HER2 mRNA Research Articles

Effect of neratinib on HER2 mRNA stability via hsa-miR-23a-5p and efficacy of CDK4/6 inhibitors in HER2-low breast cancer.

e12550 Background: Traditional HER2-negative breast cancer sometimes includes HER2-low components, and CDK4/6 inhibitors are the first- and second-line treatments for advanced HER2-low breast cancer. Nonetheless, little is understood about how the stability of HER2 mRNA in HER2-low breast cancer affects the effectiveness of CDK4/6 inhibitor therapy and the associated intervention techniques. Methods: In order to investigate the impact of HER2-low on the effectiveness of CDK4/6 inhibitor in treating breast cancer, we first used HER2-0 and HER2-low breast cancer cells/mice as the study object and assessed the anti-proliferation effect of CDK4/6 inhibitor using CCK8 technique and monoclonal formation experiment. Second, we increased the treatment of anti-HER2 inhibitor neratinib and assessed its impact on the efficacy of HER2 low breast cancer CDK4/6 inhibitors. By reducing the expression of HER2 in HER2 low breast cancer cells, we analyzed and determined whether HER2 is the primary pathway that influences the poor efficacy of HER2 low breast cancer CDK4/6 inhibitors; At last, actinomycin D was used to detect the mRNA stability of HER2 in the treatment of HER2-low breast cancer by neratinib against CDK4/6 inhibitors, and then microRNA sequencing and luciferase reporter gene detection technology were used. To investigate the specific mechanism of action of neratinib on the mRNA stability of HER2 in the treatment of HER2-low breast cancer by CDK4/6 inhibitors. Results: Our study found that treatment with CDK4/6 inhibitors was significantly less effective against HER2-low breast cancer compared to HER2-0 subtype (P=0.005). Notably, in HER2-low subtype cells, HER2 knockdown significantly inhibited cell proliferation (P<0.001), G1 phase cell cycle arrest (P=0.001), and downregulated cyclin D1 expression and CDK4 protein levels (P=0.007, P=0.003). Second, it was found that in the cell and animal models of HER2-low breast cancer, the addition of the CDK4/6 inhibitor drug neratinib effectively reduced the stability of HER2 mRNA (P=0.001), inhibited the cell cycle of G1 phase (P=0.005). In addition, we observed that lowering the dose of neratinib to 1/2 (20 mg/kg) remained effective and well tolerated (P=0.002). Finally, we found that neratinib can reduce the mRNA stability of HER2 by binding hsa-miR-23a-5p to the 3'UTR region of HER2 (P<0.001), thereby improving the efficacy of CDK4/6 inhibitors in HER2-low breast cancer. Conclusions: We found that low expression of HER2 reduced the efficacy of CDK4/6 inhibitors in HER2-low breast cancer; Neratinib reduces the mRNA stability of HER2 by binding hsa-miR-23a-5p to the 3 'UTR region of HER2, thereby improving the efficacy of CDK4/6 inhibitors in HER2-low breast cancer. These findings provide a tolerable and effective two-drug combination regimen for patients with clinical HER2-low breast cancer.

Characterization of HER2 alterations in lung adenocarcinoma.

8534 Background: Mutations in the human epidermal growth factor receptor 2 (ERBB2; HER2) are oncogenic in lung adenocarcinoma (LUAD). An FDA-approved drug, trastuzumab deruxtecan, is now available as second line treatment (rx) of patients (pts) with HER2 mutated NSCLC. HER2 activation occurs via gene mutation (mt), gene amplification (amp), or protein overexpression. Further information is needed about HER2 alterations (alt) and the potential association with rx response. The purpose of this study is to describe the genomic landscape of a cohort of pts with HER2 alt LUAD in the American Association for Cancer Research (AACR) Project Genomics Evidence Neoplasia Information Exchange (GENIE) Biopharma Collaborative (BPC). Methods: In a cohort of 16,241 NSCLC LUAD pts available in GENIE v.13.0, we analyzed the association of HER2 alt (mt/amp) with alt in other driver genes. Also, we assessed the relationship between HER2 alt and expression levels using TCGA and CPTAC cohorts (566 and 110 pts), and prognosis using GENIE NSCLC BPC cohort (1,846 pts). Results: LUAD pts were classified as having KRASmt (K; 6426 pts; 31.9%), EGFRmt (E; 5256 pts; 26%) or EML4-ALK fusion (A; 102 pts; 0.5%). The remainder were classified as “KRAS/EGFR/ALK other (KEAother).” HER2mt (6.06%) and amp (2.06%) were more frequent in the KEAother cohort(p<0.001). HER2 amp were also found more frequently in the E deletion(del) mt cohort (1.63%), compared to KA cohorts (p<0.001). Additionally, there was significant co-occurrence of HER2 mt and HER2 amp (OR 6.49; p<0.001) in the KEAother cohort. HER2mt/amp had significant odds of co-occurrence with TP53 alt (OR 1.43; p<0.001), CDKN2A del (OR 2.15; p<0.001), TERT amp (OR 2.37; p<0.001), RB1 alt (OR 1.6; p<0.001). Additionally, HER2 amp co-occurred with TP53 alt (OR 4.0; p<0.001), CDKN2A del (OR 3.44; p<0.001), while HER2 mt co-occurred with CDKN2A del (OR 2.03; p<0.001), TERT amp (OR 2.43; p<0.001). BRAF mts were mutually exclusive with HER2 mt/amp (OR 2.31; p<0.001) and HER2 mt (OR 1.93; p<0.001). In the E cohort, there was a significant rate of HER2 mt or amp co-occurring with RB1 alt (OR 2.95; p<0.001). Despite limited survival data in the GENIE database, HER2 mt and amp had a trend toward inferior mOS (HER2mt 29.4 months vs HER2amp 27.8 months vs HER2wt 51.0 months), but this difference was not statistically significant. High HER2 mRNA expression is associated with increased age, while low expression is associated with stage IV disease. Pts with high HER2 expression that never smoked, have a trend toward inferior mOS (p=0.19), but this is based on a limited number of pts (n=18). Conclusions: HER2 mts are found in 1-4% of pts with NSCLC but can be identified in up to 6% of KEAother NSCLC. In contrast to prior reports, there was a significant rate of co-occurring HER2 mt and HER2 amp. BRAF mts were mutually exclusive with both HER2 mt and amp. Further analysis using the Caris database is planned to evaluate clinical outcomes by HER2 alt, co-mts, and prior rx received.

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 PO1-24-08: Targeting of HER3 potentiates the antitumor activity of paclitaxel against triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) overexpression. This aggressive form of breast cancer carries a higher risk of recurrence compared to other subtypes, presenting a significant clinical challenge. Although chemotherapy is commonly used as the primary treatment for a substantial number of TNBC patients, it is often accompanied by drug resistance and frequent tumor recurrence. Urgent efforts are needed to identify novel molecular targets specific to TNBC and develop effective therapies to combat this aggressive disease. The ultimate objective is to discover treatments capable of overcoming drug resistance and improving the overall survival of patients with TNBC. Methods: Immunohistochemistry was performed to examine the expression of HER3 in TNBC samples. Western blots were used to assess protein expression and activation. Cell proliferation and viability were determined by cell growth (MTS) assays. TCGA databases were analyzed to correlate HER3 mRNA expression with the clinical outcomes of TNBC patients. Specific shRNA was used to knockdown HER3 expression. LIVE/DEAD Cell Imaging was to detect live and dead cells. Orthotopic tumor models were established in nude mice to determine the capability of TNBC cells forming tumors and to test if our newly developed anti-HER3 monoclonal antibody (mAb) 4A7 could potentiate the antitumor activity of paclitaxel in vivo. Results: The chemo-resistant subline HCC1806-TR was obtained by subjecting TNBC HCC1806 cells to prolonged treatment with paclitaxel. In comparison to the parental cell line HCC1806-P, HCC1806-TR exhibited enhanced HER3 expression and activation. It is noteworthy that approximately half of the tested TNBC specimens and cell lines displayed elevated expression of HER3. Analyzing TCGA databases revealed that TNBC patients with high levels of HER3 mRNA expression in their tumors had significantly poorer overall survival (OS) and relapse-free survival (RFS) compared to those with low HER3 expression. Specifically knockdown of HER3 markedly inhibited TNBC cell proliferation and mammosphere formation in vitro and tumor growth in vivo. Blockade of HER3 signaling with our mAb (4A7) in combination with paclitaxel exerted significant antitumor activity against TNBC in vitro and in vivo. Conclusions: Our data demonstrate that increased HER3 is an effective therapeutic target for TNBC and our anti-HER3 mAb (4A7) may enhance the efficacy of paclitaxel in TNBC. Keywords: HER3, Anbibody, Chemotherapy, Triple Negative Breast Cancer Citation Format: Hui Lyu, Sanbao Ruan, Congcong Tan, Ann Thor, Bolin Liu. Targeting of HER3 potentiates the antitumor activity of paclitaxel against triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-08.

Abstract PO5-01-08: The impact of Human Epidermal growth factor Receptor 2 (HER2) expression by RT-PCR on tumor characteristics and outcome in estrogen receptor (ER) positive, HER2 negative early-stage breast cancer

Abstract Background: The role of HER2 expression in HER2 negative breast cancer is evolving. Whether HER2 expression has an impact on ER positive, HER2 negative early-stage breast cancer in unclear. Oncotype DX assay Recurrence Score comprises of standardized quantitative HER2 mRNA expression levels by RT-PCR. The objective of this study was to investigate the impact of Oncotype HER2 score on tumor characteristics and outcome. Methods: All women assigned to the Clalit Health Services registry between 2006 to 2011 with ER positive, HER2 negative early-stage breast cancer whose tumor were sent for oncotype were identified. Patients in which HER2 score was reported in the oncotype results were included. Differences in age, tumor characteristics and chemotherapy administration between lower HER2 score (H1), i.e. HER2 score ≤ 9.1, to higher HER2 score (H2), i.e. 9.2-10.7, were analyzed. 5-year Kaplan–Meier for distant recurrence free survival (DRFS) and overall survival (OS) were estimated and differences between H1 and H2 were evaluated. Analyses were repeated separately for node negative and for node positive (N mic and 1-3 lymph nodes) disease. Results: A total of 1535 patients were included, 948 patients with node negative disease and 587 with node positive disease. Of these, 814 and 721 patients were categorized to the H1 and H2 groups, respectively. Median follow-up was 5.4 years. The distribution of oncotype RS was different, with significantly lower proportion of patients with RS between 0-10 and higher proportion of RS above 25 in the H1 group compared to the H2 groups. This difference was identified both for patients with node negative and node positive disease (Table). The distributions of age, tumor size and histological subtype were comparable between the H1 and the H2 groups, both for node negative and node positive disease. Within each RS group, administration of chemotherapy was comparable between H1 and H2 groups, regardless of nodal involvement. DRFS and OS were not associated with HER2 score. For RS 0-25, node negative disease, 5-year DRFS was 96.2% in H1 and 96.4% in H2 groups (p=0.92) and 5-year OS was 97.2% in H1 and 98.1% in H2 groups (p=0.39). For RS >25, node negative disease, 5-years DRFS was 89% in H1 and 90.8% in H2 groups (p=0.33) and 5-year OS was 90.6% in H1 and 90.8% in H2 groups (p=0.7). Similarly, for node positive disease, DRFS and OS were comparable between H1 and H2 group in all subgroups. Conclusions: Low HER2 mRNA expression by Oncotype RT-PCR assay was associated with significantly high Oncotype RS. Patients' outcome was not directly affected by HER2 score and was determined by RS. Further study is desired to clarify the role of HER2 expression in early stage, luminal breast cancer. Table. Comparison of age and tumor characteristics between low (H1) and high (H2) HER2 score Citation Format: Hadar Goldvaser, Raz Mutai, Salomon Stemmer, Iryna Kuchuk, Margarita Tokar, Shani Paluch-Shimon, Rinat Yerushalmi, Karen Drumea, Ella Evron, Amir Sonnenblick, Einav Gal-Yam, Gil Bar Sela, Ayelet Shai, Rotem Merose, Avital Bareket-Samish, Lior Soussan-Gutman. The impact of Human Epidermal growth factor Receptor 2 (HER2) expression by RT-PCR on tumor characteristics and outcome in estrogen receptor (ER) positive, HER2 negative early-stage 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 PO5-01-08.

Abstract PO3-01-05: Enhancing HER2 Evaluation: Correlation between APIS Breast Cancer Subtyping Kit and IHC/ISH for Accurate HER2 Quantification

Abstract Background: The quantification of HER2-low in breast cancer (BC) has garnered considerable interest due to emerging evidence of the efficacy of targeted therapies in this patient subgroup. Trastuzumab deruxtecan (T-Dxd), an antibody-drug conjugate targeted at HER2, has recently gained approval in the USA and Europe for treating HER2-low BC, which is currently defined as immunohistochemical (IHC) scores of 1+ or 2+ without HER2/ERBB2 in situ hybridization (ISH) amplification. HER2 quantification using IHC/ISH was primarily designed to identify tumors overexpressing HER2, rather than differentiate between HER2-low and the absence of expression. Consequently, the adequacy of these assays in accurately detecting HER2- low remains uncertain. Improving the accuracy of HER2 evaluation is crucial to prevent the incorrect selection of patients for T-Dxd treatment. There is a clear need to develop HER2 testing methods that are more precise, reliable, and capable of detecting HER2-low expression with greater sensitivity and reproducibility. This is particularly important as the minimum threshold of HER2 expression required for T-Dxd efficacy is still under investigation, with clinical data suggesting the potential activity of T-Dxd even in patients with IHC 0 HER2 score. Here, we evaluated the correlation between HER2 mRNA expression levels, detected by APIS Breast Cancer Subtyping Kit, and IHC HER2 classification. Methods: Formalin-fixed paraffin-embedded (FFPE) tumour tissue sections (n=642) obtained by core needle biopsy or resection underwent histological analysis in accordance with laboratory’s standard of care methods. Samples with a HER2 score of 2+ were referred to ISH to determine ERBB2 amplification. To evaluate the diagnostic performance, the concordance between HER2 IHC score, and APIS Breast Cancer Subtyping Kit RNA expression level was reported in terms of Overall Percent Agreement (OPA), Negative Percent Agreement (NPA), and Positive Percent Agreement (PPA), along with their corresponding 95% confidence intervals (CI). Results: HER2 expression detected by APIS Breast Cancer Subtyping Kit showed strong correlation with IHC/ISH, with an OPA of 94.2% (95% CI: 92.2-95.8), PPA of 89.2% (95% CI: 80.1-94.4) and NPA of 94.9% (95% CI: 92.8-96.4). The expression of HER2 was detected by APIS Breast Cancer Subtyping Kit in a subset of patients with 0 and 1+ IHC HER2 scores, suggesting its potential as a more sensitive and reliable method for detection of HER2 expression, and providing the opportunity to enhance HER2 stratification. Conclusions: APIS Breast Cancer Subtyping Kit can accurately detect HER2 expression and has the potential to further stratify the HER2-low patients. Further studies examining the relationship between HER2 expression and the response to anti-HER2 therapies could yield valuable insights into treatment administration and identify patients who could benefit from such therapies. Incorporating continuous quantification of HER2 could optimize patient outcomes. Citation Format: Anna Gasior, Joanna Gorniak, Sara Rollinson, Leanne Gough, Anne-Sophie Wegscheider, Axel Niendorf. Enhancing HER2 Evaluation: Correlation between APIS Breast Cancer Subtyping Kit and IHC/ISH for Accurate HER2 Quantification [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 PO3-01-05.

Lanthanide Complex for Single-Molecule Fluorescent in Situ Hybridization and Background-Free Imaging.

Traditional single-molecule fluorescence in situ hybridization (smFISH) methods for RNA detection often face sensitivity challenges due to the low fluorescence intensity of the probe. Also, short-lived autofluorescence complicates obtaining clear signals from tissue sections. In response, we have developed an smFISH probe using highly grafted lanthanide complexes to address both concentration quenching and autofluorescence background. Our approach involves an oligo PCR incorporating azide-dUTP, enabling conjugation with lanthanide complexes. This method has proven to be stable, convenient, and cost-effective. Notably, for the mRNA detection in SKBR3 cells, the lanthanide probe group exhibited 2.5 times higher luminescence intensity and detected 3 times more signal points in cells compared with the Cy3 group. Furthermore, we successfully applied the probe to image HER2 mRNA molecules in breast cancer FFPE tissue sections, achieving a 2.7-fold improvement in sensitivity compared to Cy3-based probes. These results emphasize the potential of time-resolved smFISH as a highly sensitive method for nucleic acid detection, free of background fluorescence interference.

Exosomal Linc00969 induces trastuzumab resistance in breast cancer by increasing HER-2 protein expression and mRNA stability by binding to HUR

BackgroundBreast cancer (BC) is the most common malignant disease in female patients worldwide. In HER-2+ BC patients, trastuzumab therapy is associated with a better prognosis. However, many HER-2+ BC patients experience recurrence or metastasis because of trastuzumab resistance. The mechanisms underlying trastuzumab resistance remain unclear. Recently, substantial evidence has suggested that exosomes are associated with drug resistance, and lncRNAs have attracted increasing attention due to their potential role in the regulation of trastuzumab resistance.MethodsWe collected the exosomes from the plasma of BC patients with and without trastuzumab resistance, sequenced the whole transcriptomes, identified differentially expressed lncRNAs, and identified lncRNA Linc00969, which was overexpressed in trastuzumab-resistant patients. Then, we established trastuzumab-resistant BC cell lines and explored the role of exosomal Linc00969 in trastuzumab resistance in vitro and in vivo by silencing or overexpressing Linc00969 and performing a series of functional analyses. Furthermore, to explore the mechanism by which exosomal Linc00969 contributes to trastuzumab resistance, we measured changes in HER-2, HUR and autophagy-related protein expression levels after regulating Linc00969 expression. In addition, we investigated the interaction between Linc00969 and HUR via pull-down and RIP assays and the effect of HUR on HER-2 expression and trastuzumab resistance after blocking HUR.ResultsWe first found that exosomal lncRNA Linc00969 was overexpressed in trastuzumab-resistant BC patients and that exosome-mediated Linc00969 transfer could disseminate trastuzumab resistance in BC. Then, we found that silencing Linc00969 could reduce trastuzumab resistance and that overexpressing Linc00969 could enhance trastuzumab resistance. Furthermore, our results showed that Linc00969 could upregulate HER-2 expression at the protein level and maintain the stability of HER-2 mRNA by binding to HUR. Additionally, we found that exosomal Linc00969 could regulate trastuzumab resistance by inducing autophagy.ConclusionsIn this study, we first identified that exosomal lncRNA Linc00969 could induce trastuzumab resistance by increasing HER-2 protein expression and mRNA stability by binding to HUR, and Linc00969 might also be involved in trastuzumab resistance by inducing autophagy. Our results elucidate a novel mechanism underlying trastuzumab resistance, and Linc00969 might be a new target for improving the treatment of HER-2+ BC patients.

HER3 functions as an effective therapeutic target in triple negative breast cancer to potentiate the antitumor activity of gefitinib and paclitaxel

BackgroundTriple negative breast cancer (TNBC) represents a significant clinical challenge. Chemotherapy remains the mainstay for a large part of TNBC patients, whereas drug resistance and tumor recurrence frequently occur. It is in urgent need to identify novel molecular targets for TNBC and develop effective therapy against the aggressive disease.MethodsImmunohistochemistry was performed to examine the expression of HER3 in TNBC samples. Western blots were used to assess protein expression and activation. Cell proliferation and viability were determined by cell growth (MTS) assays. TCGA databases were analyzed to correlate HER3 mRNA expression with the clinical outcomes of TNBC patients. Specific shRNA was used to knockdown HER3 expression. IncuCyte system was utilized to monitor cell growth and migration. LIVE/DEAD Cell Imaging was to detect live and dead cells. HER3 recognition by our anti-HER3 monoclonal antibody (mAb) 4A7 was verified by ELISA, flow cytometry, and co-immunoprecipitation assays. Orthotopic tumor models were established in nude mice to determine the capability of TNBC cells forming tumors and to test if our mAb 4A7 could potentiate the antitumor activity of paclitaxel in vivo.ResultsElevated expression of HER3 was observed in approximately half of the TNBC specimens and cell lines tested. Analyses of TCGA databases found that the TNBC patients with high HER3 mRNA expression in the tumors showed significantly worse overall survival (OS) and relapse-free survival (RFS) than those with low HER3 expression. Specific knockdown of HER3 markedly inhibited TNBC cell proliferation and mammosphere formation in vitro and tumor growth in vivo. Our mAb 4A7 abrogated heregulin (a ligand for HER3), but not SDF-1 (a ligand for CXCR4)-induced enhancement of TNBC cell migration. Combinations of 4A7 and the EGFR-tyrosine kinase inhibitor (TKI) gefitinib dramatically decreased the levels of phosphorylated HER3, EGFR, Akt, and ERK1/2 in TNBC cells and potently induced growth inhibition and cell death. Moreover, 4A7 in combination with paclitaxel exerted significant antitumor activity against TNBC in vitro and in vivo.ConclusionsOur data demonstrate that increased HER3 is an effective therapeutic target for TNBC and our anti-HER3 mAb (4A7) may enhance the efficacy of gefitinib or paclitaxel in TNBC.

Molecular Characterization of HER2-Low Invasive Breast Carcinoma by Quantitative RT-PCR Using Oncotype DX Assay.

HER2 immunohistochemistry (IHC) reproducibility is suboptimal for HER-low cases (IHC 1+ or 2+). The Yale cohort included 214 stages I-II estrogen receptor positive breast cancers with IHC scores 0, 1+, and 2+ and routine Oncotype DX Recurrence Score (RS) results. The Exact Sciences (ES) cohort included 9 57 624 patients who had an Oncotype DX RS assay that assigns HER2-negative, equivocal, or positive status based on HER2 mRNA levels. HER2 mRNA levels varied across IHC categories but with increasing medians of 9.10 (n = 89), 9.20 (n = 71), and 9.45 (n = 54) in IHC 0, 1+, and 2+, respectively. 22.4% of HER2-low (1+/2+) cancer had RS > 25. Over 98% of HER-low cancers were HER2-negative by Oncotype DX assignment. Cancers with higher mRNA levels exist within IHC 0 and low categories, most of the HER2-low patients by IHC have low RS indicating no benefit from current adjuvant chemotherapies.

Invasive Breast Cancer with HER2 ≥4.0 and

To investigate the HER2 status and clinicopathological features in invasive breast cancer with HER2 ≥4.0 and <6.0, which has always been controversial. Forty breast cancer cases with HER2 ≥4.0 and <6.0 by fluorescence in situ hybridization (FISH) were collected and classified into two groups based on the HRE2/CEP17 ratio (Group A: ≥2.0, n=22; Group B: <2.0, n=18). Clinicopathological characteristics, HER2 status, risk classification, and molecular typing were further analyzed and compared by 21-Gene expression assay and MammaPrint plus BluePrint test. The majority of cases in both groups were invasive carcinoma (NOS), with histological grade II, HR+, Ki-67 ≥20%, HER2 2+, and a high risk of recurrence, although younger patients and lymph node metastases were more common in Group A. Surprisingly, all HR+ breast cancers in both groups were classified as luminal-type, HR- cases were all basal-type or unknown, and the index of HER2 in all cases was <0.000 using the BluePrint test, which indicated that HER2 status should be negative. Furthermore, the level of HER2 mRNA expression in all cases of both groups was <10.7, which was defined as HER2 negative by the 21-Gene expression assay. In addition, 10 patients of Group A received anti-HER2 neoadjuvant therapy; only one patient with HR- achieved Grade 5 based on the Miller-Payne system, whereas none of the patients achieved pathological complete response (pCR) based on the Residual Cancer Burden system. Group A breast cancer, which has always been unquestionably diagnosed as HER2 amplification, was more likely to be HER2 negative and derived less benefit from anti-HER2 neoadjuvant chemotherapy. Group A breast cancer should be distinguished from classical HER2-positive breast cancers when assessing HER2 FISH, and a larger cohort of Group A patients should be included in further studies.

Cardiac Troponin T (TNNT2) plays a potential oncogenic role in colorectal carcinogenesis

PurposeColorectal cancer (CRC) is the third most common cancer in the world. The purpose of this study was to investigate the role of TNNT2 in the proliferation, migration and invasion of CRC cells and its expression in CRC tissues to better understand the regulatory role of TNNT2 in CRC.MethodsWestern blotting (WB) and qPCR were used to detect the expression of TNNT2 in colorectal cancer tissues and paracancerous tissues. CCK-8, colony formation, Transwell and other experiments were used to clarify the role of TNNT2 in the proliferation, migration and invasion of colorectal cancer cells. Changes in TNNT2, EGFR and HER2 mRNA transcription levels were detected by SYBR Real-Time PCR assay, and the effects of TNNT2 overexpression or knockdown on the expression of EGFR, HER2 and EMT-related proteins in CRC cells were determined by WB. TNNT2 and EGFR intreaction was carried out in HCT116 cells by coimmunoprecipitation experiments.ResultsThe protein and mRNA expression level of TNNT2 in CRC tissues were higher than those in paracancerous tissues. The CCK-8 results suggested that overexpression of TNNT2 significantly promoted the proliferation of HCT116 and RKO cells, and TNNT2 konckdown gets the opposite result; and the colony formation results were the same as tthose of CCK-8 assay. Transwell invasion and migration experiments showed that overexpression of TNNT2 promoted the migration and invasion of HCT116 and PKO cells, and TNNT2 konckdown suppressed the migration and invasion of the these cells. The SYBR Green I real-time PCR method revealed that them RNA levels of TNNT2, EGFR and HER2 in the TNNT2 overexpression group were higher than those in RKO cells. WB showed that overexpressing TNNT2 increased the expression of EGFR and HER2 in HCT16 and RKO cells,decreased the expression of EMT marker E-cadherin, and increased the expression of Vimentin and N-cadherin. Konckdown of TNNT2 decreased the expression of EGFR and HER2, increased the expression of E-cadherin, and decreased the expression of Vimentin and N-cadherin in HCT16 and RKO cells. The immunocoprecipitation experiment showed that there was an interaction between EGFR and TNNT2.ConclusionTNNT2 can promote the proliferation, invasion and metastasis of colorectal cancer cells. There is an interaction between TNNT2 and EGFR protein. TNNT2 can upregulate EGFR and HER2-related proteins in colorectal cancer cells and promote the occurrence of EMT. Therefore, TNNT2 can promote the invasion and metastasis of CRC cells through the EGFR/HER2/EMT signal axis, suggesting that TNNT2 is a potential target of CRC treatment.

Comprehensive Analysis of Human Epidermal Growth Factor Receptor 2 Through DNA, mRNA, and Protein in Diverse Malignancies.

Human epidermal growth factor receptor 2 (HER2) expression (protein immunohistochemistry [IHC] or gene amplification [copy-number variation, CNV]) predicts anti-HER2 therapy responsiveness, although recently it was shown that even low HER2-expressing breast cancers benefit from trastuzumab-deruxtecan. Little is known about HER2 transcriptomic (mRNA) expression, and comparisons between genomic, mRNA, and protein HER2 assays. HER2 status was evaluated using clinical-grade IHC (protein), quantitative reverse transcription polymerase chain reaction (mRNA), and next-generation sequencing (NGS; amplifications). Multi-institutional HER2 testing was performed on 5,305 diverse cancers including non-small-cell lung (n = 1,175), breast (n = 1,040), and colon cancers (n = 566; N = 3,926 tested for CNV; N = 1,848, mRNA; N = 2,533, IHC). Overall, 4.1% (161/3,926) had NGS HER2 amplification; 33.3% (615/1,848) had mRNA overexpression; and 9.3% (236/2,533) were IHC-positive. In 723 patients with all three tests (CNV/mRNA/IHC), various amplification/expression patterns occurred: 7.5% (54/723) had all three HER2 tests positive; 62.8% (454/723) had all three tests negative. Discrepant patterns between amplification and overexpression emerged. For instance, 20% (144/723) of patients had mRNA overexpression alone with negative CNV and IHC. A range in values for only mRNA+ cases occurred in different tumor types (eg, 16.9%, breast; 5%, hepatobiliary). There were 53 patients with various tumors from our institution who had all three assays attempted; 22 tested positive for HER2, and seven received anti-HER2 therapy: two patients achieved response: one (esophageal cancer), complete response (≥42 months); one (cholangiocarcinoma), who only had HER2 mRNA positivity (tissue was inadequate for IHC and CNV assessment), partial response (≥24 months) on HER2-based regimens. We demonstrate variability of HER2 (protein and mRNA) expression and amplification using comprehensive assays (CNV, mRNA, and IHC) among diverse cancers. As HER2-targeted therapy indications expand, the relative importance of these modalities merits further evaluation.

ECOG-ACRIN E2197: Comparison of HER2 gene expression by RT-PCR across all HER2 immunohistochemistry groups with recurrence analysis.

515 Background: Accurate assessment of HER2-low [immunohistochemical (IHC) score of 1+ or 2+ and in-situ hybridization negative] breast cancer is clinically relevant following DESTINY-Breast04. In hormone receptor positive breast cancers, Oncotype DX assay Recurrence Score results are widely used to guide adjuvant chemotherapy selection, and standardized quantitative HER2 mRNA reference-normalized expression levels by RT-PCR are included in patient reports. Previously, a high degree of concordance between central IHC and quantitative HER2 by RT-PCR was demonstrated in E2197. Here, we compare quantitative HER2 gene expression using Oncotype DX assay within all IHC subgroups and risk of recurrence in E2197. Methods: Data analyzed were from a case-control sample from E2197 (no adjuvant anti-HER2 therapy) of 755 patients. Central IHC for HER2 used duplicate 1.0 mm microarrays (HercepTest, Dako) and scored per 2007 ASCO/CAP guidelines. Based on quantitative reference-normalized RT-PCR measures of HER2 expression, cases were assigned: positive ≥11.5 units, equivocal ≥10.7 to &lt;11.5 units, and negative &lt;10.7 units (1 unit increase represents ~2-fold increase in RNA). Spearman’s rank correlation was used to measure strength of association between the 2 methods. Cox proportional hazards regression was used to evaluate the association between quantitative HER2 expression and recurrence (defined as invasive breast cancer in local, regional, or distant sites). Analyses were weighted to account for study design. Results: 48% were IHC 0, 11% were IHC 1+, 23% were IHC 2+, and 18% were IHC 3+. 85% were HER2 negative, 3% equivocal, and 11% positive by RT-PCR. While there was moderately strong correlation between HER2 IHC and HER2 gene expression by RT-PCR [Spearman’s rho: 0.63 (95% CI: 0.59, 0.67); p&lt;0.001], there were wide and overlapping ranges of HER2 gene expression across all IHC categories with almost identical medians and inter-quartile ranges for HER2 IHC 1+ and 2+ (n=248; see table). An exploratory analysis showed 86% of IHC 0, 97% of IHC 1+, and 99% of IHC 2+ had HER2 &gt;8 units. After controlling for baseline clinicopathologic features (age, hormone receptor status, grade, size, and nodal status), continuous quantitative HER2 expression was associated with time to recurrence in HER2 IHC 0 to 2+ [HR 1.17 per 1-unit increase in HER2 (95% CI 1.01, 1.35; p=0.036)]. Conclusions: There was a wide range of quantitative HER2 gene expression using Oncotype DX assay across all IHC categories; continuous HER2 expression is independently associated with recurrence. Quantitative HER2 expression may be useful for identification of HER2-low breast cancer. Further studies are needed. [Table: see text]

3MO HER2 expression and early response to patritumab deruxtecan (HER3-DXd) in early-stage hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC): A correlative analysis from SOLTI-TOT-HER3 trial

HER3-DXd and trastuzumab deruxtecan (T-DXd) are antibody-drug-conjugates (ADCs) directed against HER3 and HER2, respectively. Both drugs have shown efficacy in patients (pt) with HR+/HER2- BC. Baseline HER3 protein or mRNA levels do not predict efficacy from HER3-DXd. Here, we aimed to evaluate the association of early response to HER3-DXd with HER2 levels. TOT-HER3 (NCT04610528), a window-of-opportunity trial, evaluated a single dose of HER3-DXd in 77 pts with untreated HR+/HER2- early BC in Part A (6.4 mg/kg) and 17 in Part B (5.6 mg/kg). Primary objective was CelTIL score [a surrogate of response that combines tumor cellularity and tumor-infiltrating lymphocytes] variation between baseline and day 21 tumor samples. Here, CelTIL response was defined as an absolute increase of 20 at day 21. HER2 expression was determined by immunohistochemistry (IHC). RNA and DNA were purified from baseline FFPE tumor samples and analyzed using the nCounter and the VHIO-300 NGS platforms, respectively. Logistic regression models and area under the ROC Curve (AUC) estimated the performance of HER2 IHC, mRNA or copy-number (CN) levels with CelTIL response. In TOT-HER3 Part A, HER2 IHC status was 32% HER2 0, 38% HER2 1+ and 30% HER2 2+. Distribution of high CelTIL responders was 40% in HER2 0, 45% in HER2 1+ and 13% in HER2 2+ (p=0.034). High ERBB2 mRNA and RNA-based HER2 amplicon signatures were significantly associated with low CelTIL response (ERBB2: Odds Ratio [OR]=0.34, p<0.001, AUC=0.71; and HER2 amplicon: OR=0.20, p=0.005, AUC=0.74). The association of ERBB2 mRNA with CelTIL response was independent of HER2 IHC expression (p=0.002), PAM50 subtype (p=0.001) or proliferation (p=0.003). High HER2 CN signal was found associated with low CelTIL response (p=0.005). The association of HER2 IHC and ERBB2 mRNA with CelTIL response will be independently validated in TOT-HER3 Part B. Low HER2 IHC, mRNA or CN levels in early-stage HR+/HER2- BC are associated with high response to HER3-DXd. HER3-DXd might be highly active in HR+ BC with very low HER2 levels, thereby offering a new therapeutic paradigm to this subset of patients.

Epstein-Barr virus immediate-early protein Zta mediates the proliferation and migration of HER2-overexpressing cancer cells.

Epstein-Barr virus immediate-early protein Zta plays an active role in altering cellular gene expression, which may be fundamentally linked to the viral life cycle, cell cycle, cell growth, and differentiation. HER2 is associated with a wide variety of human cancers, and its knockdown significantly reverses the malignant features of HER2-positive cancers. The aim of this study was to investigate the potential role of Zta in regulating HER2 expression and phenotype changes of MDA-MB-453 cells. Our results indicate that ectopic expression of Zta resulted in downregulation of the HER2 protein in cancer cells (MDA-MB-453, SKBR-3, BT474, and SKOV-3). The Zta protein significantly decreased HER2 mRNA and protein expression in MDA-MB-453 cells in a dose-dependent manner. Mechanistically, Zta recognized and targeted the promoter of HER2 gene, reducing the transcriptional activity of the HER2 gene. Zta induced G0/G1 arrest of MDA-MB-453 cells, inhibiting their proliferation and migration activity. These data suggest that Zta may act as a transforming suppressor of the HER2 gene.

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.

Abstract LB042: Targeting ATR enhances the antitumor efficacy of patritumab deruxtecan (HER3-DXd)in tamoxifen-resistant ER+ breast cancer cells by inducing DNA damage and apoptosis

Abstract Background: HER3, a member of the ERBB family of receptor tyrosine kinases that activates multiple oncogenic signaling pathways, is overexpressed in 50-70% of breast cancers (BC). HER3 mRNA expression is highest in luminal (ER+) breast tumors. Approximately 30% of ER+ breast tumors are de novo resistant to tamoxifen. Therefore, therapeutically targeting HER3 with HER3-DXd, an antibody-drug conjugate (ADC) composed of a fully human anti-HER3 IgG1 monoclonal antibody (patritumab) covalently linked to a topoisomerase I (TOP I) inhibitor payload (deruxtecan) via a tetrapeptide-based cleavable linker, can be an effective treatment for tamoxifen-resistant (TMR) ER+ BC. After assessing HER3-DXd’s efficacy as a single agent, we sought to identify a synergistic partner to maximize its antitumor activity in HER3+/ER+ TMR BC. Methods: Whole-genome high-throughput siRNA screening (Ambion Silencer Select Human Genome siRNA Library V4) was performed to identify synergistic partners for maximizing HER3-DXd’s antitumor efficacy. The synergistic antitumor effects were assessed in vitro using a soft agar colony formation assay and a clonogenic assay in TMR HER3+/ER+ MCF7 and T47D BC cells and in vivo using xenograft mouse models of these cells. Targeting specificity was determined using siRNA. Treatment effects on cell cycle progression, DNA damage, apoptosis, and expression of proteins of interest were assessed by flow cytometry, comet assay, staining with annexin V-PE and 7-AAD, and Western blotting, respectively. Results: HER3-DXd inhibited the anchorage-independent growth of HER3+/ER+ cells by &amp;gt;50% at 5 nM and their colony formation at 5-25 nM (P &amp;lt; 0.05). Among the synergistic targets identified by whole-genome high-throughput siRNA screening, inhibiting ATR with siRNA or BAY1895344 showed the greatest synergistic effect with HER3-DXd in TMR HER3+/ER+ BC cells. In contrast, no synergistic effect was observed with the combination of BAY1895344 plus patritumab or control ADC (IgG-DXd), suggesting its dependence on HER3-DXd-mediated delivery of DXd. To further confirm the targeting specificity, we knocked down ATR or TOP I expression using siRNA in TMR HER3+/ER+ BC cells and then treated the cells with HER3-DXd or BAY1895344, respectively. A synergy was also observed, indicating that the drugs achieve the synergy by targeting ATR and TOP I. The combination of HER3-DXd plus BAY1895344 reprogrammed cell cycle progression from G2/M arrest to sub-G1 arrest by inhibiting both ATR/Chk1/cyclin A2/CDK2 and ATR/Chk1/cyclin E/CDK2 signaling. The combination also induced DNA damage, which was further confirmed by the reduced expression of H2AX, an ATR substrate that contributes to DNA repair, and the increased expression of γH2AX (phospho-H2AX at Ser139), an indicator of DNA damage. HER3-DXd and BAY1895344 synergistically inhibited the growth of both TMR HER3+/ER+ MCF7 (P &amp;lt; 0.0001) and T47D (P &amp;lt; 0.01) xenografts in mice. Conclusion: The combination of HER3-DXd plus ATR inhibitors has therapeutic potential for overcoming tamoxifen resistance in HER3+/ER+ BC. Citation Format: Xuemei Xie, Jangsoon Lee, Jon A. Fuson, Huey Liu, Young J. Gi, Thanasis Poullikkas, Pang-Dian Fan, Kumiko Koyama, Debu Tripathy, Naoto T. Ueno. Targeting ATR enhances the antitumor efficacy of patritumab deruxtecan (HER3-DXd)in tamoxifen-resistant ER+ breast cancer cells by inducing DNA damage and apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB042.

Molecular imprinting of miR-559 on a peptide-immobilized poly L-DOPA/silica core-shell and in vitro investigating its effects on HER2-positive breast cancer cells.

In a significant percentage of breast cancers, increased expression of the HER2 receptor is seen and is associated with the spread and worsening of the disease. This research aims to investigate the effect of miR-559 (which targets HER2 mRNA) on SKBR3 breast cancer cells and the possibility of their effective delivery with polymeric nanoparticles and tumor-targeting peptides. L-DOPA monomers were polymerized on the surface of silica nanoparticles in the presence of miR-559 (as a molecular template for molecular imprinting) then an anti-HER2 peptide coupled to the surface of these polymeric nanocomposites (miR-NC-NL2), and the effects of this construct against a HER2-positive breast cancer cells (SKBR3 cells) investigated in vitro conditions. The results showed that miR-NC-NL2 is selective for HER2-positive cells and delivers the miR-559 to them in a targeted manner. miR-NC-NL2 decreased the proliferation of SKBR3 cells and reduced the expression and production of HER2 protein in these cells. Effective and targeted delivery of miR-559 to HER2-positive cancer cells by the miR-NC-NL2 promises the therapeutic potential of this nascent structure based on its inhibitory effect on cancer growth and progression. Of course, animal experiments require a better understanding of this structure's anti-tumor effects.