HER2 Expression Levels Research Articles

The design, preclinical study and phase I dose escalation plan of a HER2 targeted immunoliposome (HF-K1) for HER2 low solid tumor treatment.

3035 Background: Doxorubicin liposome formulations are being used extensively in the clinic in anthracycline chemotherapy with reduced cardiotoxicity. However, they did not improve clinical outcome because the cancer cell uptake of PEG-coated liposomes is poor. So, the concept of coating antibodies on the liposome surface was initiated and they were named immunoliposomes. There have been numerous studies conducted world-wide for the development of immunoliposomes but none have progressed to late-stage development. Our work draws on the experiences of these prior studies with a new perspective. Since the mechanism of immunoliposomes is similar to those of ADCs, we sought to compare immunoliposomes with ADCs containing the same cancer cell binding domain, for target cell binding capability, binding affinity as well as in vivo distribution, tumor tissue accumulation, and anti-tumor activities. The lessons learned from the success of many ADC products may be applicable to the development of immunoliposomes. Methods: The antibody to liposome ratio (ALR) and the drug to lipid ratio (DLR) of the immunoliposomes were selected by in vitro and in vivo anti-cancer activity screening. Cell lines with different HER2 expression levels were used to study immunoliposome binding, cell uptake, drug delivery and cytotoxicity. PK, drug distribution, efficacy and toxicities studies were conducted in respective CDX and PDX mice and non-rodent models. Based on these studies, a phase I dose escalation study plan was designed and the study is now ongoing. Results: HF-K1 was designed to have an average of 10 anti-HER2 Fab on each liposome surface and approximately 2300 doxorubicin molecules inside. The equivalent drug to antibody ratio (DAR) is 230. HF-K1 can bind to cancer cells with different levels of HER2 expression with similar kinetics and induces cells death at IC50s of 0.35-6.46 μg/ml Fab concentration. It has a long circulation behavior and enhanced permeability and retention (EPR) in tumor tissues. The clearance T1/2 of HF-K1 in mice and monkey is 12.78 h and 47.12 h, respectively. Evaluation of HF-K1 activity in vivo showed significant tumor growth inhibition >95% at the equivalent antibody dose of 3.6 mg/kg in various mouse tumor models including HER2 low and HER2 very low (HER2-). Similar activities were shown by ADCs including T-DM1 and DS-8201a at about 10 mg/kg. Conclusions: The encouraging preclinical data supported a clinical trial starting with dose escalation at equivalent antibody doses of 0.72 mg/m2, 2.16 mg/m2, 5.4 mg/m2, 10.8 mg/m2, 16.2 mg/m2, and 21.6 mg/m2. The study is ongoing to determine the safety, tolerability, PK, and preliminary antitumor efficacy in participants with HER2 positive or HER2 low expression advanced solid tumors (NCT05861895). Clinical trial information: NCT05861895 .

Trastuzumab deruxtecan (T-DXd) in patients (pts) with HER2-expressing head and neck tumors: Outcomes from DESTINY-PanTumor02 (DP-02).

6037 Background: In DP-02, T-DXd demonstrated an objective response rate (ORR) by investigator (INV) of 37.1% (95% CI 31.3, 43.2) and clinically meaningful survival outcomes in 267 pretreated pts with HER2-expressing tumors. In this post-hoc analysis, we report outcomes and characterize those with an objective response (OR) in a subset of pts with head and neck cancers (previously included in the ‘other tumor’ cohort), most of which were salivary gland tumors. Methods: This open-label, Phase 2 study (NCT04482309) evaluated T-DXd (5.4 mg/kg Q3W) in pts with HER2-expressing (immunohistochemistry [IHC] 3+/2+ by local or central testing) locally advanced/metastatic disease after ≥1 systemic treatment or without treatment options. The primary endpoint was confirmed ORR by INV. Secondary endpoints included duration of response (DOR), progression-free survival (PFS), disease control rate (DCR), and safety. Exploratory endpoints included efficacy outcomes by HER2 expression. Results: At data cutoff (June 2023), 24 pts with head and neck tumors (n=19 salivary gland, n=3 squamous cell carcinoma [SCC], n=1 adenoid cystic carcinoma, n=1 lacrimal gland) had received treatment (median [m] follow up: 20.8 months [range: 4.7–31.6]). Of these pts, 15 (62.5%) had received ≥2 prior treatment regimens. 10/24 pts (41.7%; 95% CI 22.1, 63.4; n=8 salivary gland, n=1 SCC, n=1 lacrimal gland) had a confirmed OR by INV; 9 responders had received prior radiation therapy, and 4 had known PD-L1 immune cell status ≥1%. The Table shows efficacy outcomes in all pts and by HER2 expression (central testing). Grade (G) ≥3 drug-related adverse events occurred in 10/24 (41.7%) pts. Adjudicated drug-related interstitial lung disease / pneumonitis occurred in 3/24 (12.5%) pts (n=1 G1; n=1 G2; n=1 G5). Conclusions: T-DXd showed clinically meaningful benefit in pretreated pts with head and neck tumors. Responses were observed across HER2 expression levels. Safety was consistent with the known profile. These data support T-DXd as a potential treatment for pretreated pts with HER2-expressing head and neck tumors. Clinical trial information: NCT04482309 . [Table: see text]

CNS efficacy of trastuzumab deruxtecan in metastatic breast cancer: A single-institution, real-world analysis.

e13117 Background: Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate that revolutionized the management of patients with HER2+ (HER2 FISH+, IHC 3+) and HER2low (HER2 FISH-, IHC 1+/2+) metastatic breast cancer (MBC). Studies have shown that T-DXd may induce antitumor activity in HER2+ MBC with brain metastases (BM). However, data is limited on the central nervous system (CNS) efficacy of T-DXd in HER2low disease. The only report thus far is from the HER2low cohorts in the DEBBRAH study with 12 patients showing an intracranial overall response rate (ORR-IC) of 50% and progression free survival (PFS) of 5.7 months. Here, we provide a real-world, retrospective analysis of the CNS efficacy of T-DXd in HER2+, HER2low, and HER2null MBC with BM. Methods: This is a single institution, retrospective analysis of patients with MBC with BM who received T-DXd between January 2020 and November 2023. Patients with HER2+, HER2low, or HER2null MBC and BM, treated or untreated at the time of T-DXd initiation, were included. All patients had baseline brain magnetic resonance imaging before T-DXd initiation. Efficacy analyses were performed using the RECIST 1.1 criteria to CNS target lesions by investigator assessment. ORR-IC and intracranial clinical benefit rate (CBR-IC) were reported. CNS PFS, defined as time until CNS progression or death of any cause, and overall survival (OS) were also evaluated. Results: 29 patients were included in the study: 10 (35%) had HER2+, 16 (55%) had HER2low, and 3 (10%) had HER2null disease. The patients had ECOG performance status ranging from 0 to 3, and received a median of 3 prior lines of therapy (range 1-8). At baseline, 3 patients (10%) had untreated BM, 6 (21%) had prior surgery, and 23 (79%) had prior radiation. 28 (97%) had concurrent extra-cranial metastases. Best CNS response and duration of response are listed in the table below. ORR-IC was 10% overall and 6% in HER2low group. CBR-IC was 58% overall and 50% in HER2low group. Median OS in all patients was 65 weeks. Median CNS PFS was not statistically different in the overall group (24 weeks) compared to the HER2low group (21 weeks) with p=0.4661 via log rank test. Conclusions: This is the first real-world study reporting the CNS efficacy of T-DXd in patients with MBC across HER2 expression levels. The CNS efficacy of T-DXd in our institutional experience was similar in HER2+ and HER2low MBC, and was much lower than previously reported in prospective clinical trials. Performance status, extent of prior treatment, and systemic disease burden likely contributed to this difference. Larger cohort and prospective studies on patients with HER2low MBC with BM are warranted. [Table: see text]

Efficacy and safety of trastuzumab deruxtecan (T-DXd) in patients (pts) with HER2-expressing solid tumors: Results from the bladder cohort of the DESTINY-PanTumor02 (DP-02) study.

4565 Background: In DP-02, T-DXd showed robust responses and clinically meaningful survival outcomes in 267 pretreated pts with HER2-expressing solid tumors; the objective response rate (ORR) by investigator (INV) was 37.1% (95% CI 31.3, 43.2). Here we report subgroup analyses in the bladder cohort (urothelial carcinoma including transitional cell carcinoma of the renal pelvis, ureter, urinary bladder, or urethra), and characterize pts with an objective response (OR). Methods: This open-label, Phase 2 study (NCT04482309) evaluated T-DXd (5.4 mg/kg Q3W) in pts with HER2-expressing (immunohistochemistry [IHC] 3+/2+ by local or central testing) locally advanced/metastatic disease after ≥1 systemic treatment (Tx), or without Tx options. The primary endpoint was confirmed ORR by INV. Secondary endpoints included duration of response (DOR), progression-free survival (PFS), disease control rate (DCR), and safety. Exploratory endpoints included efficacy outcomes according to HER2 expression. Results: At data cutoff (June 2023), 41 pts with urothelial cancers had received T-DXd (median [m] follow up [range]: 12.65 [0.4–26.8] months); 27 (65.9%) had received ≥2 prior Tx regimens. 16/41 pts (39.0%; 95% CI 24.2, 55.5) had a confirmed OR by INV; 12 responders had received ≥2 prior Tx regimens, 14 had received prior immunotherapy Tx, and 14 had known PD-L1 immune cell status ≥1%. The Table shows efficacy outcomes in all pts and by HER2 expression (central testing). Grade (G) ≥3 drug-related adverse events occurred in 17/41 (41.5%) pts. Adjudicated drug-related interstitial lung disease / pneumonitis occurred in 4/41 (9.8%) pts (n=1 G1; n=3 G2). Conclusions: T-DXd showed clinically meaningful responses in pretreated pts with urothelial cancers, including across HER2 expression levels (IHC 3+ and 2+). Safety was consistent with the known profile. These data support further evaluation of T-DXd as a potential Tx for pretreated pts with HER2-expressing urothelial cancers. Clinical trial information: NCT04482309 . [Table: see text]

Amide Proton Transfer-Weighted Imaging in Assessing the Aggressive and Proliferative Potential of Bladder Cancer.

Ki-67 and human epidermal growth factor receptor 2 (HER2) are known oncogenes involved in bladder cancer (BCa) patient risk stratification. Preoperative assessment of their expression level can assist in clinical treatment decision-making. Recently, amide proton transfer-weighted (APTw) MRI has shown promising potential in the diagnosis of several malignancies. However, few studies reported the value of APTw imaging in evaluating Ki-67 and HER2 status of BCa. To investigate the feasibility of APTw MRI in assessing the aggressive and proliferative potential regarding the expression levels of Ki-67 and HER2 in BCa. Retrospective. 114 patients (mean age, 64.78 ± 11.93 [SD] years; 97 men) were studied. APTw MRI acquired by a three-dimensional fast-spin-echo sequence at 3.0 T MRI system. Patient pathologic findings, included histologic grade and the expression status of Ki-67 and HER2, were reviewed by one uropathologist. The APTw values of BCa were independently measured by two radiologists and were compared between high-/low-tumor grade group, high-/low-Ki-67 expression group, and high-/low-HER2 expression group. The interclass correlation coefficient, independent sample t-test, Mann-Whitney U test, Spearman's rank correlation, and receiver operating characteristic curve (ROC) analysis were used. P < 0.05 was considered statistically significant. Significantly higher APTw values were found in high-grade BCa patients (7.72% vs. 4.29%, P < 0.001), high-Ki-67 expression BCa patients (8.40% vs. 3.25%, P < 0.001) and HER2 positive BCa patients (8.24% vs. 5.40%, P = 0.001). APTw values were positively correlated with Ki-67 (r = 0.769) and HER2 (r = 0. 356) expression status. The area under the ROC curve of the APTw values for detecting Ki-67 and HER2 expression status were 0.883 (95% CI: 0.790-0.945) and 0.713 (95% CI: 0.592-0.816), respectively. APTw MRI is a potential method to assess the biological and proliferation potential of BCa. Stage 2.

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&lt;0.001). HER2 amp were also found more frequently in the E deletion(del) mt cohort (1.63%), compared to KA cohorts (p&lt;0.001). Additionally, there was significant co-occurrence of HER2 mt and HER2 amp (OR 6.49; p&lt;0.001) in the KEAother cohort. HER2mt/amp had significant odds of co-occurrence with TP53 alt (OR 1.43; p&lt;0.001), CDKN2A del (OR 2.15; p&lt;0.001), TERT amp (OR 2.37; p&lt;0.001), RB1 alt (OR 1.6; p&lt;0.001). Additionally, HER2 amp co-occurred with TP53 alt (OR 4.0; p&lt;0.001), CDKN2A del (OR 3.44; p&lt;0.001), while HER2 mt co-occurred with CDKN2A del (OR 2.03; p&lt;0.001), TERT amp (OR 2.43; p&lt;0.001). BRAF mts were mutually exclusive with HER2 mt/amp (OR 2.31; p&lt;0.001) and HER2 mt (OR 1.93; p&lt;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&lt;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.

HERTHENA-PanTumor01: A global, multicohort, phase 2 trial of HER3-DXd in relapsed/refractory metastatic solid tumors.

TPS3164 Background: Human epidermal growth factor receptor 3 (HER3) is a member of the HER/EGFR family of receptor tyrosine kinases. HER3 is expressed in numerous solid tumors, including melanoma, head and neck squamous cell cancer (HNSCC), and gastric cancer, and high levels of HER3 expression in multiple tumor types are associated with poor clinical outcomes. HER3-DXd is a potential first-in-class HER3-directed antibody-drug conjugate (ADC) composed of a fully human anti-HER3 monoclonal antibody (patritumab) covalently linked to a potent topoisomerase I inhibitor payload via a tumor-selective cleavable linker. HER3-DXd has shown antitumor activity and a manageable safety profile in previously treated EGFR-mutated metastatic NSCLC and metastatic breast cancer. Based on clinical data and exposure-response analyses, HER3-DXd 5.6 mg/kg IV every 3 weeks (Q3W) was selected as the monotherapy dose for investigation in new indications. HERTHENA-PanTumor01 (NCT06172478) is a global, multicohort, open-label, single-arm, phase 2 trial evaluating the efficacy and safety of HER3-DXd in previously treated patients (pts) with cutaneous melanoma, HER2-negative gastric cancer, or HNSCC. Methods: Each cohort of HERTHENA-PanTumor01 will enroll 40 pts. Study sites are in Australia, Belgium, France, Japan, South Korea, Spain, Taiwan, the UK, and the US. Key inclusion criteria for melanoma include cutaneous or acral subtype, BRAF–wild-type or -mutant status, and progression on or after ≥1 anti–PD-(L)1 therapy. Key inclusion criteria for HNSCC include HPV+ or negative status and progression after platinum-based chemotherapy (PBC) ± anti–PD-(L)1 therapy; pts with nasopharyngeal cancer are excluded. Key inclusion criteria for gastric or gastroesophageal junction cancer include confirmed HER2-negative tumor status (immunohistochemistry [IHC] 0/1+ or IHC 2+/in situ hybridization negative) and progression after PBC ± anti–PD-(L)1 therapy; pts with HER2+ cancer are excluded. Other key exclusion criteria for all cohorts include any history of, suspected, or current interstitial lung disease; active brain metastases (treated/asymptomatic brain metastases are allowed); or prior treatment with an anti-HER3 antibody and/or ADC containing an exatecan derivative. A fresh biopsy or pretreatment archival biopsy performed since progression on or after treatment with the most recent cancer therapy is required for all cohorts. Pts will be treated with HER3-DXd 5.6 mg/kg IV Q3W; tumor assessments will occur every 6 wks for the first 48 wks, then every 12 wks until disease progression, adverse events, or other discontinuation criteria are met. The primary endpoint for each cohort is ORR by investigator per RECIST version 1.1. Secondary endpoints include safety and tolerability, DOR, DCR, PFS, overall survival, pharmacokinetics, and the correlation between HER3 IHC protein expression and efficacy. Clinical trial information: NCT06172478 .

Prevalence of “HER2 ultra-low” among patients with advanced breast cancer with historical IHC0 status.

e13156 Background: Breast Cancer (BC) patients are classified as HER2-positive (IHC3+ or IHC2+/ISH+) or HER2-negative (IHC0, IHC1+ and 2+/ISH-). Within the HER2-negative category, patients who meet HER2-low (IHC1+ or IHC2+/ISH-) criteria can benefit from HER2 targeted therapy like trastuzumab deruxtecan. Patients who meet IHC0 criteria with incomplete and faint membrane staining in &gt;0 but ≤10% of tumor cells — defined as “HER2 Ultra-low”, may also benefit from HER2 targeted therapy. This study aimed to assess the prevalence of “HER2 Ultra-Low” expression based on re-scored HER2 IHC biopsy slides. Methods: This study utilized EHR data from all three US Mayo Clinic campuses. A total of 300 patients with advanced BC (stages III-IV), and clinically documented HER2 IHC0 status between Jan 2017 and Jan 2023, were identified. One biopsy slide per patient (IHC assay: VENTANA Pathway anti-HER2/neu (4B5) rabbit monoclonal primary antibody) was digitized and scanned at 40x magnification. Two Mayo Clinic pathologists independently scored and reported HER2 status according to 2023 ASCO-CAP guidelines and included tumor stain percentage for each slide. Patients were considered “HER2 Ultra-Low” if their slide was scored as IHC0 with &gt;0% but ≤10% staining. Cohen’s κ was used to quantify agreement between pathologists. Results: The re-scored patients (n=300) had a mean age of 57.8 years (SD=13.5), with ~97% having IHC0 documentation within 30 days of the reviewed biopsy slide. A total of 285 (95%) patients remained classified as IHC0 by at least one pathologist. Of these, 60% (n=171) were determined as “HER2 Ultra-low” by at least one pathologist. The rate of “HER2 Ultra-Low” per pathologist ranged from 43% (n=121) to 45% (n=104). The overall inter-pathologist concordance was 57%; patients with no observable HER2 IHC staining accounted for majority of the concordant patients (Table). Conclusions: About 3 in 5 advanced BC patients originally classified as HER2 IHC0 met the “HER2 Ultra-Low” criteria by at least one pathologist, implying that significant number of additional patients may benefit from HER2 targeted therapy. The relatively low concordance between HER2 expert pathologists in identifying lower levels of HER2 expression suggests the need for increased precision enabled by digital tools, leveraging AI, training for community practice pathologists, and continuing to follow best practice recommendations in determining HER2 IHC status. [Table: see text]

Community oncology preparedness and perceptions of challenges in care with HER2/HER3 therapy for NSCLC.

e21010 Background: The rapid pace of advances in molecular testing and targeted treatment for non-small cell lung cancer (NSCLC) can make it difficult to stay up to date with new developments. Accelerating the integration of practice-changing data requires clarification of actionable vs non-actionable mutations, relevance for therapeutic decisions, and treatment options for patients. To address this need further an educational program was designed in 2023. Methods: A one-hour online, video-based program was designed for clinicians and their teams and hosted on OMedLive from February 2023. Knowledge and competence questions were administered pre-, and immediate post-activity. and the intersection of patient and clinician perspectives, including behavioral impact, and qualitative insights. Behavioral impact, perception, and practice pattern questions were also assessed immediately post-activity. Results: More than 1,700 participants have engaged in the educational program to date. Of the participants in the clinician program, 94% were physicians and 93% noted their specialty as oncology with 67% identifying as treaters seeing an average of 8 patients with NSCLC on a monthly basis. Low baseline (8% - 40%) knowledge and competence was observed for awareness of NGS-based approaches to detect ERBB2 mutations, trial eligibility criteria and outcomes, time to onset of drug-related toxicities, and ability to identify patients who would benefit from clinical trials with HER3 investigational therapies. Only 28% were confident in their ability to appropriately test for HER2 mutational status and 47% likely to incorporate testing for HER2 expression level or amplification prior to the selection of targeted therapy. The greatest challenges in managing patients with HER2-mutated NSCLC was identified as managing side effects (33%), affordability of therapy for patients (27%), and adherence to treatment schedules (17%). Conclusions: The outcomes of this educational program demonstrate a need for greater alignment and education among pathology and oncology team members on HER2 testing for patients with NSCLC. Additional education is needed relevant to investigational HER3 therapeutics, the appropriate patient to receive these therapies, and the potential application within the current treatment landscape. As these agents deepen their impact in the NSCLC landscape, it is important to prepare teams for timely management of adverse events.

Human epidermal growth factor receptor 2 expression level and combined positive score can evaluate efficacy of advanced gastric cancer.

Although treatment options for gastric cancer (GC) continue to advance, the overall prognosis for patients with GC remains poor. At present, the predictors of treatment efficacy remain controversial except for high microsatellite instability. To develop methods to identify groups of patients with GC who would benefit the most from receiving the combination of a programmed cell death protein 1 (PD-1) inhibitor and chemotherapy. We acquired data from 63 patients with human epidermal growth factor receptor 2 (HER2)-negative GC with a histological diagnosis of GC at the Cancer Hospital, Chinese Academy of Medical Sciences between November 2020 and October 2022. All of the patients screened received a PD-1 inhibitor combined with chemotherapy as the first-line treatment. As of July 1, 2023, the objective response rate was 61.9%, and the disease control rate was 96.8%. The median progression-free survival (mPFS) for all patients was 6.3 months. The median overall survival was not achieved. Survival analysis showed that patients with a combined positive score (CPS) ≥ 1 exhibited an extended trend in progression-free survival (PFS) when compared to patients with a CPS of 0 after receiving a PD-1 inhibitor combined with oxaliplatin and tegafur as the first-line treatment. PFS exhibited a trend for prolongation as the expression level of HER2 increased. Based on PFS, we divided patients into two groups: A treatment group with excellent efficacy and a treatment group with poor efficacy. The mPFS of the excellent efficacy group was 8 months, with a mPFS of 9.1 months after excluding a cohort of patients who received interrupted therapy due to surgery. The mPFS was 4.5 months in patients in the group with poor efficacy who did not receive surgery. Using good/poor efficacy as the endpoint of our study, univariate analysis revealed that both CPS score (P = 0.004) and HER2 expression level (P = 0.015) were both factors that exerted significant influence on the efficacy of treatment the combination of a PD-1 inhibitor and chemotherapy in patients with advanced GC (AGC). Finally, multivariate analysis confirmed that CPS score was a significant influencing factor. CPS score and HER2 expression both impacted the efficacy of immunotherapy combined with chemotherapy in AGC patients who were non-positive for HER2.

Abstract PO3-13-11: Quantitative Multiplex Immunofluorescence Assay for TROP2 and HER2 Expression in Breast Cancer: Towards Guiding Patient Selection for Antibody Drug Conjugate Therapies

Abstract Emerging antibody drug conjugate (ADC) therapies targeting human trophoblast cell-surface antigen (TROP2) and human epidermal growth factor receptor 2 (HER2) are transforming the treatment landscape for breast cancer. Sacituzumab govitecan (SG) and trastuzumab deruxtecan (T-DXd) have gained approval for an overlapping set of "HER2-low" metastatic breast cancers, including hormone receptor (HR)-positive HER2 non-amplified and "triple-negative" subtypes. Nevertheless, the optimal selection of patients and treatment sequencing for these ADC therapies remains a clinical challenge. Clinical trial objective response rates to SG are approximately 30%, compared to 30-90% for T-DXd depending on HER2 expression levels. While both drugs are thought to be targeted therapies, the value of measuring the target and the best methods to do so are still not established. We believe that quantitative measurement of TROP2 and HER2 antigen expression levels could establish thresholds for responders, enabling more effective patient selection for ADC therapies. Here, we present a TROP2, high-sensitivity HER2, and cytokeratin (CK) quantitative immunofluorescence (QIF) multiplex assay. Using a ten-cell line standard array and proteomic mass spectrometry, we can convert tumor compartment QIF intensity to protein concentration in fmol/mm2 for tissue specimens. Anti-HER2, anti-TROP2 antibodies, and fluorescence detection systems were titrated and combined to maximize signal-to-noise ratio on our cell line standard array and breast cancer tissue microarrays (TMA). The multiplex assay was designed for automated slide stainers (Leica BOND Rx) and fluorescence slide scanning (Rarecyte CyteFinder II HT). We perform our analyses in QuPath using an image processing plugin developed for automated QIF/IHC analysis (Qymia). Reproducible TROP2 and HER2 QIF scoring (R² &amp;gt; 0.95) was achieved across multiple staining batches using serial sections of breast cancer TMAs and cell standard arrays. This assay has a TROP2 linear range between 0.63 - 9.17 fmol/mm2 (about 1 million TROP2 receptors/cell) and HER2 linear range between 0.09 - 0.565 fmol/mm2 (about 60,000 HER2 receptors/cell). We then applied this multiplex assay to two serial retrospective primary breast cancer cohorts from Yale University to quantitatively measure TROP2 and HER2 expression (338 clinical cases). We find a weak negative correlation between TROP2 and HER2 expression in our breast cancer cohorts (Pearson r = -0.14, p = 0.0097, n = 338). TROP2 expression levels were above the limit of detection (LOD) in 90.2% of cases, with 4.1% exceeding the limit of linearity (LOL), and a mean TROP2 expression of 4.05 fmol/mm2. For HER2, 67.2% of cases were above the LOD, with 7.1% exceeding the LOL, and a mean HER2 expression of 0.186 fmol/mm2. Both TROP2 and HER2 were below the LOD in 3.0% of cases, which we define as “negative”. We found 29.9% expressed TROP2 and were HER2-negative, and 6.8% expressed HER2 and were TROP2-negative. Our future studies will aim to quantitatively define expression thresholds for T-DXd and SG response with the goal to produce a clinical grade assay for ADC patient selection and determine the value of the assay to help select which ADC to give first. HER2 and TROP2 protein expression summary in Yale breast cancer cohort Table 1: Summary of HER2 and TROP2 protein expression levels in serial retrospective primary breast cancer cohort of 338 cases using our high-sensitivity HER2 and TROP2 multiplex immunofluorescent assay Citation Format: Charles Robbins, Mengni He, Revekka Khaimova, Katherine Bates, Nay Nwe Nyein Chan, Daniel Liebler, Regan Fulton, David Rimm. Quantitative Multiplex Immunofluorescence Assay for TROP2 and HER2 Expression in Breast Cancer: Towards Guiding Patient Selection for Antibody Drug Conjugate Therapies [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-13-11.

Abstract PS09-04: The benefit of adjuvant pertuzumab and trastuzumab according to estrogen receptor and HER2 expression: sub-analysis of the APHINITY trial

Abstract Background: Some studies suggest that response to HER2-targeting agents may differ according to estrogen receptor (ER) and HER2 expression levels. This study aimed to investigate the magnitude of benefit from the addition of pertuzumab to trastuzumab and chemotherapy according to ER and HER2 expression levels in the APHINITY trial. Methods: APHINITY (NCT01358877; BIG 4-11) was a randomized, double-blind, phase III study comparing the addition of pertuzumab or placebo to adjuvant trastuzumab and chemotherapy in an ITT population of 4804 patients with HER2-positive early breast cancer. The primary objective of this exploratory, unplanned analysis was to compare whether the addition of pertuzumab to trastuzumab had an impact in invasive disease-free survival (IDFS) within different subgroups defined by HER2 FISH amplification ratio and/or ER percentage of positivity on immunohistochemistry (IHC) (centrally assessed). Patients with a FISH ratio less than 2 were excluded from this analysis, leaving 4782 patients. IDFS was defined as the time from randomization until the date of the first occurrence of an IDFS event. HER2 FISH amplification ratio was categorized as low (2≤ FISH ratio &amp;lt; 5) vs. high (FISH ratio ≥5). ER expression on IHC was categorized as negative ( &amp;lt; 1%) vs. positive (≥1%). A subgroup analysis using Cox proportional hazards regression models was used to model IDFS, assessing four categories formed by cross classifying the FISH ratio and ER groups. The model was adjusted for randomized arm, adjuvant chemotherapy regimen received, and a combined variable of nodal status and protocol version. Results: The 4782 patients had a median age of 51 years old (IQR 44-59), with a median follow up time of 73.6 months (IQR 63.0-75.2). Most patients received an anthracycline-based chemotherapy (n=3712, 77.6%). ER expression was positive in 3047 patients (63.7%). HER2 FISH amplification ratio was high in 2479 patients (51.8%). All FISH ratio/ER subgroups seem to derive IDFS benefit from the addition of pertuzumab (HR &amp;lt; 1) (Table 1). The HER2 FISH ratio-low/ER positive subgroup had the largest reduction in risk of an IDFS event, 30% for the pertuzumab arm (HR=0.70, 95% CI 0.51-0.95) versus placebo. Smaller differences in IDFS events were observed between the treatment arms in the other subgroups. The subgroup of tumors with HER2 FISH ratio-high/ER-negative expression showed the numerically smallest benefit in IDFS events with the addition of pertuzumab (HR 0.85, 95% CI 0.59-1.25), see Table 1. Conclusions: In this APHINITY sub-analysis, patients treated with pertuzumab/trastuzumab derive similar benefit regardless of ER and HER2 expression levels. More research is needed to find predictive biomarkers to escalate or de-escalate treatments. Table 1. Abbreviations: FISH: fluorescence in situ hybridization, ER: estrogen receptor; IDFS: invasive disease-free survival; CI: confidence interval; HR: hazard ratio Citation Format: Evandro de Azambuja, Elisa Agostinetto, Faye Samy, Serena Di Cosimo, Philippe Aftimos, Noam Pondé, Daniel Eiger, Matteo Lambertini, David Cameron, Astrid Kiermaier, Andrew Bailey, Giuseppe Viale, Sherene Loi, Martine Piccart. The benefit of adjuvant pertuzumab and trastuzumab according to estrogen receptor and HER2 expression: sub-analysis of the APHINITY trial [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 PS09-04.

Abstract PO4-03-08: Enhanced Assessment of Clinical Behavior of TNBC and TPBC using Quantified Levels of HER2, Estrogen and Progestin Receptor Proteins and Expression of Their Cognate Genes

Abstract Background: Renewed interest in clinical relevance of low-HER2 protein in breast cancer management prompted a retrospective investigation of a unique de-identified Database of quantified biomarkers associated with patient outcomes. Currently, IHC of ER, PR and HER2 proteins provides semi-quantitative results, at times complicated by variation in methods and interpretation (cf. ASCO/CAP Guidelines). Our goal was to ascertain relationships of these clinical analytes to predict cancer relapse when quantified for protein products and cognate gene expression. Methods: Database contained 2756 quantified HER2 results by ELISA (Oncogene Sciences) or by EIA (Triton Diagnostics) of which 198 patients had associated clinical outcomes. No definition of HER2-low has been accepted for IHC, although &amp;gt; 50% of biopsies may be categorized as HER2-low (cf. ASCP CE Programs). Microarray results obtained for ~ 22,000 genes using RNA purified from LCM-procured carcinoma cells of 247 primary breast biopsies were analyzed. ESR1, PGR and ERBB2 gene expression levels in 276 biopsies had been validated by RTqPCR. To assess distribution and dispersion of HER2 protein, cumulative relative frequency distribution analyses were employed, focusing on cut-offs within interquartile range. Permutations and combinations of the 3 biomarkers based on IHC definitions (cf. ASCO/CAP Guidelines) were examined. Tertiles and quartiles of data sets were used to explore association between biomarker levels and clinical outcomes. Box plots depicted protein and gene expression levels, Progression-Free Survival (PFS), or Overall Survival (OS) across ER/PR/HER2 categories and compared differences using a Kruskal-Wallis ANOVA Rank Test. Cox regressions were used to contrast hazard ratios among the ER/PR/HER2 groups. Kaplan-Meier plots visualized survival rates among groups and a log-rank test detected statistical discrepancies. We investigated differences between groups for categorical variables using chi-squared tests (Fisher's exact test for non-normally distributed data), while for ordinal variables, ANOVA tests were used (Kruskal-Wallis for non-normally distributed data). Categorical variables were summarized as count (%), and ordinal variables were summarized as median [IQR]. Analyses were performed using R version 4.2.2 (Innocent and Trusting). Results: Neither quantified ER or PR alone nor ER/PR collectively were related to HER2 oncoprotein levels. Influence of menopausal status on biopsy biomarker profiles was investigated. With a median cutoff for HER2 protein, patients with ER+/PR+/HER2+ (TPBC) exhibited increased OS, whereas those with TNBC had decreased OS, of 8 combinations possible. Using tertiles and quartiles, TPBC was exhibited by 34-37% of specimens compared to TNBC, which represented 9.1-12.7% of biopsies when biomarker proteins were quantified. After qPCR data were stratified by ER/PR/HER2 status with a 50% quantile cut for HER2, 39% were TPBC and 10% were TNBC. Using microarray data with the same cutoffs, only 14% were TPBC and 17% were TNBC. Once TPBC were identified by either microarray or by qPCR, increased ESR1, PGR and ERBB2 expression collectively was associated with increased PFS and OS of patients. In general, biopsies that were TNBC or other combinations of biomarker’s gene expression correlated with poorer prognosis and overall survival. Conclusions: Quantifying levels of ER, PR and HER2 proteins as well as of ESR1, PGR and ERBB2 expression were correlated with prediction of clinical outcomes of TNBC, TPBC and patients with other biomarker subsets. Results support quantitative assessment of these biomarkers in biopsies to assess utility of the HER2-low type in personalizing breast cancer management and prediction of risk of relapse. Citation Format: James Wittliff, Michael Daniels. Enhanced Assessment of Clinical Behavior of TNBC and TPBC using Quantified Levels of HER2, Estrogen and Progestin Receptor Proteins and Expression of Their Cognate 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 PO4-03-08.

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

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

Abstract PO1-27-07: Is HER2-low a biologically distinct breast cancer (BC) subtype? Prognosis and pathological complete response rate after neoadjuvant treatment (NAT) in early breast cancer (BC) HER2 negative

Abstract Background: Overexpression of HER2 is a significant prognostic and predictive factor in early breast cancer. HER2 positive tumors are those scored by immunohistochemistry (IHC) 3+ or 2+ with amplification by in situ hybridization (ISH). These tumors may benefit from HER2-targeted treatment and additionally, in early HER2-positive BC, achieving a complete pathological response after neoadjuvant treatment improves patient survival. In recent years, new anti-HER2 antibody-drug conjugates have proven effective for HER2 low BC (defined as IHC score 1+ or 2+ not amplified) in the metastatic disease setting; however, in HER2-low early BC, studies testing novel anti-HER2 antibody-drug conjugates as neoadjuvant therapy (NAT) are ongoing. Currently, we don´t know exactly whether HER2 expression can influence the pathological response rate after NAT or disease-free survival (DFS) in these patients compared to HER2-zero (IHC 0). The aim of our study is to determine the impact on response rate to NAT and survival outcomes in early HER2-negative BC. Methods: We conducted a retrospective study in two hospitals in Andalucia, Spain. Patients with early HER2 negative BC treated with NAT from January 2015 to June 2022 were included. Patients were divided into HER2 low patients (IHC 1+ or 2+ not amplified) and HER2 0 (IHC 0). We collected clinical and pathological characteristics, pathological response rate using the Residual Cancer Burden (RCB) system where a complete pathological response (pCR) was defined as ypT0/ypTis and ypN0, and survival outcomes. The primary objective of the study was to analyze the pCR rate after NAT according to HER2 score, and secondary objectives were to assess disease-free survival and overall survival (DFS and OS rates). Results: 574 patients were included (100% women). 397 patients had hormone receptors (luminal BC) on these tumors, and 177 did not have them, i.e., triple-negative BC (TNBC). 225 patients were HER2-zero, and 312 patients were HER2-low (253/312 patients had luminal BC and 59/312 patients were TNBC). The pCR in HER2-low patients was 17.3% and 23.1% in HER2-zero patients (p=0.047). For luminal BC patients, the pCR was 16.7% in HER2-low tumors and 13.7% in HER2-zero tumors (p=0.29). Similarly, there was no difference in pCR rates in TNBC patients (45.8% in HER2-low and 53.8% in HER2-zero tumors, respectively, (p=0.958). The overall survival (OS) at 96 months was 88.7% (95% CI 85.6%-92%). Regarding survival outcomes, neither HER2 expression level was associated with higher 8-year DFS: 81.1% in HER2-low patients (95% CI 75.7%-86.8%) and 74.9% in HER2-zero patients (95% CI 62.3%-90.1%) p=0.64. There was no difference between histological subtypes at 8-years regarding DFS rate: 80.5% (95% CI 74.3%-87.2%) in luminal HER2-low patients and 76.3% (95% CI 60.8%-95.7%) in luminal HER2-zero patients, p=0.22; 83.8% in TNBC HER2-low (95% CI 74.5%-94.2%) and 77.5% in TNBC HER2-zero patients (95% CI 69.4%-86.6%) p=0.26. We also didn´t find significant differences in terms of OS. For HER2-low patients, the 8-year OS was 90.9% (95% CI 87%-94.9%) and for HER2-zero patients, it was 86.4% (95% CI 80.8%- 92.5%) p=0.46. There were also no differences in OS for different histological subgroups with an 8-year OS in luminal HER2-low breast cancer patients of 92.5% (95% CI 88%-96.7%) and 87% in HER2-zero (95% CI 78.9%-96.1%) p=0.91. In patients with triple-negative breast cancer phenotype, the OS was 84.2% for HER2-low (95% CI 74.5%-95.2%) and 85.3% for HER2-zero (95% CI 77.9%-93.4%) p=1. Conclusions: We found no difference in survival outcomes and response rate to conventional NAT between HER2-low and HER2-zero expression levels. Therefore, our data do not support the hypothesis of HER2-low as a biologically specific breast cancer subtype. Further research on this approach with HER2 antibody-drug conjugate schemes as an alternative to traditional NAT schemes is warranted. Citation Format: Ana Gil-Torralvo, Yeray Rodriguez, Alejandro Falcon, David Morales, Mónica Cejuela, Isabel Miras, Marta Amérigo, Manuel Ruíz - Borrego, Juan Bayo, Francisco Javier Salvador Bofill. Is HER2-low a biologically distinct breast cancer (BC) subtype? Prognosis and pathological complete response rate after neoadjuvant treatment (NAT) in early breast cancer (BC) HER2 negative [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 PO1-27-07.

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

Abstract BACKGROUND: Half of the primary breast cancers reported as HER-2 negative have low HER-2 expression, defined as an immunohistochemical (IHC) score of 1+ or 2+, with negative in situ hybridization. Among these cases, 37% correspond to the triple-negative immunophenotype. The primary objective of this study was to evaluate HER-2 expression in patients with triple-negative breast cancer and compare the clinicopathologic features and response to neoadjuvant chemotherapy between HER-2 negative (0) and HER-2 low (1+/2+) subtypes. METHODS: We retrospectively analyzed 157 patients with early-stage triple-negative breast cancer who underwent neoadjuvant chemotherapy followed by surgery with curative intent and met the study's inclusion criteria. These patients were treated at the National Institute of Neoplastic Diseases between 2010 and 2021. HER-2 analysis was performed using immunohistochemistry (IHC), and in cases of HER-2 (2+) amplification, in situ hybridization was performed. RESULTS: Of all the included patients, 107 were classified in the HER-2 negative group (68%), and 50 were classified in the HER-2 low group (32%). Seventy-four percent of patients with HER-2 negative were in the age range of 40-64 years, compared to 68% in the HER-2 low group. A significant difference was found between the HER2-negative and HER2-low groups regarding age and Ki67. The median age of HER-2 low patients is significantly higher than the median age of HER-2 negative patients (52 years vs. 45 years, respectively). No significant association was found between HER-2 status and clinical stages or histological grade. However, the median Ki67 percentage of HER-2 negative patients is significantly higher than the median Ki67 of HER-2 low patients (70% vs. 50%, respectively (p=0.035)). Thirty-two percent of patients with HER-2 negative achieved a pathological complete response (pCR) compared to 18% of patients with HER-2 low. There were no differences in the pCR rate in the HER-2 1+ and 2+ subgroups. Among the patients included in both groups, who achieved pCR, no disease progression was observed at the time of analysis. CONCLUSIONS: Patients in the HER-2 negative group had a higher rate of complete pathological response to neoadjuvant chemotherapy compared to those in the HER-2 low group. No differences were observed in the pCR rate between HER-2 1+ and 2+ subgroups, suggesting that HER2 expression levels within the low range may not significantly impact treatment response. Table. Table. Citation Format: Zaida Morante, Iris Otoya, Natalia Valdivieso, Cindy Calle, Hugo Fuentes, Silvia Neciosup, Carlos Castañeda, Tatiana Vidaurre, Martin Falla, Valeria Colomo, Henry Gómez. HER2 Expression in Triple-Negative Breast Cancer: Clinicopathologic Features and Response to Neoadjuvant Chemotherapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-03-10.

Abstract PO5-16-03: RT LAMP assay for the subtyping of breast cancer

Abstract Introduction Our previous study showed that direct reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay can be employed as a substitute in order to detect tumor involvement of lymph nodes within breast cancer patients. In this research, we aimed to investigate the potential applicability of RT-LAMP for the classification of breast cancer subtype. Method Breast Cancer Cell line RNA extraction Total RNA from the cell lines was isolated from cultured cells using RNAiso Plus (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions; MCF10A, MCF7, T47D, BT474, SKBR3, MB468, HCC1806, MB231 Primer design For the Primer design, ER, PR, HER2, Ki 67, CK7 and GATA were targeted. LAMP primers targeted at ER were created between exons 5 and 6, PR were created between exons 6 and 7, HER2 were created between exons 17 and 18, KI67 were created between exons 2 and 3, CK7 were created between exons 5 and 6, and GATA3 were created between exons 2 and 3 utilizing Primer Explorer V4 (http://primereplorer.jp/elamp4.00/index.html). RT-LAMP assays RT-LAMP assays were carried out within a 25 µL reaction which included 12.5 µL of 2 × reaction buffer, 2µL of each primer, 1 µL of enzyme mixture (16U Bst DNA polymerase and 120U M-MLV reverse transcriptase) 8 µL of DEPC-treated water, and 2 µL of the template RNA. DEPC-treated water was also employed for purposes of a negative control. The reaction mixture was conducted within qRT-PCR. Results ER, PR, HER2, Ki-67, CK7, GATA3 expression level were checked for each breast cancer cell line using RT-LAMP. The luminal cell line exhibited elevated levels of ER and PR expression, while the her2 cell line demonstrated a high level of expression for her2. In the RT LAMP assay, there is a direct correlation between shorter detection time and higher expression levels of the substance. Specifically, in the luminal type, ER/PR was detected within a timeframe of 10 to 12 seconds, while HER2 was detected within a timeframe of 15 to 16 seconds. The expression levels of ER, PR, and HER2 were compared between MCF7 (luminal A) and MDA-MB-231 (TNBC) cell lines. RT-LAMP was performed to examine the RNA expression levels, while RT-PCR was applied to confirm the cDNA expression levels. MCF7 and MDA-MB-231 were mixed at 1:9, 5:5 and 9:1 ratios, respectively, and the difference in ER/PR/HE2 expression level was confirmed (Table1, Figure1). The presence of ER was detected in the MCF7 cell line at 14.44 minutes, and PR was observed at 18.84 minutes. HER2 was detected at a similar time (approximately 15 minutes) in both the MCF7 and MB 231 cell lines. Conclusion This study demonstrated that ER, PR, and HER2 can be quantified using RT-LAMP. Ongoing experiments are being conducted to classify breast cancer using the RT-LAMP method, aiming to enhance sensitivity in the quantification of ER, PR, and HER2. Additionally, new primers are being developed specifically for HER2 detection. RT-LAMP offers the advantages of being a point-of-care test that can be conducted on-site and reducing the discrepancies between pathologists. Citation Format: In Hee Lee, Byeongju Kang, Jeeyeon Lee, Jin Hyang Jung, Ho Yong Park, Nora Jee-Young Park, Ji-Young Park, Eun Ae Kim, Jieun Kang, Yee Soo Chae, Soo Jung Lee. RT LAMP assay for the subtyping of 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 PO5-16-03.

Abstract PO1-04-07: Disitamab vedotin, a clinical stage HER2-directed antibody-drug conjugate, shows potent antitumor activity as a monotherapy and in combination with tucatinib in preclinical breast cancer models

Abstract Disitamab vedotin (DV, RC48-ADC) is an antibody-drug conjugate (ADC) that targets cancers expressing HER2. DV consists of an anti-HER2 monoclonal antibody, disitamab, conjugated with the microtubule-disrupting agent monomethyl auristatin E (MMAE) via a cleavable vedotin linker. DV has multimodal antitumor mechanisms of action that include direct cytotoxicity of HER2-expressing cancer cells and bystander effect-based cytotoxicity of neighboring cells, both of which are mediated by the intracellular release of MMAE. Released MMAE can also induce immunogenic cell death, which promotes immune cell recruitment to the tumor. In addition, DV stimulates Fc-gamma receptor mediated antibody-dependent cellular cytotoxicity, which can lead to target cell death. DV also inhibits HER2-activated downstream signaling pathways, further blocking cell growth, survival, and proliferation. Previously, we showed the cytotoxic activity of DV against a panel of breast cancer cell lines with varying levels of HER2 expression, including the HER2-low range. Here, we sought to further characterize the preclinical antitumor activity of DV in patient-derived 3D models and patient-derived xenographs (PDX). In cultured breast cancer cells, DV was more potent and internalized to a greater magnitude than the HER2-directed ADCs trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd). In patient-derived 3D models, DV had superior activity compared to T-DXd. We further explored whether dual HER2 targeting with DV in combination with the HER2-selective oral TKI tucatinib improved the antitumor outcomes. In PDX models with varying HER2 IHC levels and models refractory to T-DXd, DV as a monotherapy and in combination with tucatinib showed significant tumor growth inhibition. This result is consistent with previous in vitro results that showed increased cytotoxicity of the combination of DV and tucatinib in cell lines with a wide range of HER2 expression, which may be mechanistically attributed to elevated HER2 cell surface levels upon treatment with tucatinib. Overall, these findings provide scientific rationale to explore DV in HER2-positive and HER2-low breast cancer patients as a monotherapy or in combination with tucatinib. Citation Format: Kelsi Willis, Renee Hein, Katie Snead, Robert Thurman, Anita Kulukian. Disitamab vedotin, a clinical stage HER2-directed antibody-drug conjugate, shows potent antitumor activity as a monotherapy and in combination with tucatinib in preclinical breast cancer models [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 PO1-04-07.

Abstract PO5-04-04: Development of a method to detect very low levels of HER2 expression in Circulating Tumor Cells (CTCs) by liquid biopsy in patients with metastatic breast cancer

Abstract Introduction Blood-based liquid biopsies are a non-invasive diagnostic approach for detecting circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) that may provide clinically actionable information for treatment decisions for metastatic breast cancer (MBC) patients when a conventional tissue biopsy is not feasible. Recently, the novel anti-HER2 antibody-drug conjugate, Trastuzumab Deruxtecan (T-DXd), has shown effectiveness against MBC with low HER2 expression detected by tissue biopsy characterized as HER2-low (immunohistochemically 1+ or 2+ and ERBB2 amplification-negative via in situ hybridization) and is being explored in an ultra-low population. Here we report on the application of a liquid biopsy platform designed to sensitively detect low levels of HER2 protein expression in CTCs from MBC patients. Methods Blood samples from 704 metastatic breast cancer patients as well as control cells lines were collected for cell-based and cell-free DNA analysis. After plasma isolation, nucleated cells deposited on glass slides went through immunofluorescent staining and imaging. CTCs were identified using Epic Sciences’ digital imaging and machine learning algorithms, and a subset of CTCs were isolated and sequenced for genomic quantification of large scale-state transitions (LSTs) as well as copy number variants (CNVs). The HER2 status was based upon on ASCO-CAP guidelines and the previous tissue biopsy results from the ordering healthcare provider, as documented on the DefineMBC™ test requisition form. Results Within a DefineMBC early-access program (EAP), 93% of patients with MBC had detectable CTCs. Among ERBB2-non-amp patients tested with the DefineMBC assay, 25% of patients with MBC had findings that were reported as consistent with HER2-low in the CAP-CLIA setting. To explore the lower boundaries of HER2 protein expression, we created and validated an MCF-7 HER2-negative ERBB2 knock-out cell line and utilized its 95% confidence level threshold (271 MFI) as a negative fluorescence cutoff for very low HER2 expression. Based upon publicly available CCLE datasets, MDA-MB-453, MCF7, and MCF7 ERBB2 KO breast cancer cell lines were selected for their high and low HER2 RNA expression levels and were corroborated via observed HER2 immunofluorescence median levels of 15740, 381, and 188 MFI respectively. In an exploratory analysis, among ERBB2-non-amp patients, we retrospectively applied the 271 MFI HER2 cutoff to the EAP cohort and identified 38% of patients with very low HER2 expression. We examined the impact of the HER2 ultra-low cutoff in patients not eligible for T-DXd treatment. Among patients reported as IHC=0 or with unknown IHC status from the most recent tissue biopsy, 44% and 42%, respectively, may qualify for treatment with T-DXd. Conclusion Using our higher sensitivity assay that combines CTC immunofluorescence and single-cell genomic analysis, we examined the lower boundary of HER2 protein expression. Feasibility data support further characterization of a very low HER2 expression phenotype, for the eventual inclusion into our comprehensive cancer profiling solution. To date, studies with biomarker expression have been limited to tissue biopsy, which may not always yield contemporaneous sampling in the metastatic setting. These results offer a liquid biopsy test that might identify patients potentially responsive to HER2-directed therapies. Citation Format: Giuseppe Di Caro, David Bourdon, Andrew Kunihiro, Alessandra Cunsolo, Ernest Lam, Megan Slade, Martin Blankfard, Lee Schwartzberg. Development of a method to detect very low levels of HER2 expression in Circulating Tumor Cells (CTCs) by liquid biopsy in patients with metastatic 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 PO5-04-04.

Abstract PO1-01-13: Clinical characterization, prognostic and predictive values of HER2-low in early breast cancer in the PALLAS trial

Abstract Background: Low levels of HER2 expression (HER2-low) have recently emerged as a therapeutic target in patients (pts) with breast cancer (BC). The aim of this study was to evaluate clinical and demographic variables associated with HER2 expression, as well as the value of HER2-low as a prognostic and predictive biomarker for palbociclib benefit in pts enrolled in the PALLAS trial. Methods: The phase III PALLAS trial investigated the addition of palbociclib to adjuvant endocrine therapy (ET) in pts with stage II-III hormone receptor-positive BC. The aims of this analysis are (1) to assess the association between the presence of low levels of HER2 expression with demographic and clinicopathological parameters, (2) to test the prognostic value of HER2-low status on invasive disease-free survival (IDFS), distant relapse-free survival (DRFS), and overall survival (OS) and (3) to assess the value of HER2 expression as a predictive biomarker of response to palbociclib. HER2-low was defined as HER2 immunohistochemistry (IHC) 1+ or IHC 2+ with negative in situ hybridization; all pathologic evaluation was performed locally. Pts with HER2-0 and Her2-low BC were included in this analysis; those with HER2-positive BC or missing HER2 status were excluded. Association of HER2 with other baseline covariates were tested with Chi-squared tests. Prognostic and predictive power of HER2 were assessed with Cox Models. Multivariable models included age, T-stage, N-stage, grade and progesterone receptor (PR) expression. Hazard ratios (HR) and 95% Confidence intervals (CI) are reported. Results: From the original PALLAS intention-to-treat population (N = 5,753), 5304 pts (92.2%) were included in this analysis. Among these, 2,254 pts (42.5%) were classified as HER2 IHC 0 (HER2-0) and 3,050 (57.5%) as HER2-low (1,838 with IHC 1+ and 1,212 with IHC 2+). For this analysis, median follow-up was 59.8 months. Compared to HER2-0, pts with HER2-low tumors had statistically higher estrogen receptor expression levels (mean expression 89.2% vs 88.2%, p = 0.019), lower PR expression levels (65.9% vs 68.2%, p = 0.007). Importantly, HER2-low status varied significantly across 21 participating countries (range 16.7% to 75.6%; p&amp;lt; 0.001) and was more frequent in pts enrolled in North America (63.1%) than in Europe (53.4%) and other regions (53.4%) (p &amp;lt; 0.001). No differences in HER2 expression were observed according to anatomic stage, T-stage, N-stage, histological grade, age, menopausal status, primary surgery type, prior radiation, prior chemotherapy, or baseline performance status. There were no statistically significant differences in IDFS, DRFS or OS according to HER2 status in univariate or multivariable Cox models (prognostic value, HR provided in the Table). There was no significant interaction between the HER2 status and the benefit to palbociclib in IDFS, DRFS or OS (Table). Similar results were obtained when HER2-0 was compared to HER2 1+ and HER2 2+ separately. Conclusions: In this large, prospective and international cohort, no differences were observed in clinical parameters, prognosis, or differential benefit from palbociclib (predictive value) between HER2-0 and HER2-low tumors. Significant geographic variability was observed in the prevalence of HER2-low status, suggesting a high degree of variation in pathologic assessment of HER2 expression without impact on outcomes. Support: AFT, Abbvie Pfizer; Clinicaltrials.gov: NCT02513394 https://acknowledgments.alliancefound.org Citation Format: Guilherme Nader-Marta, Christian F. Singer, Dominik Hlauschek, Angela DeMichele, Paolo Tarantino, Georg Pfeiler, Miguel Martín, Justin Balko, Zbigniew Nowecki, Marija Balic, Adam Brufsky, Arlene Chan, Patrick G. Morris, Tufia Haddad, Sibylle Loibl, Yuan Liu, Lidija Sölkner, Christian Fesl, Erica Mayer, Michael Gnant. Clinical characterization, prognostic and predictive values of HER2-low in early breast cancer in the PALLAS trial [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 PO1-01-13.