membrane HER2ecs of tumor cell was 3.6 ± 7.8% (mean ± standard deviation), while that of non-tumor cell was 0.9 ± 2.9% (p <0.001 vs. tumor cell), suggesting, even in HER2 score 0 cases, tumor cell exhibits HER2 expression beyond non-specific HER2 staining intensity. Based on our internal validation dataset, membrane-specific HER2ecs of 6.0% was the optimal threshold discriminating HER2 no-staining cells versus HER2 incomplete and faint/barely perceptible cells (AI-HER2-weak) per ASCO/CAP guideline. Among HER2 score 0 by pathologists, when applying this cutoff, the proportion of WSIs showing AI-HER2-weak or higher membrane-specific HER2ecs in over 10% of all tumor cells was 23.6% (82/347). Interestingly, that of WSIs among HER2 score 1+ by pathologists was 51.9% (28/54), which is comparable to 52.3%, objective response rate of trastuzumab deruxtecan in HER2-low patients in DESTINY-Breast04. Conclusions: AI-powered subcellular-level analysis enables identification of 23.6% HER2 ultra-low WSIs among HER2 score 0 by pathologist, which is defined by AI-HER2-weak or higher membrane-specific HER2 expression ≥ 10%. Clinical validation of the AI-powered HER2 ultra-low definition is warranted.

Peony exploratory analyses of biomarker changes: From baseline to surgical samples in patients with HER2+ breast cancer.

e15136 Background: PEONY (NCT02586025) is a randomized, multicenter, double-blind, placebo-controlled, phase III trial evaluating neoadjuvant and adjuvant pertuzumab/placebo with trastuzumab and docetaxel in HER2-positive early/locally advanced breast cancer. We previously reported that pertuzumab showed an improved efficacy vs. placebo in terms of total pathological complete response (tpCR), event-free survival and disease-free survival (DFS), as well as in pre-defined biomarker subgroups. Limited data are available on biomarker changes after neoadjuvant treatment in the Asian population. Thus, we aim to (1) describe the biomarker changes from baseline to surgical samples after HER2 targeted neoadjuvant therapy, and (2) explore the association of surgical biomarkers and DFS. Methods: This study will focus on biomarkers of tumor tissue samples collected at baseline and surgery among 218 non-tpCR patients, due to limited samples collected at disease progression. Samples were tested using Immunohistochemistry (IHC) for HER3, PTEN, CD8 and PD-L1. Both HER2/HER3 mRNA expression and PIK3CA mutation were assessed using a PCR based methodology. The statistical analyses were using R (4.1.3). Biomarkers were categorized as high/low either by median derived from baseline samples, or with well-established cutoffs, e.g. HER2 IHC of 3+ vs. 1/2+ and PD-L1 at 1%. The changes of biomarkers were examined among the paired samples using Fisher’s exact test and depicted by alluvial plots. Cox proportional hazard regression analysis was performed to examine the association between surgical biomarkers and DFS in pooled arms. Results: Since the surgical biomarkers are only evaluable among patients with residual disease, patients in this analysis had a higher stage, and were more likely to be node+. Of 166 paired samples, more patients show a decrease in HER2 and HER3 mRNA/IHC, PTEN IHC from baseline to surgery. PIK3CA mutation status remained the same among the majority of patients (n = 154, 95%). Immune-related biomarkers, PD-L1 and CD8, are more enriched in surgical samples compared to baseline. All these observations are similar between the two arms. No clear association has been shown in most of the biomarkers, except for PIK3CA, with DFS. Surgical tumor samples with PIK3CA mutation detected trended with a worse DFS than those with no PIK3CA mutation detected [HR: 1.81 (95% CI: 0.94 – 3.49)]. Conclusions: Of baseline and surgery paired samples, most biomarkers showed a change, except for PIK3CA mutation status. In pooled analyses, we only observed a trend of PIK3CA mutation detected in surgical samples with a worse DFS, which is in line with the association observed with baseline PIK3CA mutation status. No other associations were found between surgical biomarkers and DFS. These findings may suggest that despite biomarker changes patients continue to benefit From HER2 therapies and dual blockade. Clinical trial information: NCT02586025 .

Dose-dense adjuvant chemotherapy in patients with HER2-low early breast cancer: An exploratory analysis of the GIM2 trial.

532 Background: Dose-dense (DD) adjuvant chemotherapy represents the standard treatment for patients with node-positive early-stage HER2-negative breast cancer, but its role in HER2-low disease is unknown. In this exploratory analysis of the Gruppo Italiano Mammella 2 (GIM2) trial, we investigated efficacy of DD chemotherapy among patients affected by HER2-negative breast cancer according to HER2 immunohistochemistry (IHC) score (low vs. zero). Methods: Patients with node-positive early breast cancer were randomly assigned to receive either DD or standard schedule anthracycline- and taxane-based chemotherapy. HER2 status was assessed locally. Tumours with HER2 score 0 were classified as HER2-zero, while those with a HER2 score 1+ or 2+ without fluorescence in situ hybridization (FISH) amplification were defined as HER2-low. Some tumours were classified as HER2-negative with unknown IHC score. Survival outcomes were compared between patients with HER2-zero vs HER2-low tumours. Results: Among 2003 patients enrolled in the GIM2 trial, 1243 subjects were eligible for this analysis. Median age was 52 years (interquartile range [IQR] 44-59) and 604 patients (48.6%) were pre-menopausal. Hormone receptor status was positive in 87.9% of cancers. 475 tumours were classified as HER2-zero (38.2%), 446 (35.9%) were defined HER2-low and 322 (25.9%) were HER2-negative with unknown IHC score. No significant differences were found in terms of baseline characteristics between these three subgroups. At a median follow-up of 14.9 years (IQR 8.4-16.2), no significant interaction was observed between treatment effect and HER2 status in invasive disease-free survival (iDFS) (p for interaction=0.46) nor in overall survival (OS) (p for interaction=0.43). Comparing patients with HER2-zero breast cancer with those affected by HER2-low tumours, no significant differences in iDFS were observed (15yr iDFS were 61% vs. 54%, respectively, p=0.25), nor OS differences (15yr OS were 75% vs. 72%, respectively, p=0.19). When investigating DD schedule efficacy among patients with HER2-zero tumours, hazard ratio (HR) for iDFS was 0.64 (95% confidence interval [CI] 0.46-0.87) and HR for OS was 0.57 (95%CI 0.38-0.88). Similarly, within patients affected by HER2-low breast cancer, HR was 0.83 (95%CI 0.62-1.11) for iDFS and 0.83 (95%CI 0.57-1.22) for OS. These results were consistent with the findings of efficacy of DD chemotherapy in the overall population included in this analysis (HR for iDFS 0.73 [95%CI 0.61-0.89]; HR for OS 0.69 [95%CI 0.54-0.88]). Conclusions: In this exploratory analysis of a large phase III adjuvant trial, HER2 status (i.e. HER2-zero vs HER2low) did not show prognostic value. In patients with high-risk early breast cancer, the benefit of DD chemotherapy was observed irrespective of HER2 status. Clinical trial information: NCT00433420 .

The benefit of chart curation of electronic health record (EHR) data: Comparing data completeness between curated and structured data.

11165 Background: The main objective of an EHR is to effectively standardize clinical workflow. However, EHR data is also utilized extensively as secondary data for clinical research and quality improvement. Healthcare data exists in different forms such as paper, digital images, and notes, only some of which are recorded as structured data in the EHR. It is therefore essential to curate detailed information from unstructured data so that the patient’s journey can be understood in depth for research and quality studies. Methods: Patients diagnosed with metastatic breast cancer (mBC) between 01-Jan-2020 and 31-Dec-2022 were identified from the Integra Connect PrecisionQ de-identified database of 3 million cancer patients across 500 sites of care across ~80% community oncology and ~20% academic practices. Manual curation was conducted for a sample of these patients and the fill rates of crucial elements were captured as part of this study. Results: A total of 13,763 mBC patients were identified during this study period. The availability of data for the different variables was assessed from structured data in 13,120 patients. Additional information was obtained by manual curation for 643 patients. Information on staging and grade were available for 62.8% in the structured data, while curation increased the availability of these details to 99.5% among those 643 patients. The fill rates for patients’ tumor size and nodal status, as defined by the T and N values, were found to be 99% in the curated data compared to 60% in the structured data. Similarly, the fill rates were more than 98% for estrogen, progesterone, and HER2 receptor by curation, while they were only approximately 65% in the structured data. HER2 low, HER2-ve, and HER2+ve status was identified in 8%, 45.2%, and 10.1% by structured data and in 57.1%, 84.1%, and 14.0%, respectively, by curated data. Furthermore, the fill rates via curation were 97.1% for HER2 immunohistochemistry tests compared to 49.0% for structured data, while the fill rates for in-situ hybridization (ISH) or fluorescent in-situ hybridization (FISH) tests were 61.3% for curated data compared to 28.4% for structured data. This discrepancy is caused by the fact that IHC and FISH/ISH tests are primarily found only in PDF format. Conclusions: This study highlights the need for curation in order to maximize the utilization of EHR data for secondary research purposes and quality studies. Natural language processing and augmented curation methods can further enhance the quality of EHR data for secondary research.

HER2-neu Expression and Survival in Colorectal Cancer in the South of Egypt; Immunohistochemistry and Genetic Study.

Colorectal cancer (CRC) is a major public health problem and one of leading cancer related death all over the world. One of the prognostic parameters that play a role in different types of cancer is HER2. However, the role of HER2 in CRC and its relation with clinicopathological features and survival is conflicting. We hypothesize that HER2 has different patterns of expression in CRC which may affect the prognosis of patients. We studied sixty specimens of colorectal carcinoma for HER2 immunohistochemistry and gene amplification and correlate it with clinicopathological features and patients` survival. Our data showed that negative HER2 expression was statistically associated with female gender (P = 0.010) and low & intermediate tumor budding (P = 0.030). There was a statistically significant relation between HER2 IHC and HER2 FISH amplification (P=0.000). Although neither HER2 immunoexpression and FISH amplification showed significant relation with overall survival nor disease free survival, HER2 amplified CRCs tended to have a worse survival compared with negative CRCs (40 months versus 50 months). The presence of male gender, lymphovascular invasion, nodal metastasis and distant metastasis (P = 0.013, 0.006, 0.006 and 0.000 respectively) were significantly statistically associated with poor overall survival. The presence of tumor grade III and high tumor budding (P = 0.035 and 0.007 respectively) were significantly statistically associated with shorter disease free survival. Our results showed that HER2 IHC 3+ staining is highly predictive of HER2 gene amplification in colorectal carcinomas. There is a tendency towards poorer prognosis in amplified HER2 CRC cases.

Abstract A007: Circulating tumor DNA and tissue staining analyses reveal heterogeneous ERBB2/HER2 status in urothelial cancer

Abstract Human epidermal growth factor receptor 2 (HER2) is encoded by the ERBB2 gene, and frequently mutated, amplified, and/or overexpressed in urothelial cancer (UC). Promising antibody-drug conjugates have led to new interest in HER2 as a UC clinical target. Patient selection for HER2-targeted therapy typically relies on immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) of tumor tissue, however DNA sequencing can identify ERBB2 genomic alterations. Accurate biomarker-driven patient selection will be critical to optimize the clinical benefit of HER2-targeted therapy. Therefore, we evaluated plasma circulating tumor DNA (ctDNA) for ERBB2/HER2 status determination in UC, as compared to IHC and FISH of metachronous tissue. 411 plasma samples from 236 metastatic UC patients were profiled with targeted sequencing across >50 genes frequently altered in UC, including dense coverage flanking the ERBB2 locus. Using an established bioinformatics workflow, we determined the presence of ERBB2 alterations (mutations, amplifications, and/or structural variants) in the 181 patients with evidence of ctDNA in ≥1 sample. Clinical records were reviewed for the availability of archival tumor tissue; in total, 81 formalin-fixed paraffin-embedded tissue specimens were retrieved from 23 patients with ctDNA ERBB2 alterations and 20 ERBB2-wildtype controls. HER2 IHC was performed with a polyclonal rabbit anti-human Her2 antibody (Dako) and scored according to gastric cancer guidelines. HER2 FISH was performed with the PathVysion HER-2 DNA Probe Kit. Protein-altering ERBB2 mutations were identified in 14% of evaluable patients, with two-thirds at known oncogenic hotspots. ERBB2 copy gain was detected in 8% of patients overall, 9% when excluding low tumor fraction samples. IHC results were assessable for 82 tissue samples from 43 patients, and 33 patients had at least one sample with positive HER2 staining (2+/3+). IHC scores varied in 16/23 patients with ≥2 tissue samples, with the variation leading to a change in classification (HER2-negative [0/1+] versus positive [2+/3+]) in half (8/16). In two patients with mixed variant histology, the urothelial component showed IHC 3+ while the scores were 2+ (plasmacytoid) and 0 (squamous) in the variant regions. Frequent focal staining patterns were observed with both IHC and FISH. ERBB2 alterations in ctDNA were correlated with IHC positivity – when considering the most recent tissue sample per patient, 79% of ctDNA ERBB2-altered patient’s tumors were also positive by IHC. Conversely, 55% of ctDNA ERBB2-wildtype cases were positive by IHC. FISH results were available from 24 samples (20 positive, 4 negative), all from patients with ctDNA ERBB2 amplification. 18/20 samples with FISH positivity were HER2 IHC 2+/3+; the remaining two samples were IHC 1+. Our results demonstrate significant spatial and/or temporal heterogeneity in ERBB2/HER2 status, with implications for the rational implementation of biomarker-directed (HER2-targeted) therapy in UC. Citation Format: Gillian Vandekerkhove, Andrew J. Murtha, David C. Müller, Kimia Rostin, Carlos Vasquez-Rios, Jussi Nikkola, Maria Stephenson, Emily Fung, Jaskirat Atwal, Karan Parekh, Cecily Q. Bernales, Gráinne Donnellan, Gang Wang, Tilman Todenhöfer, Piet Ost, Peter C. Black, Kim N. Chi, Bernhard J. Eigl, Alexander W. Wyatt. Circulating tumor DNA and tissue staining analyses reveal heterogeneous ERBB2/HER2 status in urothelial cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A007.

Novel approach to HER2 quantification using phosphor-integrated dots in human breast invasive cancer microarray.

HER2 expression in breast cancer is evaluated to select patients for anti-HER2 therapy. With the advent of newly approved HER2-targeted drugs for low HER2 expression breast cancer, more solid evidence on the whole spectrum of HER2 expression is needed. In this study, we quantitatively assessed HER2 expression from the whole core by combining high-intensity phosphor-integrated dot (PID) immunostaining and whole slide imaging (WSI) analysis. Two types of staining were performed using a 170-core tissue microarray of invasive breast cancer. First, HER2 was stained by immunohistochemistry (IHC), and IHC scores were determined by two practicing pathologists according to the ASCO/CAP HER2 guideline. Second, HER2 was stained with PID, and tentative PID scores were determined by quantitative analysis. The results show that PID can numerically classify HER2 expression status into scores 3+, 2+, 1+, and 0. The HER2 value quantified by PID strongly correlated with the 3, 3'-diaminobenzidine (DAB) IHC score determined by pathologists (R2 = 0.93). PID IHC score 1+ cases included both DAB IHC score 1+ and 0 cases, and low HER2 expression cases appeared to be often evaluated as DAB IHC score 0. Therefore, digital image analysis by PID and WSI can help stratify HER2 IHC. It may also help classify low HER2 expression.

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.

Quantitative HER2 mRNA assay in breast cancer with HER2 immunohistochemistry 0

Objective: To investigate HER2 mRNA expression in breast cancer with HER2 immunohistochemistry (IHC) 0 and to analyze the feasibility of distinguishing between the tumor with HER2 μltra-low expression and the one without expression of HER2 (no staining by IHC) by HER2 mRNA level preliminarily. Methods: HER2 mRNA was analyzed by reverse transcription digital PCR in 41 cases of formalin-fixed paraffin-embedded surgical tissue samples of invasive breast cancer obtained between January 2020 and March 2023 at Peking Union Medical College Hospital. The cohort included 21 HER2 IHC 1+ and 20 IHC 0 (12 ultra-low and 8 non-expression of HER2). HER2 mRNA expression level was quantitatively evaluated by the FAM (HER2)/VIC (reference gene) ratio. Results: The expression of HER2 mRNA for the cases with 1+, ultra-low, and non-expression of HER2 by IHC was 0.30 to 1.78 (average 0.90, median 0.82), 0.55 to 1.51 (average 0.93, median 0.90) and 0.22 to 0.78 (average 0.41, median 0.36), respectively. For the mean and median HER2 mRNA levels, there was no significant difference between HER2 IHC 1+ and HER2 ultra-low expression diseases (P=0.757). A remarkable difference in HER2 gene expression was found between the tumors with 1+ and non-expression of HER2 by IHC (P=0.002). And, HER2 ultra-low cases contained statistically higher levels of HER2 mRNA compared with non-expression of HER2 subgroup by IHC (P=0.001). Conclusions: Based on HER2 mRNA, HER2 non-expression and HER2 weak expression (including HER2 IHC 1+ and ultra-low) belong to two different types of the tumor and the disease with HER2 IHC 1+ and HER2 ultra-low expression may be the same. It is necessary to further test the performance of HER2 mRNA detection for stratifying the HER2 weak expression subgroup and to determine the threshold.

Abstract PO2-14-05: COMPARISON OF HER2 mRNA PCR LEVELS AND IMMUNOHISTOCHEMISTRY (IHC) IN HORMONE RECEPTOR POSITIVE (HR+)/HER2 NEGATIVE (HER2-) EARLY BREAST CANCER

Abstract BACKGROUND IHC assays were developed to identify HER2-positive breast cancers deemed to be treated with trastuzumab-based therapies. However, these assays lack sensitivity to reliably identify tumors with low levels of HER2 protein on the cell surface. HR+/HER2-low may benefit from Trastuzumab Deruxtecan (TDxd) and therefore accurate quantification of HER2 expression levels becomes a critical issue. As a result, additional technologies are needed to achieve a quantitative and reproducible detection of HER2 in the low range expression compared with the standard IHC testing. METHODS We conducted a retrospective study of all incident stage I-II HR +/HER2- breast cancer patient whose genomic risk was assessed with the Oncotype 21 gene assay from 2009 to 2022 in Hospital Clínico San Carlos (Spain). Local HER2 expression (IHC w/wo cerbB2 FISH) was performed in all patients according to ASCO/CAP guidelines available during the different periods. HER2 mRNA expression levels by RT-PCR were obtained from OncotypeDx patient reports. The primary objective was to determine the distribution of HER2 mRNA levels across the different HER2 expression groups. Other objectives were to compare the clinicopathological characteristics, management and OncotypeDX Recurrence Score (RS) in the different HER2 expression groups. Qualitative variables are presented with their frequency distribution. Quantitative variables are summarized in their mean and standard deviation (SD). Welch’s F-test ANOVA was used to measure association between the two HER2 measuring methods. RESULTS 500 early breast cancer patients with HER2 IHC 0, 1+ and 2+ (FISH negative) and HER2 mRNA expression by RT-PCR tumors were included. Among them, 169 (34%) were IHC 0, 211(42%) were IHC 1+ and 120 (24%) were IHC 2+ (FISH negative). The distribution of clinicopathological characteristics was similar between different IHC HER2 expression groups, and the distribution of chemotherapy treatment was also similar among them (Table 1). All patients had undergone surgery, and most of them had received hormone therapy (487, 97%) and radiotherapy when indicated (375, 77%). The mean OncotypeDx® recurrence score across HER2 IHC 0, 1, 2 showed not significant difference between groups (16,85, 17,36 and 17,89 respectively). The mean OncotypeDx® HER2 gene score expression across the different levels of HER2 (0, 1+, 2+) determined by IHC were 9.07, 9.25 and 9.51, respectively. One-way ANOVA and Games-Howell post-hoc test revealed that differences between all the groups were significant. The ranges of HER2 gene score data for the 3 IHC groups were widely overlapped, with IQR of [8,5-9,5], [8,83-9,7] and [9,07-10] respectively. CONCLUSIONS Our data show statistically significant differences between HER2 mRNA expression levels and IHC groups. This suggests that RT-qPCR could complement the data obtained by IHC. However, the wide range and dispersion of mRNA expression within groups limits its clinical utility. Clinicopathological characteristics and management between different IHC HER2 expression groups. Citation Format: Julia Tejerina-Peces, Marta Amann-Arévalo, Pablo Ballestín, Beatriz González-Diez, Mateo Paz-Cabezas, Alejandro Pascual, Alicia de Luna, Vanesa García-Barberán, Alfonso López de Sá, Jose Angel García-Sáenz, Fernando Moreno. COMPARISON OF HER2 mRNA PCR LEVELS AND IMMUNOHISTOCHEMISTRY (IHC) IN HORMONE RECEPTOR POSITIVE (HR+)/HER2 NEGATIVE (HER2-) EARLY 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 PO2-14-05.

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.