Evaluation Of HER2 Expression Research Articles

Precision HER2: a comprehensive AI system for accurate and consistent evaluation of HER2 expression in invasive breast Cancer

BackgroundWith the development of novel anti-HER2 targeted drugs, such as ADCs, it has become increasingly important to accurately interpret HER2 expression in breast cancer. Previous studies have demonstrated high intra-observer and inter-observer variabilities in evaluating HER2 staining by human eyes. There exists a strong requirement to develop artificial intelligence (AI) systems to achieve high-precision HER2 expression scoring for better clinical therapy.MethodsIn the present study, we collected breast cancer tissue samples and stained consecutive sections with anti-Calponin and anti-HER2 antibodies. High-quality digital images were selected from immunohistochemical slides and interpreted as HER2 3+, 2+, 1+, and 0. AI models were trained and assessed using annotated training and testing sets. The AI model was trained to automatically identify ductal carcinoma in situ (DCIS) by Calponin staining and myoepithelial annotation and filter out DCIS components in HER2-stained slides using image-overlapping techniques. Furthermore, we organized two-phase validation studies. In phase one, pathologists interpreted 112 HER2 whole-slide images (WSIs) without AI assistance, whereas in phase two, pathologists read the same slides using the AI system after a washing period of 2 weeks.ResultsOur AI model greatly improved the accuracy of reading (0.902 vs. 0.710). The number of HER2 1 + patients misdiagnosed as HER2 0 was significantly reduced (32/279 vs. 65/279), and they benefitted from ADC drugs. In addition, the AI algorithm improved the intra-group consistency of HER2 readings by pathologists with different years of experience (intra-class correlation coefficient [ICC]: 0.872–0.926 vs. 0.818–0.908), with the improvement most pronounced among junior pathologists (0.885 vs. 0.818).ConclusionsWe proposed a high-precision AI system to identify and filter out DCIS components and automatically evaluate HER2 expression in invasive breast cancer.

Multiparametric MR characterization for human epithelial growth factor receptor 2 expression in bladder cancer: an exploratory study.

To investigate the application value of multiparametric MRI in evaluating the expression status of human epithelial growth factor receptor 2 (HER2) in bladder cancer (BCa). From April 2021 toJuly 2023, preoperative imaging manifestations of 90 patients with pathologically confirmed BCa were retrospectively collected and analyzed. All patients underwent multiparametric MRI including synthetic MRI, DWI, from which the T1, T2, proton density (PD) and apparent diffusion coefficient (ADC) values were obtained. The clinical and imaging characteristics as well as quantitative parameters (T1, T2, PD and ADC values) between HER2-positive and -negative BCa were compared using student t test and chi-square test. The diagnostic efficacy of parameters in predicting HER2 expression status was evaluated by calculating the area under ROC curve (AUC). In total, 76 patients (mean age, 63.59years ± 12.84 [SD]; 55 men) were included: 51 with HER2-negative and 25 with HER2-positiveBCa. HER2-positive group demonstrated significantly higher ADC, T1, and T2 values than HER2-negative group (all P < 0.05). The combination of ADC values and tumor grade yielded the best diagnostic performance in evaluating HER2 expression level with an AUC of 0.864. The multiparametric MR characterization can accurately evaluate the HER2 expression status in BCa, which may further guide the determination of individualized anti-HER2 targeted therapy strategies.

Evaluating antitumor activity and response determinants to trastuzumab deruxtecan in pediatric solid tumors.

10049 Background: Trastuzumab deruxtecan (T-DXd) is an anti-HER2 antibody-drug conjugate (ADC) linked to a topoisomerase inhibitor FDA-approved for several indications, including the treatment of HER2+ breast and gastric cancer. Although HER2 amplification is uncommon in pediatric cancers, recent demonstration of T-DXd efficacy in HER2-low breast cancer patients prompted us to examine the potential clinical relevance of T-DXd in pediatrics by evaluating HER2 expression and activity of T-DXd in preclinical pediatric solid tumor models. Methods: Cell viability was assessed in a panel of histologically diverse pediatric solid tumor cell lines and 2 HER2-amplified adult cancer cell lines treated with T-DXd, payload (DXd), and a control IgG-ADC. HER2 protein expression in patient-derived xenograft (PDX) tumors was evaluated by immunohistochemistry (IHC) using 5 clinically validated anti-HER2 antibodies. RNAseq was performed on clinical (n=290) and PDX (n=184) tumors to determine relative expression of ERBB2. In vivo activity of T-DXd was evaluated in osteosarcoma (OS, n=10), Wilms tumor (WT, n=1) and malignant rhabdoid tumor (MRT, n=1) PDX models and a desmoplastic small round cell tumor (DSRCT) cell line xenograft model. Differences in tumor volume and time to disease progression was assessed and compared across treatments and models. Responses were correlated with HER2 expression by IHC. Results: In vitro, HER2-amplified control cell lines demonstrated a &gt;30-fold reduction of IC50 when comparing T-DXd to ADC control. In contrast, the ADC control IC50 was nearly identical to T-DXd across all pediatric cancer cell lines, consistent with the absence of HER2 amplification in these models. We observed focal and membranous staining of HER2 by IHC in PDXs but was quite variable across antibodies tested: WT 0-17% HER2+ cases (n=6), MRT 0-40% (n=5), DSRCT 0-33% (n=24), OS 0-7% (n=30). ERBB2 gene expression was highest in DSRCT followed by WT, OS and MRT. In vivo efficacy studies demonstrated complete and partial responses in OS, WT, and DSRCT and improved disease control rates (OS: 67%, p=0.006, Mann-Whitney; WT: 100%; DSRCT: 100%). However, T-DXd and ADC control demonstrated similar activity in all tumor types with no consistent correlation between in vivo response and HER2 expression. Consistent with these preclinical studies, 4 patients with progressive DSRCT were treated with T-DXd via compassionate/off-label access with signs of clinical and radiographic responses. Conclusions: T-DXd shows significant preclinical antitumor activity across multiple pediatric solid tumors but little correlation with HER2 expression suggesting a mechanism of action similar to the clinical activity observed in HER2-low breast cancer. Xenograft efficacy studies and preliminary clinical experience with T-DXd in DSRCT patients warrant formal clinical trial investigation in this largely incurable disease.

Abstract PO5-13-11: Quantitative Assessment of HER2 Expression in Intraductal Neoplasms of the Breast

Abstract HER2 (ERBB2) is an established prognostic and predictive marker for patients with invasive breast cancer and used in HER2-targeted therapy. The clinical and biological significance of expression of HER2 protein in patients with intraductal neoplasms of the breast is not well characterized. In this study, we used our HS-HER2 (High Sensitivity-HER2) which is a CAP/CLIA certified laboratory derived test (LDT) using quantitative immunofluorescence (QIF). We evaluated HER2 expression in DCIS (ductal carcinoma in situ), LCIS (lobular carcinoma in situ) and benign lesions to correlate with their clinicopathologic characteristics. Thirty- eight cases of DCIS, and fourteen cases of LCIS and thirty benign cases were selected from the Yale Pathology department archives and 252 ROIs (regions of interest) were annotated by a board-certified pathologist. DCIS is classified according to the three-tier nuclear grading system: low, intermediate, and high-nuclear grade. LCIS is classified as PLCIS (pleomorphic variant) and CLCIS (classic LCIS) according to the WHO classification of breast tumors. Serial sections of FFPE tumor specimens were used to accurately quantify the HER2 expression by the HS-HER2 assay and the acquisition by QuPath v.04 with Qymia extension. According to the LOD (limit of detection,3 amol/mm2), LOQ (limit of quantification, 9 amol/mm2) and LOL (limit of linearity, 23 amol/mm2) of the optimized HS-HER2 assay as performed in our CLIA lab shows a broad range of HER2 expression in both DCIS and LCIS lesions, ranging from 0.7 amol/mm2 to 111.4 amol/mm2 in DCIS and 3.6 amol/mm2 to 49.9 amol/mm2 in LCIS. High grade DCIS lesions express higher average HER2 levels (111.4 + 0.7 amol/mm2) than combined low and intermediate grade DCIS (77.9 +1.9 amol/mm2) with no significant difference between low/intermediate and high-grade DCIS. CLCIS lesions express higher average HER2 level (49.9 + 3.6 amol/mm2) than PLCIS (35.1 + 5.4amol/mm2) but again this is not statistically significant. HER protein expression is relatively low in benign lesions ranging from 0.8 to 8.4 amol/mm2. Using the HS-HER2 assay, our results show quantitative level HER2 levels in DCIS and LCIS breast lesions. These findings may be important as HER2 targeted therapies that work in gene-unamplified cancer (HER2 conjugated ADCs) gain broader usage in the clinic. Citation Format: Nay Nwe Nyein Chan, Haiying Zhan, Revekka Khaimova, Thazin Aung, Charles Robbins, Patricia Gaule, David Rimm. Quantitative Assessment of HER2 Expression in Intraductal Neoplasms of the Breast [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 PO5-13-11.

Abstract PO3-13-03: RNAScope: a practical approach and promising alternative to immunohistochemistry to quantify HER2 expression in breast cancer

Abstract T-DXd has been approved by the FDA to treat patients with metastatic HER2-low and -positive breast cancer. The utility of current HER2 immunohistochemistry (IHC) assays in evaluating HER2-low tumors is not clear. A simple and objective method to evaluate HER2 expression in breast cancer is urgently needed. RNAScope can detect HER2 RNA levels by in situ hybridization using one regular unstained FFPE slide and processed using the Leica BOND-III autostainer that is readily available in many clinical laboratories. RNA level detected by RNAScope can be quantified by dots/cell using publicly available software. Therefore, RNAScope is a practical assay and could be a promising alternative to IHC to quantify HER2 levels in breast cancer. We evaluated HER2 levels in 605 breast cancer tissue microarray cores using RNAScope and the two most commonly used FDA approved HER2 IHC assays: Ventana PATHWAY (PATHWAY) and Dako HercepTest (HercepTest). Clinical data were available for 505 cores from 347 patients. RNA level (dots/cell) by RNAScope was quantified using publicly available software QuPath. IHC assays were scored as 0, ultralow (UL, &amp;gt;0% but ≤10% incomplete membranous staining), 1+, 2+ and 3+. In addition, HER2 protein levels (AQUA protein level) were quantified from 100 cores through regression analysis, using AQUA score against cell line arrays with pre-calibrated HER2 protein levels determined by mass spectrometry. We used ANOVA to assess the differences in RNAScope results among the five IHC scores, and linear regressions to correlate RNAScope with HER2 AQUA protein levels. We finally evaluated 41 RNAScope whole-slide images (IHC 1+: n=5; 2+: n=26; 3+: n=10) of metastatic tumors from 31 patients treated with T-DXd. No significant differences of RNAScope results were observed among the 0, UL, and 1+ cores in both IHC assays, indicating the current IHC assays cannot differentiate HER levels in HER2-low tumors. However, statistically significant differences (p &amp;lt; 0.0001) were found among the IHC 1+, 2+, and 3+ cores and higher RNAScope dots/cell was associated with higher stage of the tumors. There was a strong correlation (R2 = 0.610) between the RNAScope results and quantitative HER2 AQUA protein level in the 100 cores. There were significantly higher HER2 RNA levels in the 41 metastatic biopsies with higher IHC scores (p &amp;lt; 0.05). When we used RNAScope to measures HER2 levels in metastases right before T-DXd treatment, there was numerically (p=0.881) higher HER2 RNA levels in responders (5.60±8.82 dots/cell) vs non-responders (5.20±5.31). Interestingly, the HER2 RNA levels in bone metastases was statistically higher (p=0.030) in non-responders (5.24±2.87, n=3) than in responders (1.55±0.81, n=5); although number of patients was low. For the non-bone metastases (esophagus, lymph node, liver, brain, lung), HER2 RNAScope values were numerically higher (p=0.261) in responders (9.65±2.87, n=5) than non-responders (5.19±5.74, n=15). In these non-bone metastatic cases, the response rates by IHC scores were 100% in 1+ cases, 24% in 2+ and 33% in 3+. When we used AI assisted categories based on RNAScope results, the response rates were 20% in RNAScope 1+ cases, 20% in 2+, 50% in 3+ cases. Our study shows that current IHC assays are unable to differentiate HER2 levels between IHC 0 and 1+ breast cancer cases, which is a critical issue in properly identifying patients who will benefit from T-DXd treatment. RNAScope results strongly correlate with HER2 protein levels and showed similar RNA levels among IHC 0 and 1+ cases. RNAScope is a simple and objective assay to quantify HER2 levels by dots/cell using publicly available software and may help better identify which patients benefit from T-DXd treatment. Other factors besides HER2 level may also contribute to the response rate in patients treated with T-DXd. RNAScope results in association with immunohistochemistry (IHC) scores by PATHWAY, HercepTest, and in biopsies from patients treated with T-DXd (T-DXd cohort). XX XX No significant (ns) difference of RNAScope dots/cell were seen among 0, UL and 1+ cases by both PATHWAY and HercepTest assays. There were significant differences of dots/cell comparing 1+, 2+ and 3+ cases. Note: ns not significant (p&amp;gt;0.05), * p&amp;lt;0.05, *** p&amp;lt;0.001, **** p&amp;lt;0.0001 Citation Format: Xiaoxian Li, Ji-Hoon Lee, Yuan Gao, Jilun Zhang, Katherine Bates, David Rimm, Huina Zhang, Geoffrey Smith, Diane Lawson, Jane Meisel, Jenny Chang, Lei Huo. RNAScope: a practical approach and promising alternative to immunohistochemistry to quantify HER2 expression 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 PO3-13-03.

Abstract PO4-03-10: HER2 Expression in Triple-Negative Breast Cancer: Clinicopathologic Features and Response to Neoadjuvant Chemotherapy

Abstract BACKGROUND: Half of the primary breast cancers reported as HER-2 negative have low HER-2 expression, defined as an immunohistochemical (IHC) score of 1+ or 2+, with negative in situ hybridization. Among these cases, 37% correspond to the triple-negative immunophenotype. The primary objective of this study was to evaluate HER-2 expression in patients with triple-negative breast cancer and compare the clinicopathologic features and response to neoadjuvant chemotherapy between HER-2 negative (0) and HER-2 low (1+/2+) subtypes. METHODS: We retrospectively analyzed 157 patients with early-stage triple-negative breast cancer who underwent neoadjuvant chemotherapy followed by surgery with curative intent and met the study's inclusion criteria. These patients were treated at the National Institute of Neoplastic Diseases between 2010 and 2021. HER-2 analysis was performed using immunohistochemistry (IHC), and in cases of HER-2 (2+) amplification, in situ hybridization was performed. RESULTS: Of all the included patients, 107 were classified in the HER-2 negative group (68%), and 50 were classified in the HER-2 low group (32%). Seventy-four percent of patients with HER-2 negative were in the age range of 40-64 years, compared to 68% in the HER-2 low group. A significant difference was found between the HER2-negative and HER2-low groups regarding age and Ki67. The median age of HER-2 low patients is significantly higher than the median age of HER-2 negative patients (52 years vs. 45 years, respectively). No significant association was found between HER-2 status and clinical stages or histological grade. However, the median Ki67 percentage of HER-2 negative patients is significantly higher than the median Ki67 of HER-2 low patients (70% vs. 50%, respectively (p=0.035)). Thirty-two percent of patients with HER-2 negative achieved a pathological complete response (pCR) compared to 18% of patients with HER-2 low. There were no differences in the pCR rate in the HER-2 1+ and 2+ subgroups. Among the patients included in both groups, who achieved pCR, no disease progression was observed at the time of analysis. CONCLUSIONS: Patients in the HER-2 negative group had a higher rate of complete pathological response to neoadjuvant chemotherapy compared to those in the HER-2 low group. No differences were observed in the pCR rate between HER-2 1+ and 2+ subgroups, suggesting that HER2 expression levels within the low range may not significantly impact treatment response. Table. Table. Citation Format: Zaida Morante, Iris Otoya, Natalia Valdivieso, Cindy Calle, Hugo Fuentes, Silvia Neciosup, Carlos Castañeda, Tatiana Vidaurre, Martin Falla, Valeria Colomo, Henry Gómez. HER2 Expression in Triple-Negative Breast Cancer: Clinicopathologic Features and Response to Neoadjuvant Chemotherapy [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 PO4-03-10.

Abstract 7509: Evaluation ofHER-2 expression on circulating tumor cells as a real time biomarker in advanced breast cancer

Abstract Introduction: Breast cancer accounts for 12.5% of all new annual cancer cases worldwide. Cases classified as HER2-positive tend to proliferate, metastasize, and often lead to relapse. HER-2 is the most valid tumor marker, used as a diagnostics and prognostic biomarker in metastatic breast cancer (MBC). Thus, analysis of expression of HER-2 on CTCs can offer a real time dynamic biomarker for treatment options between endocrine therapy versus chemotherapy in advanced breast cancer. Functional assays on CTCs with HER-2 biomarker offer evolving biomarkers especially when the tissue is not accessible and inadequate. Methods: Retrospectively, 179 breast cancer patients' blood was analyzed for the presence of CTCs using the OncoDiscover® platform approved by Drug Controller General of India (CDSCO). Consisting of immunomagnetic multi-components mediated by anti EpCAM antibodies. The isolated cells were immunostained consisting of a nuclear dye DAPI, CK-18, CD45 and HER-2 antibodies. CTCs were identified by the presence of HER-2 + ve, CK 18 + ve, DAPI + ve and CD 45 -ve staining. Validation of HER-2 expression on the CTCs was analyzed based on the linear intensity gradients of the fluorescence signals. CTCs were termed as HER-2 -ve based on weak or no detectable fluorescence signal and termed +ve based on high fluorescence signal. Results: Among the cohort of 179 patient samples, 63.68 % samples showed the presence of CTC (CTC ranged from 1-7). 47.84% of these CTCs showed the expression of HER-2 signal. The mean value for fluorescence intensity for expression of HER-2 in CTCs was found to be 3.23. The observed mean fluorescence intensity for HER-2 expression further emphasizes the robustness of CTCs as a viable source for molecular characterization. Conclusion: Integration of HER-2 analysis on CTCs into the clinical assessment of metastatic breast cancer, may offer a non-invasive, real-time strategy for profiling and possibly paving the way for more precise and tailored therapeutic interventions. Citation Format: Jayant Khandare, Gourishankar Aland, Saloni Andhari, Ganesh Khutale, Prathamesh Patil, Tanvi Deshpande, Meghana Garbhe, Vikas Jadhav, Aravindan Vasudevan, Sreeja Jayant. Evaluation ofHER-2 expression on circulating tumor cells as a real time biomarker in advanced breast cancer [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 7509.

Correlation of HER2 Protein Level With mRNA Level Quantified by RNAscope in Breast Cancer

Trastuzumab deruxtecan (T-DXd) has been approved by the US Food and Drug Administration (FDA) to treat patients with metastatic HER2-positive and HER2-low breast cancer, and clinical trials are examining its efficacy against early-stage breast cancer. Current HER2 immunohistochemical (IHC) assays are suboptimal in evaluating HER2-low breast cancers and identifying which patients would benefit from T-DXd. HER2 expression in 526 breast cancer tissue microarray (TMA) cores was measured using the FDA-approved PATHWAY and HercepTest IHC assays, and the corresponding RNA levels were evaluated by RNAscope. HER2 protein levels by regression analysis using a quantitative immunofluorescence score against cell line arrays with known HER2 protein levels determined by mass spectrometry were available in 48 of the cores. RNAscope was also performed in 32 metastatic biopsies from 23 patients who were subsequently treated with T-DXd, and the results were correlated with response rate. HER2 RNA levels by RNAscope strongly correlated with HER2 protein levels (P < .0001) and with HER2 IHC H-scores from the PATHWAY and HercepTest assays (P < .0001). However, neither protein levels nor RNA levels significantly differed between cases scored 0, ultralow, and 1+ by PATHWAY and HercepTest. The RNA levels were significantly higher (P = .030) in responders (6.4 ± 8.2 dots/cell, n = 12) than those in nonresponders (2.6 ± 2.2, n = 20) to T-DXd. RNAscope is a simple assay that can be objectively quantified and is a promising alternative to current IHC assays in evaluating HER2 expression in breast cancers, especially HER2-low cases, and may identify patients who would benefit from T-DXd.

The prevalence and clinical significance of HER2 expression in prostate adenocarcinoma

AimsAbnormalities in HER2 are well-established oncogenic drivers and are approved therapeutic targets in various malignancies. Prior studies evaluating HER2 expression in prostate cancer (PCa) have yielded variable results. Most of these studies used immunohistochemistry scoring systems based on breast cancer data. The goal of this study was to determine the prevalence and clinical significance of HER2 expression using a scoring system that better reflects the HER2 staining pattern observed in PCa. MethodsWe randomly selected similar numbers of localized low risk (AJCC stage I), locally advanced (AJCC stages II & III), and metastatic (AJCC stage IV) PCa patients treated at the DC VA Medical Center between 2000 and 2022. Among them, we included patients who had sufficient PCa tissue samples and clinical information required for this analysis. Two experienced pathologists independently scored HER2 expression (Ventana Pathway anti-HER2) according to a modified gastric cancer HER2 scoring system. ResultsOut of the 231 patients included, 85 % self-identified as Black. 58.9 % of patients expressed HER2 (1+: 35.5 %; 2+: 18.2 %; 3+: 5.2 %). Validity of the results was confirmed using the HercepTest antibody. Higher HER2 expression was associated with a higher Gleason Score/Grade Group and advanced disease. ConclusionsOur findings support the adverse prognostic impact on HER2 in PCa. We propose the use of a modified scoring system to evaluate HER2 expression in PCa. The observed high prevalence of HER2 expression supports the consideration of novel HER2-targeted therapies addressing even low levels of HER2 expression in future PCa trials.

HER2-low breast cancer: evolution of HER2 expression from primary tumor to distant metastases

BackgroundBreast cancer (BC) with low human epidermal growth factor receptor 2 (HER2) expression is attracting much attention due to the breakthrough progress of novel anti-HER2 antibody-drug conjugates. HER2 expression is examined in patients with HER2-low BC and their distant metastases in this study, so as to further clarify the dynamic characteristics of HER2 low status in the process of disease progression.MethodsPatients diagnosed with HER2 low breast cancer (defined as IHC1+ or IHC2+/ISH-) between 2012 and 2021 were included in this study. We evaluated HER2 expression of primary sites and metastatic sites, compared the impact of different clinicopathological parameters on HER2 status of metastases and compared the overall survival and disease-free survival of patients with different HER2 status in metastases.ResultsNinety-eight patients were included. All HER2 IHC scores were confirmed and the consistent rate with the original pathological report was 81.1%. 27.6% of the patients showed different HER2 status in metastases. The HER2 discordance rate differed among different metastatic sites (p = 0.040). The higher the T stage of the primary BC, the higher the rate of HER2 discordance was observed (p = 0.042). For the specimen type of metastasis, HER2 discordant rate was higher in surgical specimen than biopsy (p = 0.050). No difference of HER2 discordance rate was found between HER2-1+ and HER2-2+ patients. But comparing HER2 IHC score, HER2-2+ patients were less likely to have consistent metastatic HER2 levels than HER2-1+ patients (p = 0.006). No difference in survival outcomes was observed between patients with different HER2 status in metastases.ConclusionsThere is a possibility of HER2 expression alteration in the metastases of HER2-low breast cancer. And the rate of altered HER2 low expression was different among different metastatic sites, different T stages of primary BC and specimen type of metastasis. No prognostic significance was observed.

A comparative study of gastric adenocarcinoma HER2 IHC phenotype and mass spectrometry-based quantification.

Gastric cancer is a highly heterogeneous malignant tumor of the digestive system. Anti-HER2 treatment can inhibit downstream signaling pathways and improve clinical treatment and outcomes in patients with HER2 protein overexpression. Currently, two standard methods for evaluating HER2 expression status are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). However, these low-throughput assays often produce discordant or equivocal results. In this study, we presented a new HER2 protein detection method based on mass spectrometry selected reaction monitoring (MS-SRM) and validated the method. We conducted a retrospective study on 118 formalin-fixed paraffin-embedded (FFPE) tissues from patients with advanced gastric adenocarcinoma in northern China, and we compared the MS-SRM results with those from IHC and correlated them with FISH. We established and validated the upper and lower detection limits (300-700 amol/μg) for abnormal HER2 protein expression in advanced gastric cancer. We also found that, among samples with mixed Lauren subtypes, those with a high level of HER2 expression had typical intestinal type features in pathology. This study demonstrated that the MS-SRM method can overcome the limitations and deficiencies of IHC, directly quantify the expression of HER2 protein in tumor cells and be used as a supplement to IHC. It has the potential to be used as a companion diagnosis for new drugs used to treat advanced gastric cancer. Large-scale clinical validation is required.

56P Association between HER2 expression level and prognosis using RT-PCR in HER2-low breast cancer

The variation and heterogeneity of conventional immunohistochemistry (IHC) or in situ hybridization (ISH) techniques for measuring HER2 has long been pointed out, and the method of testing for HER2 has been discussed for the selection of appropriate treatment. Therefore, we compared HER2 expression in RT-PCR using OncotypeDX have correlation with conventional IHC, and evaluated HER2 expression in RT-PCR as a prognostic factor in HER2-negative early breast cancer. ER+/HER2- patients who underwent breast cancer surgery between 2004 and 2016 and underwent OncotypeDX testing were included. Data from 632 patients were collected and analyzed by retrospective chart review at 9 centers to determine if there was a correlation between HER2 expression by IHC and RT-PCR. HER2 expression was classified as negative, HER2-low, and positive when HER2 mRNA levels were ≤ 9.1, 9.2-11.4, and11.5≤, respectively, based on the median mRNA level in HER2 negative in IHC. Survival curves were estimated using the Kaplan-Meier method with a log-rank test between the three groups. The median age of the 632 patients was 51 years. Of the 632 patients, 311(49.2%) were pStage I, the 309 (48.9%) were pStage II, and 11(1.7%) were pStage III. 127 (20%) patients received postoperative chemotherapy, and 54 (8.5%) patients had relapse. HER2 IHC rates were HER2 0: 166 (26.3%), 1+: 304 (48.1%), and 2+: 162 (25.6%). There was a weak correlation between the IHC and RT-PCR methods for HER2 expression (Spearman rank correlation P<0.001, r=0.33). The recurrence rates of IHC 0, 1+, and 2+ were 1.8%, 3.9%, 2.6%, respectively. There was no statistically significant difference in DFS between the three groups of HER2 expression by the IHC method (P=0.59). In addition, in the study of HER2 expression and prognosis by RT-PCR, the patients were divided into HER2 negative: 269, HER2-low: 357, and HER2 positive: 1. There was no statistically significant difference between the three groups (P=0.832). The recurrence rates were 3.9%, 4.4%, and 0%, respectively. In the analysis of HER2 expression, there was a weak correlation between IHC and RT-PCR mRNA expression levels; in the HER2-negative population, RT-PCR mRNA expression of HER2 was not a prognostic factor in early breast cancer.

Establishment of a murine model of breast cancer expressing human epidermal growth factor receptor 2 (4T1-HER2).

Although people with HER2-positive breast cancer benefit from approved HER2-targeted therapy, acquiring resistance to the therapies occurs. Animal models can play a part in gaining a deep understanding of such a process and addressing questions concerning developing and improving immunotherapy approaches. To develop such a model, we transfected murine 4T1 cells with the pCMV6-Neo-HER2 construct and evaluated HER2 expression and its effects on the established cell line behavior in vitro and in vivo. Data illustrated that human HER2 protein was expressed on isolated 4T1-HER2 clones in vitro and in vivo. Except for proliferation over 48 hours, such expression did not change 4T1-HER2 characteristics compared to 4T1 in vitro. Notwithstanding the reduction in proliferation, the rate of tumorigenicity was 90% in challenged mice and Herceptin therapy significantly decreased tumors' growth and metastasis compared to the control group. We describe a murine model for HER2-positive breast cancer not only helping shed light on the mechanisms by which the tumor evades antitumor immunity but also playing a key role in making breast cancer more sensitive to novel immunotherapy modalities.

Human Epidermal Growth Factor Receptor 2 (HER2) Expression by Immunohistochemistry and Its Clinical Significance in Hepatocellular Carcinoma: A Single-Center Analysis.

Human epidermal growth factor receptor 2 (HER2) is a member of the tyrosine kinase receptor family. It has been identified as an oncogene and is associated with poor outcomes in multiple tumortypes. In hepatocellular carcinoma (HCC), there are contradictory data regarding the HER2 expression and its role in tumor development and progression. Some studies have identified HER2 expression as an early event during tumorigenesis, which decreases with progression and metastasis. Additional data provided evidence that treatment with anti-HER2 therapy resulted in local response and reduction in the metastasis rate in HCC micemodels. Patients with histological diagnoses of HCC between 2010 and 2020 were included. HER2 staining was performed by immunohistochemistry (IHC), and scoring was done in accordance with the gastric cancer guidelines as 0, 1+, 2+, and 3+. Clinicopathological features were accessed by medical records. This study aims to evaluate HER2 expression by IHC in HCCand to correlate this expression with some clinicopathological features such as Barcelona Clinic Liver Cancer (BCLC) staging,number of hepatic lesions,alpha-fetoprotein level, underlying liver disease, presence of livercirrhosis, Child-Pugh score, and tumor recurrence. A total of 57 specimens from 54 patients were included. Of the patients, 85% were men, and the median age at diagnosis was 71 years (interquartile range: 59-75 years). Regarding stage, 61% were at stage 0-A of BCLC. Of the patients,57% had a solitary HCC nodule. Concerning treatment, surgery was performed in 50% of the patients.HER2 expression was identified in seven patients: five in the membrane and two in the cytoplasm. Concerning the membrane staining, HER2 expression was scored as 1+/2+ in 7.4% (n = 4 patients). Of the patients with HER2 expression, fourhad a BCLC stage of 0-A and a single HCC nodule; alpha-fetoprotein was <400 ng/mL in all cases. There was no correlation to clinicopathological features. In one patient with HER2 2+ expressionat diagnosis, this expression was not identified at tumor progression. Median disease-free survival in HER2 with IHC scores 1+/2+ and cytoplasmatic was 38 months versus 22 months in HER2 with a score of 0 (p = 0.604). HER2 expression is a rare event in HCC. It was not possible to identify any relation to clinicopathological features. However, when we relate our data to previous trials, HER2 appears to be an early event in the course of HCC.

HER2 status is positively associated with vessel invasion of colorectal cancer: a retrospective large cohort study

PurposeHER2-positive colorectal cancer was drawn increasing attention in recent years. Accumulating evidence showed HER2-positive metastatic colorectal cancer could benefit from HER2-targeted therapy. While HER2 expression and the relationship between HER2 status and clinicopathological characteristics of overall colorectal cancer remains largely unknown. The aim of this study was to evaluate HER2 expression in colorectal cancer and compare the clinicopathological features between HER2-positive and HER2-negative colorectal cancer.MethodsWe retrospectively analyzed 3910 primary colorectal cancer patients treated in our institution from January 2016 to December 2019. Medical records and pathology reports after surgery were collected to provide information about HER2 status and other clinicopathological characteristics.ResultsWe identified 3347 HER2-negative and 79 HER2-positive colorectal cancer patients in our cohort. The chi-square test showed that vessel invasion was significantly more common in HER2-positive colorectal cancer patients. Crude analysis showed HER2 positive was associated with vessel invasion in colorectal cancer [OR and 95% CI 0.534 (0.341, 0.835), p = 0.006]. After adjusting for N stage, a significant association was still observed between HER2 status and vessel invasion in colorectal cancer [OR and 95% CI 0.550 (0.322, 0.941), p = 0.029]. Survival analysis showed that there was no significant difference in 3-year overall survival rate between HER2 positive and HER2 negative group (p = 0.603).ConclusionOur findings indicate that the rate of HER2 positivity in colorectal cancer was relatively low, and HER2 status was strongly associated with vessel invasion while having no significant influence on the 3-year overall survival rate in colorectal cancer patients.

Artificial intelligence-powered human epidermal growth factor receptor 2 (HER2) analyzer in breast cancer as an assistance tool for pathologists to reduce interobserver variation.

e12543 Background: Human epidermal growth factor receptor 2 (HER2) expression is a predictive marker for HER2-targeted therapy in breast cancer patients. Interobserver variation in the interpretation of HER2 levels exists among pathologists, thus a method to increase the consistency of evaluation is needed. This study aimed to evaluate the performance of the artificial intelligence (AI)-based Lunit SCOPE HER2 in assisting pathologists to evaluate HER2 expression levels in breast cancer. Methods: Lunit SCOPE HER2 was developed with a 1.04 x 1010 μm2 area and 7.31 x 105 tumor cells from 1,133 HER2 immunohistochemistry stained whole-slide images (WSI) of breast cancer, annotated by 113 board-certified pathologists. The AI model was developed based on a semantic segmentation algorithm, which consists of two atrous spatial pyramid pooling blocks for tissue level classification and for tumor cell level classification. To validate the model, a total of 209 HER2 WSIs diagnosed with breast cancer were obtained from Kyung Hee University Hospital in Korea and were assigned as an external validation set. Three board-certified pathologists evaluated slide level HER2 expression (3+, 2+, 1+, and 0) twice, first without AI assistance and second, with it. The second reading was performed for WSIs where the pathologist's reading showed discrepancy with the AI model. Results: In the external validation set, all pathologists scored the same HER2 grade in 103 WSIs (49.3%), and the Fleiss kappa value was 0.512. The HER2 grade from the AI model and pathologists was the same in 151 WSIs (72.2%), and the weighted kappa value was 0.844. The pathologists re-evaluate 43, 63, and 83 WSIs, respectively. After AI assistance, all pathologists scored the same HER2 grade in 156 WSIs (74.6%), and the Fleiss kappa value increased to 0.762 (Table). Conclusions: This study demonstrates that an AI-powered HER2 analyzer can help achieve consistent HER2 expression level evaluation in breast cancer by reducing interobserver variability. Thus, the AI model can be applied as an assistance tool for pathologists in HER2 grade evaluation.[Table: see text]

Triple negative phenomenon in endometrial cancer: recognition criteria and impact on survival.

Endometrial cancer is the most common malignant cancer of female reproductive organs. The number of diagnosed cases of endometrial cancer is increasing from year to year. Endometrial cancer is a neoplasm with a good survival rate. However, there are also cases with a fast, aggressive course. In recent years, the triple negative phenomenon (TNP) has been identified as one of the factors determining shorter survival in patients with endometrial cancer. The study covered 265 patients with histopathologically confirmed endometrial cancer. Patients were divided into two groups: 1) patients with endometrial cancer with TNP; 2) patients with endometrial cancer without TNP. Tissue microarrays (TMA) were examined with immunohistochemistry to evaluate the expression of estrogen, progesterone and HER2 receptors. In several cases FISH method was used to assess HER2. The expression was evaluated by computer image analysis using the Nuclear Image Analysis virtual microscopy system. The evaluation of HER2 expression was performed manually. The criterion for TNC diagnosis was H-Score < 50 or < 75 and Allred score < 4. Depending on the scoring system used, TNP was found in from 10.19% to 15.09% of cases. Regardless of the criteria employed in endometrial cancer, the presence of TNP was neither a factor increasing the risk of death nor it affected the patients' survival. The proportion of TNP diagnosed in endometrial cancer depends on the examined population and the diagnostic criteria. The incidence of TNP did not affect the survival of patients.

Abstract P2-02-05: Dynamic circulating tumor cell changes in enumeration and HER2 expression during systemic therapy for metastatic breast cancer

Abstract Introduction: CTCs are tumor cells that circulate in the blood of patient with primary and MBC and, are responsible for seeing of metastasis. The monitoring of CTCs in MBC emerged as strong prognostic and possible predictive biomarker in oncology over the past couple of decades. Meanwhile, overexpression of HER2 protein has been associated with rapid cell division and worse prognosis of MBC. Here we report a significant correlation between dynamic CTCs enumeration and CTCs-HER2 expression during the systemic therapies, with potential implication to understand treatment resistance. Methods: A total of 298 whole blood samples (7.5ml/each) were collected from 149 patients with stage IV breast cancer (2016-2020) at the Northwestern University Robert H Lurie Comprehensive Cancer Center, before (Baseline) and 3 months after (Time point 2) initiation of systemic treatment. CTC enumerations were performed using the FDA approved CellSearch™ system (Menarini) which is specific for the intracellular protein cytokeratin (CK) in epithelial cells, DAPI stains the cell nucleus, anti-CD45-APC is specific for leukocytes, and anti-HER-2/neu-FLU is specific for HER-2/neu antigen. The CTCs were classified as CK+, EpCAM+, DAPI+ and CD45-. We developed a criteria for evaluation of HER2 expression by 4 different categories (0,1+,2+,3+) based on expression intensity in our lab (present in 2021 ASCO). In this study we included CTCs with all intensities of HER2 expression (1+ to 3+) which was standardized in our lab. Mann-Whitney U test was used for statistics. Results: Of the 149 baseline samples, CTC≥1 were found in 101 patients (67.8%). A change in CTCs between baseline and time point 2 for these 101 patients: Three groups were identified: Group 1: 33patients (33%) with increase CTCs; Group 2: 64 patients (63%) with decreased CTC; Group 3: 4 patients (4%) with no change The median increase of total CTCs and HER2+ CTCs in Group 1 were 7.0 and 2.0, respectively; the median decrease of total CTCs and HER2+ CTCs in Group 2 were 9.5 and 2.0, respectively. The change of HER2+ CTCs was significantly correlated with the change of total CTCs after systemic therapy, with the correlation coefficient as rs=0.662 (p&amp;lt;0.001) for these 101 patients. A significant positive correlation between HER2+ CTCs and total CTCs were found in both Group 1 and Group 2, rs=0.717 (p&amp;lt;0.001) and rs=0.604 (p&amp;lt;0.001) respectively. Furthermore, the ratio of HER2+ CTCs (HER2+ CTCs/total CTCs) in both Group 1 and Group 2 was also significantly correlated with total CTCs after therapy with the corresponding correlation coefficient as rs=0.536 (p&amp;lt;0.001) and rs=0.388 (p&amp;lt;0.001) in Group 1 and Group 2, respectively. Conclusion: Our study demonstrated that dynamic changes of CTCs after systemic therapies, are positively correlated with the HER2 expression in CTCs in different levels of baseline CTCs amounts. Observation of HER2 expression and ratio in CTCs during the course of systemic therapy is useful in monitoring therapy efficacy and potential disease progress. Citation Format: Qiang Zhang, Weijun Qin, Paolo D'Amico, Andrew A. Davis, Jianhua Jiao, Lorenzo Gerratana, Saya L. Jacob, Youbin Zhang, Jeannine Donahue, Wenan Qiang, Ami N Shah, Amir Behdad, Lisa Flaum, William Gradishar, Leonidas C Platanias, Massimo Cristofanilli. Dynamic circulating tumor cell changes in enumeration and HER2 expression during systemic therapy for metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-02-05.

Abstract P1-05-05: Prognostic value of HER2 expression levels in non-metastatic triple-negative breast cancer and correlation with other biomarkers

Abstract Background: HER2 low breast cancer (BC), presenting 1+ or 2+ (without gene amplification) IHC staining, is an emerging subtype due to dedicated treatments that are now tested. Very few data have been reported on this specific subtype, especially in the triple-negative breast cancer (TNBC) group. Our aim was to evaluate HER2 expression and its prognostic value in a large retrospective series of patients with non-metastatic TNBC and a long follow-up, characterized for Basal-like (BL) or molecular apocrine-like (MA) IHC profiles as well as TILs infiltrate, PDL1 expression and PIK3CA/PTEN mutations. Patients and methods: HER2 expression (0, 1+, 2+) was evaluated on the clinical sample with the HercepTestTM immunohistochemical (IHC) assay, using the 2018 ASCO-CAP guidelines for HER2 scoring, in a series of 296 patients with non-metastatic TNBC treated in our center between 2002 and 2012. HER2 2+ IHC cases were evaluated by FISH and amplified cases were excluded. BL and MA profiles were defined as IHC CK5/6 and/or EGFR expression, and Androgen-receptor and FOXA1 IHC expression, respectively, in a previously used Tissue MicroArray. HER2 expression levels were tested for their association with baseline clinicopathological variables, and for Relapse-Free Survival (RFS) and Overall survival (OS). Results: A total of 296 TNBC tumors was selected for this analysis (248 HER2=0 (83.8%), 40 HER2=1+ (13.5%), 8 HER2=2+ (2.7%)). Considering these percentages, HER2 1+ and 2+ cases were grouped for statistical analysis. Median age was 57.7 years (range 28.5-98.6). 45.8% of tumors were classified pT1 and 63.5% pN0. We found 81.9% of patients had ductal carcinomas, 5.5% lobular carcinomas and 12.6% other histological types. Histological grade 1-2 represented 21% of all tumors. A BL phenotype was observed in 63.8% of cases, and an MA profile in 40.2% of the cases. Adjuvant chemotherapy (ACT) was delivered in 75.3% of patients. Compared to HER2 0 tumors, HER2 1+/2+ tumors exhibited a lower nuclear grade (1/2) (35.4% vs. 18.2%, p=0.007) and MA profile (57.5% vs. 36.7%, p=0.008). With a median follow-up of 9.7 years, HER2 2+ tumors (compared to HER2 0/1+ tumors) were associated, in univariate analysis, with a worse RFS (HR=3.16, 95%CI [1.27; 7.85], p=0.034). No statistically significant OS or RFS difference was found when using the HER2 0 vs. 1+/2+ grouping. Multivariate analyses results are summarized in Table 1. Conclusions: HER2 1+/2+ expression is associated with lower histological grade and MA profile, a specific subgroup of TNBC with a worse prognosis. Considering the current development of anti-androgens for MA tumors and anti-HER2 antibody-drug conjugates in this HER2 1+/2+ population, this clinicopathological association could have therapeutic implications. While rare (2.7% of cases in our series), HER2 2+ TNBC seems to be affected with a worse RFS, advocating for a dedicated evaluation of this subgroup. VariablesOS (N=263)RFS (N=285)HR95% CIP-valueHR95% CIP-valueTumor size &amp;lt;0.001 T11 T2/T3/T42.551.49 – 4.37 Nodal status 0.004 &amp;lt;0.001N-11 N+2.011.26 - 3.224.472.71 - 7.37HistologyDuctal10.001Other0.340.16 – 0.72Adjuvant chemotherapy &amp;lt;0.001 0.001No11 Yes0.330.21 - 0.530.400.24 - 0.66Molecular Apocrine profileNo10.041Yes1.611.02 – 2.54TILs (%) 0.024 0.002≤ 51 1 &amp;gt; 5 0.570.34 – 0.95 0.440.25 - 0.77HER2 (0 vs 1+/2+)010.46610.3001+/2+0.810.45 – 1.451.370.77 - 2.42 Citation Format: William Jacot, Aurélie Maran-Gonzales, Oceane Massol, Charlotte Sorbs, Caroline Mollevi, Séverine Guiu, Florence Boissière-Michot, Jeanne Ramos. Prognostic value of HER2 expression levels in non-metastatic triple-negative breast cancer and correlation with other biomarkers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-05-05.

Prognostic Value of HER2-Low Expression in Non-Metastatic Triple-Negative Breast Cancer and Correlation with Other Biomarkers.

Simple SummaryHER2-low breast cancer is an emerging subtype for which very few data are available, especially within the triple-negative breast cancer (TNBC) group. Our aim was to evaluate HER2 expression using the 2018 ASCO/CAP guidelines for HER2 scoring and its prognostic value in a large retrospective series of 296 patients with non-metastatic TNBC. In this large, homogeneous series of patients with early TNBC, HER2 1+/2+ expression was associated with a lower histological grade and molecular apocrine profile, and a specific subgroup of androgen receptor-expressing TNBC with worse long-term prognosis. Considering the current development of anti-androgens for molecular apocrine-like tumors and anti-HER2 antibody–drug conjugates for this HER2 1+/2+ population, this clinicopathological association could have therapeutic implications. Although rare (2.7% of patients in our series), HER2 2+ TNBC seems to have worse relapse-free survival, advocating a dedicated clinical, biological and therapeutic evaluation of this subgroup.HER2-low breast cancer (i.e., HER 1+ or 2+, without gene amplification) is an emerging subtype for which very few data are available, especially within the triple-negative breast cancer (TNBC) group. Our aim was to evaluate HER2 expression and its prognostic value in a large retrospective series of patients with non-metastatic TNBC (median age: 57.7 years; range: 28.5–98.6). Among the 296 TNBC samples, 83.8% were HER2 0, 13.5% were HER2 1+, and 2.7% were HER2 2+ (HercepTestTM and 2018 ASCO/CAP guidelines for HER2 scoring). CK5/6 and/or EGFR-expressing androgen receptors and FOXA1-expressing tumors were classified as basal-like (63.8%) and molecular apocrine-like (MA, 40.2%), respectively. Compared with HER2 0 tumors, HER2 1+/2+ tumors exhibited a lower histological grade (1/2) (35.4% vs. 18.2%, p = 0.007) and MA profile (57.5% vs. 36.7%, p = 0.008). Moreover, patients with HER2 1+/2+ tumors were older (p = 0.047). After a median follow-up of 9.7 years, HER2 2+ tumors (compared with HER2 0/1+ tumors) were associated with worse relapse-free survival (RFS) (HR = 3.16, 95% CI [1.27; 7.85], p = 0.034) in a univariate analysis. Overall survival (OS) and RFS were not different in the HER2 0 and 1+/2+ groups. HER2 levels were not significantly associated with OS or RFS in a multivariate analysis.