HER2 Gene Copy Research Articles

Significance of concurrent evaluation of HER2 gene amplification and p53 and Ki67 expression in gastric cancer tissues.

The primary objective of this study is to explore the significance of concurrent evaluation of HER2 gene amplification and p53 and Ki67 expression in gastric cancer tissues. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) methodologies were used to detect HER2 gene amplification, as well as the expression levels of HER2, p53, and Ki67 proteins, across a group of 78 gastric cancer cases. The expression rate of the HER2 protein was determined to be 43.6% (34/78), with 17.9% (14/78) categorized as HER2 protein 3 + , 14.1% (11/78) as HER2 protein 2 + , and 11.5% (9/78) as HER2 protein 1 + . Using FISH technology, the HER2 gene amplification rate was identified as 19.2% (15/78), including 3 cases of HER2 gene cluster amplification, 5 cases of large granular amplification, 4 cases of punctate amplification, and 3 cases of high polysomy. The positive rate of p53 in gastric cancer cells was 52.6% (41/78), with 62.8% (49/78) of patients exhibiting a ki67 proliferation index ≤ 30, and 37.2% (29/78) accounting for a ki67 proliferation index > 30. The expression rates of the HER2 gene, p53, and ki67 in gastric cancer tissues were significantly associated with both gastric cancer staging and lymph node metastasis (P < 0.05). The HER2 gene amplification rate and gene copy number exhibit a positive correlation with the expression rates of p53 and ki67. Combining these assessments can provide crucial insights into the assessment of metastatic potential, disease progression, and prognosis of gastric tumor cells. This holds paramount importance in steering the formulation of individualized treatment strategies.

Abstract PO5-13-06: Classifying HER2-low breast cancer using a combination of ERBB2 mRNA expression and altered genes

Abstract Background It has been recently demonstrated that some HER2-low, defined as immunohistochemistry (IHC) 1+ or 2+ with no gene amplification by FISH, breast cancer (BC) patients respond to trastuzumab deruxtecan (T-DXd). Results of the DAISY trial also suggested clinical activity with T-DXd in some of those classified as HER2-0 cancers by IHC, indicating an unmet need to better identify cancers that may truly respond to T-DXd. We compared ERBB2 (HER2) mRNA expression obtained from whole-transcriptome sequencing with IHC/FISH assignment of HER2 status. Furthermore, we constructed a classifier using ERBB2 expression and alterations in other genes as a proof of concept to illustrate classification of BC samples into HER2-0 versus HER2-low status. Methods We chose consecutive HR positive/HER2-negative BC samples that were submitted for OncoExTraTM testing between April 2020 and October 2022. The OncoExTra test uses tumor-normal whole exome and whole transcriptome sequencing to detect somatic single base substitutions, indels, copy number alterations, gene fusions and alternative transcripts. In addition, we investigated the expression profile of select genes for this analysis. We utilized OncoExTra data to examine the relationship between HER2 IHC status and gene expression, mutation and copy number profiles of selected genes, and built a classifier to predict HER2 status. Results A total of 279 BC samples were analyzed, including 106 HER2-IHC 0, 120 HER2-IHC 1+ and 53 HER2-IHC 2+. Of these, 273 were primary and 6 were metastatic samples. The distributions of patient age and tumor fraction were similar among these categories (p=0.54 and p=0.41, respectively; one-way ANOVA). ERBB2 expression differed among all three HER2-IHC categories and was lowest in HER2-0 and highest in HER2-2+ (HER2-0 vs HER2-1+, p&amp;lt; 0.001; HER2-0 vs HER2-2+, p&amp;lt; 0.0001; HER2-1+ vs HER2-2+, p=0.011; HER2-0 vs HER2-low, p&amp;lt; 0.0001; Mann-Whitney U test in all comparisons). Five genes were mutated in more than 10% of samples: PIK3CA (45.5%), GATA3 (15.4%), CDH1 (12.5%), MAP3K1 (11.1%) and TP53 (10.8%). Mutations in two genes, GATA3 and PTEN, appeared to differ in frequency between the HER2-0 and HER2-low categories (Table 1). We selected 4 genes with mutations, 2 genes with copy number changes, and expression from ERBB2, ESR1, CD274, HLA-A, HLA-B and HLA-C to train a logistic regression classifier to predict HER2 IHC status. The classifier was trained on 163 samples, hyperparameter tuning was done using 55 samples, and testing was performed on 61 samples. Preliminary results indicated that of the 28 HER2-IHC 0 samples in the test set, 17 (65%) were classified as HER2-low. Further development and evaluation of the classifier using a blinded testing set is currently in progress. Conclusion In this cohort of BC samples, ERBB2 mRNA expression was positively associated with HER2 protein IHC status. Additional studies are required to determine whether a classifier including ERBB2 mRNA expression could be used to identify patients for T-DXd therapy. The relative enrichment of PTEN alterations in HER2-IHC 0 samples suggests that mTOR/AKT inhibitors may sometimes be appropriate in patients who are not eligible for T-DXd therapy, and further investigation should be considered. Table 1. Number (frequency) of altered genes by HER2 IHC status. Citation Format: Gargi Basu, Niru Chennagiri, Turgut Dogruluk, David Hall, Jess Hoag, Cynthia Flannery, Snehal Thakkar, Melanie Palomares, Frederick Baehner, Lajos Pusztai. Classifying HER2-low breast cancer using a combination of ERBB2 mRNA expression and altered genes [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 PO5-13-06.

Abstract 3705: Development and analytical validation of a novel assay for HER2 assessment on circulating tumor cells using Parsortix® isolation and BioView imaging technologies

Abstract Background. HER2 testing in tissue biopsies is extensively performed in breast cancer (BC) patients. However, tissue biopsy is invasive and repeated examination is impractical. Additionally, lack of concordance between primary tumor and metastatic sites are often observed. Liquid biopsy, on the other hand, enables less invasive and recurrent sample collection for ongoing monitoring. In this study, we developed an end-to-end Research Use Only assay for concomitant evaluation of HER2 gene amplification and protein overexpression in circulating tumor cells (CTCs). Methods. Blood was drawn from healthy volunteers into Streck Cell-Free DNA tubes, spiked with SKBR3 (HER2+) and Hs 578T (HER2-) breast cancer cells and processed on ANGLE's Parsortix® PC1 System, a microfluidic device that captures CTCs from metastatic breast cancer patients’ blood based on their size and deformability, without relying on specific markers. Captured cells were harvested and spun onto positively charged slides and stained with the Portrait HER2 immunofluorescence (IF) panel, consisting of a nuclear dye, epithelial and mesenchymal markers, leukocytes’ markers, and an antibody targeting HER2. Slides were then de-stained and subjected to HER2 FISH using a commercially available kit. All slides were imaged twice using BioView Allegro Plus, a platform equipped with artificial intelligence for automated imaging, CTC candidate identification and reporting. First, an automated scan after IF was performed to identify CTCs based on marker expression and morphology profiles. Second, an automated relocation of the CTCs based on IF results and reimaging using higher magnification to ensure precise analysis of the FISH signals in single cells. The workflow is being implemented in a cohort of BC patients. Results. Following IF, HER2 protein expression in SKBR3 cells was significantly higher (p&amp;lt;0.0001) than that of HER2-negative cells, with 100% analytical specificity and sensitivity. Following FISH, 100% of the cells were de-stained and presented sharp HER2/CEP17 foci, with 96% of the SKBR3 cells and only 4% of the Hs 578T cells displaying HER2 amplification. Additionally, in a small cohort of metastatic BC patients’, 100% of the identified CTCs are retained and evaluable from IF to FISH. Conclusions. An assay integrating HER2 identification by IF and FISH to characterize CTCs in metastatic BC patients was successfully developed. The assay enables the identification of both HER2 protein and gene copy information from each target cell with minimal cell loss, with the potential for minimally invasive patient monitoring during treatment and better classification of patients who may qualify and benefit from HER2-targeted therapies. An added value of the assay is the practicality of utilizing preserved blood samples, allowing streamline batch shipping of clinical samples. Citation Format: Mariacristina Ciccioli, Alex Young, Laila Vlietinck, Chassidy Johnson, Anne-Sophie Pailhes-Jimenez. Development and analytical validation of a novel assay for HER2 assessment on circulating tumor cells using Parsortix® isolation and BioView imaging technologies [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 3705.

Effect of HER2/CEP17 ratio on survival in metastatic HER2-positive gastric cancer, multicenter study.

HER2-positive metastatic gastric cancer is still a highly fatal disease despite advances. We aimed to investigate the relationship between HER2/CEP17 ratio and survival in patients with HER2-positive metastatic gastric cancer. A total of 99 patients from 8 different centers in Turkey were included in the study. Patients with HER2-positive metastatic gastric cancer and whose HER2/CEP17 ratio was examined were included in the study. Patients were divided into two groups according to HER2/CEP17 values, and survival analysis was performed. The median age was 64 (24-83) years. There were 74 (74.8%) male and 25 (25.2%) female patients. OS in the high HER2/CEP17 ratio group was 21.97months (95% CI: 16.36-27.58), and in the low ratio group was 16.17months (95% CI: 10.95-21.38) (p = 0.015). OS was 17.7months (95% CI: 7.02-28.37) in the high HER2 gene copy number group and 10.13months (5.55-14.71) in the group with low copy number (p = 0.03). PFS was 10.94months (95% CI: 7.55-14.33) in the group with high HER2 gene copy number and 7.56months (4.62-10.49) in the low copy number group (p = 0.06). Patients with both high HER2 gene amplification and high HER2/CEP17 ratio had better OS. The PFS of the group with high HER2 gene amplification was also better. To our knowledge, this is the first study in the literature showing that the HER2/CEP17 ratio affects survival in patients with metastatic gastric cancer.

High HER2 Intratumoral Heterogeneity Is a Predictive Factor for Poor Prognosis in Early-Stage and Locally Advanced HER2-Positive Breast Cancer.

Breast cancer tumors frequently have intratumoral heterogeneity (ITH). Tumors with high ITH cause therapeutic resistance and have human epidermal growth factor receptor 2 (HER2) heterogeneity in response to HER2-targeted therapies. This study aimed to investigate whether high HER2 heterogeneity levels were clinically related to a poor prognosis for HER2-targeted adjuvant therapy resistance in primary breast cancers. This study included patients with primary breast cancer (n = 251) treated with adjuvant HER2-targeted therapies. HER2 heterogeneity was manifested by the shape of HER2 fluorescence in situ hybridization amplification (FISH) distributed histograms with the HER2 gene copy number within a tumor sample. Each tumor was classified into a biphasic grade graph (high heterogeneity [HH]) group or a monophasic grade graph (low heterogeneity [LH]) group based on heterogeneity. Both groups were evaluated for disease-free survival (DFS) and overall survival (OS) for a median of ten years of annual follow-up. Of 251 patients with HER2-positive breast cancer, 46 (18.3%) and 205 (81.7%) were classified into the HH and LH groups, respectively. The HH group had more distant metastases and a poorer prognosis than the LH group (DFS: p < 0.001 (HH:63% vs. LH:91% at 10 years) and for the OS: p = 0.012 (HH:78% vs. LH:95% at 10 years). High HER2 heterogeneity is a poor prognostic factor in patients with HER2-positive breast cancer. A novel approach to heterogeneity, which is manifested by the shape of HER2 FISH distributions, might be clinically useful in the prognosis prediction of patients after HER2 adjuvant therapy.

CCQM-K176 breast cancer biomarker HER2 copy number variation (CNV) measurement

Main textBreast cancer is the most common malignant tumor in women all over the world. Breast cancers with amplification of the human epidermal growth factor receptor 2 (HER2) gene (referred to as HER2-positive breast cancer) accounts for about 20% ~ 30% of invasive breast cancer, and this type of tumor is characterized by high invasiveness, high risk of recurrence, rapid progression and poor prognosis. The determination of HER2 gene expression or copy number variant (CNV) (HER2 copy number normalized to cell number or a reference gene) in tumor cells of breast cancer patients is beneficial to the choice of treatment, through targeted therapies such as trastuzumab, and prognosis. Testing of the core competencies of laboratories to deliver services of CNV measurements for different genetic analytes has not been covered previously. Agreement was received in the October 2019 meeting of the NAWG in Torino to conduct the HER2 CNV measurement as a key comparison. Evidence of successful participation in formal, relevant international comparisons is needed to support calibration and measurement capability claims (CMCs) made by national metrology institutes (NMIs) and designated institutes (DIs).Ten National Metrology Institutions participated in the Track A Key Comparison: CCQM-K176 "Breast cancer biomarker HER2 copy number variation (CNV) measurement". Participants were requested to evaluate the copy number concentration (expressed in [μL-1]) and copy number ratio of defined genomic sequences in eukaryotic intact genomic DNA at an indicative concentration of 90~130 ng/μL. Participation in K176 required assay selection/design and quantitative detection of the analyte in the buffer. The objectives of the key comparison were to measure 1) HER2 copy number concentration; 2) the copy number concentration of the single copy reference gene ribonuclease P RNA component H1 (RPPH1); 3) the HER2/ RPPH1 copy number ratio, using two defined sequences representative of the two genes (HER2 and RPPH1) in mixed genomic DNA samples, and 4) provide evidence for CMC claims by participating laboratories when measuring target sequence content in purified genomic DNA. All participants performed measurements by digital PCR (dPCR), with alternative assays and pre-treatment of samples (for example, restriction enzyme digestion). Interlaboratory reproducibility (%CV) was less than 13% and 5% for copy number concentration and HER2/RPPH1 copy number ratio, respectively, demonstrating very high interlaboratory reproducibility in CNV measurements.Successful participation in CCQM-K176 demonstrates the following measurement capabilities in determining copy number ratio (of a defined target sequence expressed relative to a reference gene sequence) in the range from 1.0 to 40.0 at a background of relatively high concentration of intact (non-fragmented) genomic DNA. The study will also support participants' claim for measurement of defined DNA sequence/gene copy number concentration in the range ~104-106 μL-1 (taking into account both reported reference gene and HER2 copy number concentration values).To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database https://www.bipm.org/kcdb/.The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

Decreased HER2 expression in endometrial cancer following anti-HER2 therapy.

Trastuzumab has demonstrated clinical efficacy in the treatment of HER2-positive serous endometrial cancer (EC), which led to its incorporation into standard-of-care management of this aggressive disease. Acquired resistance remains an important challenge, however, and its underlying mechanisms in EC are unknown. To define the molecular changes that occur in response to anti-HER2 therapy in EC, targeted next-generation sequencing (NGS), HER2 immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH) were performed on pre- and post-treatment tumour samples from 14 patients with EC treated with trastuzumab or trastuzumab emtansine. Recurrent tumours after anti-HER2 therapy acquired additional genetic alterations compared with matched pre-treatment ECs and frequently showed decreased HER2 protein expression by IHC (7/14, 50%). Complete/near-complete absence of HER2 protein expression (score 0/1+) observed post-treatment (4/14, 29%) was associated with retained HER2 gene amplification (n = 3) or copy number neutral status (n = 1). Whole-exome sequencing performed on primary and recurrent tumours from the latter case, which exhibited genetic heterogeneity of HER2 amplification in the primary tumour, revealed selection of an early HER2-non-amplified clone following therapy. Our findings demonstrate that loss of target expression, by selection of HER2-non-amplified clones or, more commonly, by downregulation of expression, may constitute a mechanism of resistance to anti-HER2 therapy in HER2-positive EC. © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Differential response of HER2-positive breast cancer to anti-HER2 therapy based on HER2 protein expression level

BackgroundIncreasing data indicate that HER2-positive (HER2 + ) breast cancer (BC) subtypes exhibit differential responses to targeted anti-HER2 therapy. This study aims to investigate these differences and the potential underlying molecular mechanisms.MethodsA large cohort of BC patients (n = 7390) was utilised. The clinicopathological characteristics and differential gene expression (DGE) of HER2+ immunohistochemical (IHC) subtypes, specifically HER2 IHC 3+ and IHC 2 + /Amplified, were assessed and correlated with pathological complete response (pCR) and survival in the neoadjuvant and adjuvant settings, respectively. The role of oestrogen receptor (ER) status was also investigated.ResultsCompared to HER2 IHC 3+ tumours, BC patients with IHC 2 + /Amplified showed a significantly lower pCR rate (22% versus 57%, P < 0.001), shorter survival regardless of HER2 gene copy number, were less classified as HER2 enriched, and enriched for trastuzumab resistance and ER signalling pathway genes. ER positivity significantly decreased response to anti-HER2 therapy in IHC 2 + /Amplified, but not in IHC 3 + BC patients.ConclusionIn HER2 + BC, overexpression of HER2 protein is the driver of the oncogenic pathway, and it is the main predictor of response to anti-HER2 therapy. ER signalling pathways are more dominant in BC with equivocal HER2 expression. personalised anti-HER2 therapy based on IHC classes should be considered.

HER2-low metastases of HER2-negative primary tumors: a single institution analysis of intertumoral and internodal heterogeneity in node-positive breast cancer.

HER2 status in breast cancer is an essential parameter in individual therapeutic decision-making and is routinely assessed in primary tumors in accordance with international recommendations. Reports of HER2 heterogeneity raise the question of basing treatment decisions on HER2 status in metastases, if present. We investigated the degree and clinical implications of HER2 heterogeneity in lymph node-positive breast cancer. Because of recent recognition of therapeutic opportunities in this group of tumors, we especially focused on cases involving low-level HER2 expression. The HER2 status of primary tumors and of corresponding lymph node metastases was determined in archived material at the protein and gene levels using the gene- protein assay and interpreted in accordance with 2018 ASCO/CAP criteria. HER2-low status was defined as protein expression levels 1+ or 2+ with negative amplification status. We analyzed a series of 43 cases of primary infiltrating breast cancer, each with at least two axillary nodes harboring macrometastases (>2 mm), in total 206 such nodes. In 7% of cases, we detected intertumoral HER2 heterogeneity. Three of nine HER2-positive primary tumors were associated with HER2-negative metastases. No cases with HER2-negative primary tumors had HER2-positive metastases, but 55% (6/11) of HER2 0 primary tumors had HER2 1+ and/or 2+ metastases, and 19% (3/16) HER2 1+ cases had exclusively HER2 0 metastases. All metastases in HER2 2+ cases showed HER2-low protein expression levels. Internodal HER2 heterogeneity at low protein expression levels (presence of HER2 0, HER2 1+, and/or HER2 2+ metastatic deposits within the same axilla) was seen in 40% (17/43) of cases. We found no statistically significant association between HER2 heterogeneity and other tumor-related parameters. Survival data indicated worse outcomes in the HER2-low group compared with the rest of the cohort. Our results indicate a substantial instability of HER2 protein expression, leading to considerable intertumoral and internodal HER2 heterogeneity in lymph node-positive breast carcinomas. This heterogeneity is particularly relevant in HER2-low tumors in which the corrective effects of HER2 gene copy number analysis definitionally is absent. Our findings suggest that determining HER2 status in metastatic lymph nodes may generate relevant information for therapeutic decision-making.

Negative selection of patients (pts) with HER2-positive and RAS wild-type (wt) metastatic colorectal cancer (mCRC) receiving dual HER2 blockade.

e15164 Background: Refining the selection of pts with HER2-positive mCRC for dual HER2 blockade is a challenge for precision oncology. Alterations in receptor tyrosin-kinase (RTK)/MAPK pathway and HER2 gene copy number (GCN) are promising predictive biomarkers for primary resistance or sensitivity, respectively. Methods: We conducted a multicentric case-control study to evaluate the negative predictive impact of a panel of candidate genomic alterations of primary resistance (PRESSING-HER panel) in pts with HER2-positive, RAS wt mCRC treated with trastuzumab-based dual HER2 blockade. Comprehensive genomic profiling was performed on archival pre-treatment tumor samples and screening sources were: the TRIUMPH trial (Oncomine Comprehensive Assay), the Italian-Spanish observational cohort (Foundation One CDx) and the MSKCC cohort (MSK-IMPACT). The panel grouped resistance alterations with a solid biological rationale as on-target, i.e. HER2 pathogenic mutations or rearrangements or off-target, i.e. mutations/amplifications in RTK/MAPK genes. Primary resistance was defined by PFS &lt; 4 months (mos), sensitivity was defined by PFS ≥4 mos. Hypothesizing a prevalence of PRESSING-HER alterations equal to 5% and 30% among controls and cases, respectively, 35 cases and 35 controls were needed to reject the null hypothesis of equally prevalent alterations, with α and β errors of 0.05 and 0.20. HER2 GCN was derived from NGS and the cut-off was calculated by ROC curve analyses using 4-month PFS rate as endpoint. Results: Sixty-eight pts were evaluable (33 resistant and 35 sensitive). In the overall population, PRESSING-HER alterations were found in 14 (20.6%) pts and included HER2 mutations in 7 (50%), HER2 rearrangements in 4 (29%), BRAF class 1 mutations in 2 (14%) and EGFR amplification in 1 (7%). PRESSING-HER alterations were significantly more frequent in pts with primary resistant (12/21, 57%) versus (vs) sensitive tumors (2/35, 6%; P= 0.004). HER2 GCN had a median value of 34.5 (IQR: 18.7-78.5) and an optimal cut-off of 33. In multivariable analyses including the main baseline clinical prognostic features, PRESSING-HER alterations were independently associated with inferior PFS (median PFS 2.0 vs 5.4 mos; adjusted HR 3.5, 95%CI 1.7-7.0; P&lt; 0.001) and OS (median OS 3.7 vs 14.8 mos; adjusted HR 3.9, 95%CI 1.8-8.3; P&lt; 0.001). HER2 GCN &lt; 33 was associated with significantly inferior median PFS (2.6 vs 5.4 mos; adjusted HR 1.8, 95%CI 1.0-3.1; P= 0.038) and numerically inferior OS (median OS 9.1 vs 15.4 mos; adjusted HR 1.3, 95%CI 0.7-2.5; P= 0.356). The predictive accuracy of PRESSING-HER was 66% and it was increased to 77% when combined with HER2 GCN. Conclusions: The combined assessment of HER2 on/off-target alterations and HER2 GCN stratifies patient outcomes to HER2 dual blockade.

Abstract P1-03-02: Efficacy of eribulin mesylate in HER2-low and HER2-0 metastatic breast cancer (MBC): Results from an analysis of two phase 3 studies

Abstract Background: Breast cancer (BC) with low-level HER2 expression (HER2-low) is defined by an immunohistochemistry (IHC) score of 1+ or 2+ without HER2 gene amplification or excess HER2 gene copy number, as measured by in situ hybridization (ISH). This represents approximately half of patients with BC overall (estimated as 55% for hormone-receptor positive [HR+] BC and 38% for triple-negative breast cancer [TNBC]; Scott, ASCO, 2021). Some data suggest that patients with HER2-low BC may respond differently to treatment than those whose BC has no HER2 expression (HER2-0). In this post hoc unplanned analysis, we analyzed data from two pivotal phase 3 studies (Studies 305 and 301) comparing eribulin with other chemotherapeutic agents (treatment of physician’s choice and capecitabine, respectively [“control”]) in patients with both HER2-low and HER2-0 MBC. Methods: Patients with MBC, 2–5 (Study 305) or &amp;lt; 2 (Study 301) prior lines of chemotherapy for advanced/metastatic disease, and who had received an anthracycline and a taxane, were analyzed. HER2-expression status was determined by IHC and/or ISH assays. Median progression-free survival (PFS) and overall survival (OS) were calculated using the Kaplan–Meier method adjusted by study; comparisons of PFS and OS between treatment groups were performed using stratified (by prior capecitabine use, geographic region, and study) log-rank tests. Hazard ratios were estimated by a stratified Cox model. For each study, median PFS and OS were also calculated for HR+ and TNBC subgroups. Results: Baseline characteristics were generally balanced between treatment groups among patients with HER2-low (n=427) and HER2-0 (n=824) BC. Patients with HER2-low or HER2-0 BC showed trends toward benefit with eribulin treatment. In patients with HER2-low and HER2-0 BC, median OS was longer with eribulin vs control (15.1 vs 12.0 months and 15.2 vs 12.5 months, respectively); median PFS by independent imaging review (IIR) was also longer with eribulin vs control (4.0 vs 3.1 months and 3.9 vs 3.1 months, respectively). Objective response rate (ORR) by IIR was also higher with eribulin vs control in patients with HER2-low and HER2-0 BC (13.7% vs 9.2% and 10.2% vs 7.4%, respectively). In a separate analysis, median OS was longer with eribulin vs capecitabine in patients with TNBC and HER2-low and HER2-0 (15.4 vs 10.3 months and 14.4 vs 8.9 months, respectively). Conclusions: In this post hoc analysis, treatment with eribulin demonstrated trends toward improved OS, PFS, and ORR compared with chemotherapy controls in patients with HER2-low or HER2-0 MBC. Funding source: This trial was sponsored by Eisai Inc., Nutley, NJ, USA. Medical writing support was provided by Oxford PharmaGenesis Inc., Newtown, PA, USA, and was funded by Eisai Inc., Nutley, NJ, USA. Citation Format: Chris Twelves, Peter A. Kaufman, Ahmad Awada, Seock-Ah Im, Bagrat Lalayan, Ran Xie, Linda T. Vahdat, Javier Cortés. Efficacy of eribulin mesylate in HER2-low and HER2-0 metastatic breast cancer (MBC): Results from an analysis of two phase 3 studies [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-03-02.

HER2 Overexpression and Cytogenetical Patterns in Canine Mammary Carcinomas.

Human epidermal growth factor receptor 2 (HER2) is a tyrosine kinase receptor that promotes tumor cell growth and is implicated in the pathogenesis of human breast cancer. The role of HER2 in canine mammary carcinomas (CMCs) is not clear. Therefore, this study aimed to examine the protein expression and cytogenetic changes of HER2 and their correlation with other clinical-pathological parameters in CMC. We retrospectively selected 112 CMCs. HER2, ER, and Ki67 were assessed by immunohistochemistry. HER2 antibody validation was investigated by immunoblot on mammary tumor cell lines. Fluorescence in situ hybridization (FISH) was performed with probes for HER2 and CRYBA1 (control gene present on CFA9). HER2 protein overexpression was detected in 15 carcinomas (13.5%). A total of 90 carcinomas were considered technically adequate by FISH, and 8 out of 90 CMC (10%) were HER2 amplified, 3 of which showed a cluster-type pattern. HER2 overexpression was correlated with an increased number of HER2 gene copies (p = 0.01; R = 0.24) and overall survival (p = 0.03), but no correlation with ER, Ki67, grade, metastases, and tumor-specific survival was found. Surprisingly, co-amplification or polysomy was identified in three tumors, characterized by an increased copy number of both HER2 and CRYBA1. A morphological translocation-fusion pattern was recognized in 20 carcinomas (22%), with a co-localized signal of HER2 and CRYBA1. HER2 is not associated with clinical-pathological parameters of increased malignancy in canine mammary tumors, but it is suitable for studying different amplification patterns.

Copy number gains of chromosome 17 identified by dual in situ hybridization in non-small cell lung cancer tissue correlate with overexpression of c-Myc.

Backgroundc-Myc regulates multiple genes involved in cell proliferation in various cancer types including non-small cell lung cancer (NSCLC). Copy number gains of cytoband 17q25.3, along with chromosome 17, have been reported in NSCLC patients, emphasizing the clinical significance as a potential molecular target for therapy. The upregulation of c-Myc has been found to accelerate tumor development associated with duplication of the syntenic human cytoband 17q25.3. This study aimed to explore and compare the correlations of chromosome 17 copy number and c-Myc expression in NSCLC with the paired-normal respiratory epithelium and to examine their role as potential molecular targets for NSCLC therapy.MethodsA total of 66 NSCLC tissue samples with paired-normal respiratory epithelium were examined. The copy number of chromosome 17 was determined by human epidermal growth factor receptor 2 (HER2)/centromeric enumeration probe of chromosome 17 (CEP17) dual in situ hybridization (DISH).ResultsCopy number gains of chromosome 17 were identified in 8 of 60 (13.3%) available NSCLC specimens. No copy number gains of chromosome 17 were demonstrated in the paired-normal respiratory epithelium. The mean HER2 (1.2±0.3) and CEP17 (1.4±0.3) copy numbers of the normal respiratory epithelium were significantly lower than those of the NSCLC tissue [1.8±1.0 vs. 2.0±0.8, respectively (P<0.001)]. Twelve of 66 (18.2%) NSCLC patients had overexpression of c-Myc. Five (41.7%) of the patients whose tumors positive for c-Myc had HER2 gene amplification [1] or copy number gain of chromosome 17 [4]. HER2 gene amplification or copy number gain of chromosome 17 and high expression of c-Myc were associated with decreased overall survival.ConclusionsBoth biomarkers deserve further investigation to identify NSCLC patients with poorer survival outcomes requiring better therapeutic approaches.

Prognostic value of HER2 expression levels for upper tract urothelial carcinoma.

557 Background: HER2 expression is an adverse prognostic factor of bladder urothelial carcinoma (UBC) while the prognostic value of HER2 expression in upper tract urothelial carcinoma (UTUC) remains to be explored. This study aims to investigate the prognostic value of different HER2 expression and gene amplification levels in UTUC. Methods: The baseline clinicopathological data of 130 patients with UTUC were reviewed. The expression level of HER2 was detected by immunohistochemistry (IHC) staining ( Anti-Her2/neu, Cat.4B5, Ventana ) from formalin-fixed paraffin-embedded (FFPE) tumor tissue of patients. The HER2 gene amplification level was detected by second-generation sequencing (NGS) (HER2 gene copy number [CN]) or Fluorescence in situ hybridization (FISH) from FFPE tumor tissue of patients. HER2 negative was defined as IHC 0. HER2 low expression was defined as IHC 2+ but CN- or FISH-, while HER2 overexpression included IHC 3+, and IHC 2+ but CN+ or FISH+. The correlation between HER2 expression levels or other clinicopathological characteristics and overall survival (OS) were analyzed. The Cox proportional-hazards model was used to analyze the independent prognostic factors of UTUC. Results: All the 130 patients provided FFPE tumor slides for the IHC detection of the HER2 expression levels including: IHC 0 (n = 47), IHC 1+ (n = 28), IHC 2+ (n = 41) and IHC 3+ (n = 14), the median OS were 27.5 months, 48.7 months, 67.3 months, and not reached, respectively, with significantly difference ( P = 0.014). Among the 41 patients with HER2 IHC 2+, 15 patients received the detection of the HER2 gene amplification level by NGS or FISH while the other 26 patients whose HER2 gene amplification levels were unknown were excluded from the analysis. The patients of HER2 negative, low expression, and overexpression had a median OS of 27.5 months, 58.0 months, and 60.0 months, respectively, but the difference was not significant ( P = 0.184). Cox proportional-hazards model also verified that the HER2 IHC expression is an independent prognostic factor on the survival, while the prognostic value of the HER2 gene amplification levels was not found. Conclusions: HER2 IHC expression is an independent factor for the prognosis of patients with UTUC. However, HER2 gene amplification levels have no impact on the prognosis of UTUC patients. HER2 expression especially the HER2 gene amplification level in UTUC needs to be explored further.

Abstract PD4-03: Characterization of HER2 amplification in the cerebrospinal fluid of patients with Leptomeningeal Disease in stage IV patients with breast cancer

Abstract Introduction. Breast cancers that metastasize to the central nervous system (BrM) and progress to leptomeningeal disease (LMD) are commonly HER2 amplified. Triple negative breast cancer (TNBC) also may also acquire HER2 amplification and/or copy number changes (HER2+) in BrM and LMD due to genetic heterogeneity. LMD has a dismal prognosis if untreated, but advances in anti-HER2 therapy have been highly effective for improving outcomes in patients with HER2+ LMD. Current methods for evaluating HER2 status in LMD are extremely limited. Cerebrospinal fluid (CSF) cytology is of limited sensitivity with poor cellular yield and preservation due to technical artifacts of preparation that severely limit evaluation of HER2 by FISH or IHC. We evaluated the ability of CNSide™ to selectively enrich CSF tumor cells (CSF-TCs) and assess HER2 gene amplification or copy number gain in breast cancer patients with LMD. Methods. CSF specimens from 77 unique breast cancer patients with suspected LMD were collected using CEE-Sure™ CSF collection tubes and analyzed using the CNSide assay, both produced by Biocept, Inc (San Diego CA). CSF tumor cells (CSF-TCs) stabilized at ambient temperature in CEE-Sure for shipping. In the CLIA laboratory CSF-TCs are labeled using a proprietary mouse anti-human antibody cocktail with secondary biotinylated anti-mouse Ig. All CSF cells are then captured in a streptavidin-coated micro-fluidic channel and fluorescently labeled with DAPI, streptavidin, keratin and CD45 to discriminate nucleated CSF-TCs that are SA+ and cytokeratin+, but CD45 negative. CSF-TCs are digitally imaged, localized and counted with specific X-Y coordinates in the channel that allow further characterization of the HER2 status of each cell using FISH probes for centromere 17 and HER2. Results. Of 77 unique patient CSF specimens, 74.0% (57/77) demonstrated the presence of metastatic tumor cells (defined as cells which were DAPI positive, streptavidin positive, and CD45 negative). HER2 cells ranged from fewer than 10 cells to tens of thousands of cells recovered from a single collection tube of CSF. Of the 57 specimens in which malignant cells were identified, 56.1% (32/57) demonstrated HER2 amplification by FISH (defined as HER2:CEP17 ≥ 2.0 or ≥ 6 HER2 signals) and 43.9% (25/57) did not demonstrate HER2 amplification by FISH. Ten (10) patients also had polysomy of chromosome 17 as well as other gene alterations. Conclusion: HER2 amplification is frequently found in breast cancer patients with LMD and can be reliably detected in tumor cells isolated from CSF. Evaluation of HER2 status in CSF-TCs allows treating physicians the opportunity to rapidly confirm the presence of this biomarker and select a more effective targeted therapy against HER2 in patients who present with life-threatening complications of breast cancer. Additional studies are needed to fully establish the clinical utility of CNSide when using various anti-HER2 therapies or therapy combinations to treat breast cancer patients with LMD. Citation Format: Michael Dugan, Deanna Fisher, Nathan Sweed, Barbara Blouw, Julie Mayer. Characterization of HER2 amplification in the cerebrospinal fluid of patients with Leptomeningeal Disease in stage IV patients with 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 PD4-03.

HER2 copy number quantification in primary tumor and cell-free DNA provides additional prognostic information in HER2 positive early breast cancer.

BackgroundThe quantitative relationship between HER2 copy number and prognosis in HER2 positive adjuvant setting remain controversial, and few studies have focused on adjuvant setting to illustrate the potential clinical relevance of HER2 in cfDNA. Our study aim to develop a novel method in HER2 quantification and explore the relationship between HER2 copy number in primary tumors or cfDNA and prognosis in HER2 positive early breast cancer.MethodsTwo hundred and two patients with early breast cancer were prospectively included in a study where primary tumors, matching non-cancer breast tissue, corresponding plasma, and the plasma from 20 healthy volunteers were collected. Cox proportional hazard analysis was employed to determine the prognostic value of HER2 gene copy number in tissue and cfDNA. Tissue based nomograms and time-dependent decision curve analysis were used to evaluate the practicality of HER2 copy number stratification.ResultsHER2 amplification by CNVplex demonstrated a robust concordance with FISH (concordance 89.2%). A three-tiered system of tissue and a two-tiered system of cfDNA classification were shown to be independent prognostic factors. A tissue copy number-based nomogram was fitted and further evaluation revealed a good performance in discrimination (c statistic 0.801) and calibration.ConclusionsWe first report CNVplex as a viable alternative for HER2 detection. Quantitative evaluation of HER2 presents tremendous potential for use in risk stratification. We also uncover the potential for using HER2 copy number in cfDNA as a biomarker for prognosis in a HER2 positive adjuvant setting.

Changes in the detection of human epidermal growth factor receptor 2 gene (Her-2) status for Her-2 fluorescent in situ hybridization testing

Changes in the detection of human epidermal growth factor receptor 2 gene (Her-2) status for Her-2 fluorescent in situ hybridization testing

The Effects of HER2 on CDK4/6 Activity in Breast Cancer

BackgroundCDK4/6 inhibitors have been used to treat hormone receptor-positive HER2-negative advanced breast cancer. Their benefit in HER2-positive breast cancer has not been determined yet. In this study, we investigated the effects of HER2 on CDK4/6 activity by assessing the level of downstream phosphorylated retinoblastoma protein (pRb) in HER2-positive breast cancer (HER2 positivity is defined by immunohistochemical study or FISH, regardless of ER status) to determine if these cases may be responsive to CDK4/6 inhibitors. Materials and MethodsOne hundred and thirty cases of breast biopsies with invasive carcinoma were collected, including 77 cases of HER2+ (39 cases of ER +PR±HER2+ and 38 cases of ER-PR-HER2+) and 53 cases of HER2- (ER-PR-HER2-) breast cancer. Immunohistochemical study of pRb was performed and the pRb level was assessed by H-score (intensity x percentage of positive cells). ResultsThe pRb H-score ranges from 3 to 270. The average H-scores for the ER-PR-HER2+, ER+PR±HER2+ and ER-PR-HER2- groups are 115.8 ± 75.8, 93.1 ± 68.6 and 63.1 ± 65.6, respectively. By comparison, HER2+ cases have significantly higher pRb levels than HER2- cases (P = .001). Among HER2+ cases, there was a trend of positive correlation between the HER2 gene copy number, and the pRb level although not statistically significant (r = 0.192, 95% CI, [-0.033, 0.399], P = .09). ConclusionIn breast cancer, HER2 positivity leads to significantly higher levels of CDK4/6 activity as reflexed by pRb. Breast cancer that is positive for HER2 may respond to CDK4/6 inhibitors and pRb may potentially be used as a biomarker to predict the responsiveness.

Retrospective observational study of HER2 immunohistochemistry in borderline breast cancer patients\xa0undergoing neoadjuvant therapy, with an emphasis on Group 2 (HER2/CEP17 ratio \u22652.0, HER2 copy number

BackgroundThe ASCO/CAP guidance on HER2 testing in breast cancer (BC) has recently changed. Group 2 tumours with immunohistochemistry score 2+ and HER2/CEP17 ratio ≥2.0 and HER2 copy number <4.0 signals/cell were re-classified as HER2 negative. This study aims to examine the response of Group 2 tumours to neoadjuvant chemotherapy (NACT).Methods749 BC cases were identified from 11 institutions. The association between HER2 groups and pathological complete response (pCR) was assessed.Results54% of immunohistochemistry HER2 positive (score 3+) BCs showed pCR, compared to 19% of immunohistochemistry 2+ FISH amplified cases. 27% of Group 2 treated with HER2 targeted therapy achieved pCR, compared to 19 and 11% in the combined Groups 1 + 3 and Groups 4 + 5, respectively. No difference in pCR rates was identified between Group 2 and Group 1 or combined Groups 1 + 3. However, Group 2 response rate was higher than Groups 4 + 5 (p = 0.017).ConclusionNo difference in pCR was detected in tumours with a HER2/CEP17 ratio ≥2.0 and a HER2 score 2+ by IHC when stratified by HER2 gene copy number. Our data suggest that ASCO/CAP HER2 Group 2 carcinomas should be evaluated further with respect to eligibility for HER2 targeted therapy.

Predictive significance of HER2 intratumoral heterogeneity, determined by simultaneous gene and protein analysis, for resistance to trastuzumab-based treatments for HER2-positive breast cancer.

Gene-protein assay (GPA), a combination of immunohistochemistry and dual in situ hybridization, allows simultaneous visualization of HER2 protein and gene on a single slide. We aimed to clarify the clinical significance of HER2 intratumoral heterogeneity (ITH) using GPA. We investigated the relationships between various HER2 ITH indicators and clinical course in 102 patients with HER2-positive breast cancer, treated with neoadjuvant trastuzumab and chemotherapy. Five representative microscopic images were captured from each GPA slide of pre-therapeutic biopsy materials. All evaluable cancer cells in the images were individually assessed for HER2 gene copy number and protein expression. Mean and coefficient of variation (CV) of both gene copy number and protein category were calculated, and each was divided into negative, equivocal, and positive. Based on their combined status, cancer cells were classified into nine types. Pathological complete response (pCR) to neoadjuvant treatments showed positive relationships to mean gene copy number (P < 0.001), mean protein category (P < 0.001), and proportion of gene- and protein-positive tumor cells (P < 0.001) and showed negative relationships to the CV of protein category (P < 0.001) and the proportion of gene-amplified but protein-negative tumor cells (P = 0.002). Two diagnostic models, created by combining clinicopathological factors and ITH indicators, showed excellent potential diagnostic ability for pCR (mean gene copy number and protein category CV; AUC = 0.837, proportion of gene- and protein-positive tumor cells; AUC = 0.831). HER2 ITH quantified by GPA is a potential predictive indicator for efficacy of HER2-targeted treatment.