HER2-amplified Breast Cancer Research Articles

Immunological responses to brain metastasis stereotactic radiosurgery in patient-matched longitudinal blood and tumour samples

BackgroundStereotactic radiosurgery (SRS) is highly effective as focal treatment for brain metastases (BrMs), but whether it can promote anti-tumour immune responses that synergise with immunotherapy remains unclear. We investigated this by examining blood samples from a clinical trial for HER2-amplified breast cancer (HER2-BC) BrMs, matched with longitudinal HER2-BC BrM samples resected from the same location in the same patient. MethodsBlood samples from 10 patients taken pre- and 7–14 days post-SRS were analysed by mass and flow cytometry. One patient received pre-operative SRS for a BrM that recurred 7 months after resection, followed by planned re-resection 8 days post-SRS. Pre- and post-SRS tumours from this patient were analysed by bulk RNAseq, multiplex immunohistochemistry (mIHC), and TCR sequencing. ResultsMonocytes, central memory CD8+ T and regulatory T cells were enriched in blood post-SRS, together with increased MHC-II expression on monocytes, conventional DCs, and monocytic MDSCs. In tumour, SRS upregulated antigen presentation, T cell proliferation and T cell co-stimulation signatures, alongside an influx of tumour-associated macrophages (TAMs) and CD4+ T cells. Specifically, TAMs and CD4+ T cells, but not CD8+ T cells, demonstrated spatial co-localisation post-SRS. These TAMs were lowly PD-L1 expressing, but CD4+ T cells showed increased PD-1 expression. A sizeable proportion of T cell clonotypes were retained post-SRS, and four clones demonstrated significant, non-stochastic expansion. ConclusionSystemic and local immunological changes in this homogenous patient cohort suggest that SRS may facilitate MHC-II-restricted T cell priming responses involving the monocyte-macrophage lineage and CD4+ T cells, which should be further explored.

Preliminary safety and efficacy of TQB2930, a HER2-targeted bispecific antibody in patients with advanced breast cancer: Results from a phase 1b study.

1026 Background: TQB2930 is a HER2-targeted bispecific antibody, binding to two distinct HER2 epitopes, the same domain as trastuzumab (ECD4) and pertuzumab (ECD2). This study aims to evaluate the safety and efficacy of TQB2930 for patients with advanced breast cancer after failure of standard therapy. Methods: This phase 1b, dose-escalation and expansion trial included patients preferred HER2-expressing or HER2-amplified breast cancer who had received standard therapy. In the dose-escalation phase, TQB2930 was given weekly (10mg/kg, QW), biweekly (20mg/kg, Q2W), or triweekly (30mg/kg, Q3W). After the dose-escalation phase, eligible patients were enrolled and dosed biweekly or triweekly in the expansion cohort. The primary endpoints were safety, maximum tolerated or maximum administered dose, and the secondary endpoints were immunogenicity, pharmacokinetics, and tumor response per RECIST v1.1. Results: As of Dec 20, 2023, 34 patients(pts) have received TQB2930 weekly (n=3), biweekly (n=16), and triweekly (n=15). All pts were female, the median age was 59. 22 pts had HER2 expression of ICH3+ or ICH2+ with FISH+, and the others had ICH2+ with FISH- or ICH1+. The median prior lines of therapies were 3 (range: 0-8), with one pt intolerance to first-line treatment enrolled, and the median prior lines of HER2-targeted therapies were 2 (range: 0-8). No dose-limited toxicity was observed. The main treatment-related adverse events (TRAEs) were infusion reaction, occult blood of urine, leukopenia, and neutropenia. 3 pts were reported grade≥3 TRAEs, including neutropenia, anemia, and hypokalemia. Of 31 response-evaluable pts, the objective response rate (ORR) was 25.8% with 8 partial responses (PRs) were observed (including 5 confirmed PRs and 3 PRs awaiting confirmation). The disease control rate was 80.6%. Moreover, 5 pts achieved PR at 30mg/kg (ORR 41.7%), including one with prior exposure to trastuzumab, pyrotinib, tucatinib, ARX788, and the other one prior to trastuzumab, pertuzumab, lapatinib, MRG002. The median progression-free survival for all pts was 5.5 months (95%CI 3.58-NE). Additionally, Pharmacokinetic analysis showed exposure (Cmax and AUC0-t) of TQB2930 increased linearly with doses within 10~30mg/kg. After dosing, the mean peak concentration reached at 2.5-4.3 h, and the elimination half-life of TQB2930 was 109-161 h. Conclusions: TQB2930 is well tolerated and has demonstrated promising single-agent anti-tumor activity in HER2-positive breast cancer who have failed standard anti-HER2 therapies, including multiple lines of prior HER2 targeted agents. These early signs of activity support the further exploration of combination therapy of TQB2930 and the study is ongoing. Clinical trial information: NCT06202261 .

Neoadjuvant targeted therapy in early resectable HER2-mutant lobular breast cancer.

TPS630 Background: Invasive lobular carcinoma (ILC) accounts for approximately 10-15% of all breast cancers and is characterized by hormone receptor positivity (HR+), low Ki-67 score, and loss of cell adhesion receptors such as E-cadherin. While these tumors tend to be indolent initially, late recurrences and distant metastasis are common, and ILC has very low rates of pathologic complete response (pCR) to both neoadjuvant chemotherapy and endocrine therapy (ET). HER2 ( ERBB2) mutations are enriched in about 10% of unselected ILC, with some reports showing the prevalence as high as 27% in pleomorphic and higher grade ILCs. HER2 mutations in ILC are associated with ET resistance and a worse prognosis. Neratinib is a HER2 tyrosine kinase inhibitor (TKI) currently FDA-approved for adjuvant treatment of HER2-amplified (HER2+) early-stage breast cancer. It has shown safety and efficacy in combination with ET in metastatic HER2-mutant HR+ breast cancer. We hypothesize that neoadjuvant treatment with neratinib and ET will improve pathologic responses in newly diagnosed HER2-mutant HR+ ILC. Methods: This study (NCT05919108) is an open label multi-site Phase II clinical trial for 30 patients with Stage I-III ILCs and a HER2 activating mutation. Patients will be identified by next generation sequencing (NGS) of the diagnostic biopsy and randomized to 4 weeks of ET +/- neratinib; a biopsy will be done at the end of the lead-in phase. All patients will then receive ET and neratinib for 20 weeks prior to proceeding to surgery. To optimize tolerability, the first cycle of neratinib will include a dose escalation and antidiarrheal prophylaxis. The primary objective of this study will be the pre-operative endocrine prognostic index (PEPI) score, which will be compared to a historical PEPI-0 rate for patients with HR+ breast cancer treated with neoadjuvant ET. Secondary objectives will include pCR, residual cancer burden, and rates of breast conserving surgery. Correlative studies will include biomarkers of tumor cell cycle arrest, apoptosis, HER2 pathway activation, and gene expression on the pre- and on-treatment (4 week) biopsy from the lead-in phase to document molecular synergy between neratinib and ET. Patient-derived organoids from the 4-week biopsies will be established and subjected to single-cell RNA, ATAC seq, and DNA sequencing to study tumor evolution and elucidate epigenetic programs and somatic alterations that allow breast cancer survival despite continuous HER2-directed therapy. This is a first-in-kind, mechanism-based, molecularly targeted combination of a HER2 TKI and ET in HER2-mutant ILC. We posit results from this study may generate a clear signal of clinical activity leading to a randomized phase III trial aimed at incorporating HER2-directed therapies as a treatment for operable, early stage ILC. This signal may also suggest for the first time the added value of NGS in patients with newly diagnosed HR+ breast cancer. Clinical trial information: NCT05919108 .

Abstract CT271: Neoadjuvant targeted therapy in stage I-III HER2-mutant lobular breast cancer

Abstract Background: Invasive lobular carcinoma (ILC) accounts for approximately 10-15% of all breast cancers and is characterized by hormone receptor positivity (HR+), low Ki-67 score, and loss of cell adhesion receptors such as E-cadherin. While these tumors tend to be indolent initially, late recurrences and distant metastasis are common, and ILC has very low rates of pathologic complete response (pCR) to both neoadjuvant chemotherapy and endocrine therapy (ET). HER2 (ERBB2) mutations are enriched in about 10% of unselected ILC, with some reports showing the prevalence as high as 27% in pleomorphic and higher grade ILCs. HER2 mutations in ILC are associated with ET resistance and a worse prognosis. Neratinib is a HER2 tyrosine kinase inhibitor (TKI) currently FDA-approved for adjuvant treatment of HER2-amplified (HER2+) early-stage breast cancer. It has shown safety and efficacy in combination with ET in metastatic HER2-mutant HR+ breast cancer. We hypothesize that neoadjuvant treatment with neratinib and ET will improve pathologic responses in newly diagnosed HER2-mutant HR+ ILC. Methods: This study (NCT05919108) is an open label multi-site Phase II clinical trial for 30 patients with Stage I-III ILCs and a HER2 activating mutation. Patients will be identified by next generation sequencing (NGS) of the diagnostic biopsy and randomized to 4 weeks of ET +/- neratinib; a biopsy will be done at the end of the lead-in phase. All patients will then receive ET and neratinib for 20 weeks prior to proceeding to surgery. To optimize tolerability, the first cycle of neratinib will include a dose escalation and antidiarrheal prophylaxis. The primary objective of this study will be the pre-operative endocrine prognostic index (PEPI) score, which will be compared to a historical PEPI-0 rate for patients with HR+ breast cancer treated with neoadjuvant ET. Secondary objectives will include pCR, residual cancer burden, and rates of breast conserving surgery. Correlative studies will include biomarkers of tumor cell cycle arrest, apoptosis, HER2 pathway activation, and gene expression on the pre- and on-treatment (4 week) biopsy from the lead-in phase to document molecular synergy between neratinib and ET. Patient-derived organoids from the 4-week biopsies will be established and subjected to single-cell RNA, ATAC seq, and DNA sequencing to study tumor evolution and elucidate epigenetic programs and somatic alterations that allow breast cancer survival despite continuous HER2-directed therapy. This is a first in kind, mechanism-based, molecularly targeted combination of a HER2 TKI and ET in HER2-mutant ILC. We posit results from this study may generate a clear signal of clinical activity leading to a randomized Phase III trial aimed at incorporating HER2-directed therapies as a treatment for operable, early stage ILC. This signal may also suggest for the first time the added value of NGS in patients with newly diagnosed HR+ breast cancer. Citation Format: Laura C. Kennedy, Nisha Unni, Ariella B. Hanker, Carlos L. Arteaga. Neoadjuvant targeted therapy in stage I-III HER2-mutant lobular breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT271.

Neoadjuvant anthracycline-based (5-FEC) or anthracycline-free (docetaxel/carboplatin) chemotherapy plus trastuzumab and pertuzmab in HER2 + BC patients according to their TOP2A: a multicentre, open-label, non-randomized phase II trial

PurposePrevious studies have reported the benefit of dual HER2-targeting combined to neoadjuvant chemotherapy in HER2-amplified breast cancer (HER2 + BC). Moreover, besides the cardiac toxicity following their association to Trastuzumab, anthracyclines chemotherapy may not profit all patients. The NeoTOP study was designed to evaluate the complementary action of Trastuzumab and Pertuzumab, and the relevance of an anthracycline-based regimen according to TOP2A amplification status.MethodsOpen-label, multicentre, phase II study. Eligible patients were aged ≥ 18 with untreated, operable, histologically confirmed HER2 + BC. After centralized review of TOP2A status, TOP2A-amplified (TOP2A+) patients received FEC100 for 3 cycles then 3 cycles of Trastuzumab (8 mg/kg then 6 mg/kg), Pertuzumab (840 mg/kg then 420 mg/kg), and Docetaxel (75mg/m2 then 100mg/m2). TOP2A-not amplified (TOP2A-) patients received 6 cycles of Docetaxel (75mg/m2) and Carboplatin (target AUC 6 mg/ml/min) plus Trastuzumab and Pertuzumab. Primary endpoint was pathological Complete Response (pCR) using Chevallier’s classification. Secondary endpoints included pCR (Sataloff), Progression-Free Survival (PFS), Overall Survival (OS), and toxicity.ResultsOut of 74 patients, 41 and 33 were allocated to the TOP2A + and TOP2A- groups respectively. pCR rates (Chevallier) were 74.4% (95%CI: 58.9–85.4) vs. 71.9% (95%CI: 54.6–84.4) in the TOP2A + vs. TOP2A- groups. pCR rates (Sataloff), 5-year PFS and OS were 70.6% (95%CI: 53.8–83.2) vs. 61.5% (95%CI: 42.5–77.6), 82.4% (95%CI: 62.2–93.6) vs. 100% (95%CI: 74.1–100), and 90% (95%CI: 69.8–98.3) vs. 100% (95%CI: 74.1–100). Toxicity profile was consistent with previous reports.ConclusionOur results showed high pCR rates with Trastuzumab and Pertuzumab associated to chemotherapy. They were similar in TOP2A + and TOP2A- groups and the current role of neoadjuvant anthracycline-based chemotherapy remains questioned.Trial registration numberNCT02339532 (registered on 14/12/14).

Abstract B002: The impact of mir-4728 on translation and pathway regulation in HER2-amplified breast cancer

Abstract MicroRNAs (miRNAs) are a class of small non-coding RNAs that have been implicated in cancer. Our research focuses on the miRNA mir-4728, which is located in an intron in the HER2 locus. The HER2 locus is amplified in approximately 15% of all breast tumors and is associated with a poor prognosis. Current therapeutic strategies for HER2-positive tumors involve targeted inhibition of the HER2 protein with monoclonal antibodies such as trastuzumab in combination with chemotherapy and surgery. However, if mir-4728 has a function independent of its host gene, targeting the HER2 protein alone may be insufficient. To investigate the role of mir-4728, we utilized antisense oligonucleotides to specifically block mir-4728 in a HER2-amplified breast cancer cell line, while leaving the protein receptor in tact. Polysome fractionation and ribosome profiling revealed that blocking the miRNA resulted in a substantial change in protein translation. We hypothesize that this effect may come as a result of mir-4728 being an indirect regulator of EIF4A, a key factor in the initiation of translation. In addition, we have observed changes in the synthesis of aromatase, a crucial enzyme in estrogen synthesis. These findings suggest that mir-4728 may play a role in the regulation of multiple pathways that contribute to cancer progression. Further investigation into this mechanism may provide valuable insight into the development of new therapeutic strategies for cancer treatment. Citation Format: Völundur Hafstad, Euisuk Han, Helena Persson. The impact of mir-4728 on translation and pathway regulation in HER2-amplified breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B002.

Anti-cathepsin D immunotherapy triggers both innate and adaptive anti-tumour immunity in breast cancer.

Triple-negative breast cancer (TNBC) has poorer outcomes than other breast cancers (BC), including HER2+ BC. Cathepsin D (CathD) is a poor prognosis marker overproduced by BC cells, hypersecreted in the tumour microenvironment with tumour-promoting activity. Here, we characterized the immunomodulatory activity of the anti-CathD antibody F1 and its improved Fab-aglycosylated version (F1M1) in immunocompetent mouse models of TNBC (C57BL/6 mice harbouring E0771 cell grafts) and HER2-amplified BC (BALB/c mice harbouring TUBO cell grafts). CathD expression was evaluated by western blotting and immunofluorescence, and antibody binding to CathD by ELISA. Antibody anti-tumour efficacy was investigated in mouse models. Immune cell recruitment and activation were assessed by immunohistochemistry, immunophenotyping, and RT-qPCR. F1 and F1M1 antibodies remodelled the tumour immune landscape. Both antibodies promoted innate antitumour immunity by preventing the recruitment of immunosuppressive M2-polarized tumour-associated macrophages (TAMs) and by activating natural killer cells in the tumour microenvironment of both models. This translated into a reduction of T-cell exhaustion markers in the tumour microenvironment that could be locally supported by enhanced activation of anti-tumour antigen-presenting cell (M1-polarized TAMs and cDC1 cells) functions. Both antibodies inhibited tumour growth in the highly-immunogenic E0771 model, but only marginally in the immune-excluded TUBO model, indicating that anti-CathD immunotherapy is more relevant for BC with a high immune cell infiltrate, as often observed in TNBC. Anti-CathD antibody-based therapy triggers the anti-tumour innate and adaptive immunity in preclinical models of BC and is a promising immunotherapy for immunogenic TNBC.

Integrative whole-genome and transcriptome analysis of HER2-amplified metastatic breast cancer

BackgroundIn breast cancer, the advent of anti-HER2 therapies has made HER2+ tumors a highly relevant subgroup. However, the exact characteristics which prohibit clinical response to anti-HER2 therapies and drive disease progression are not yet fully known. Integrative whole-genome and transcriptomic sequencing data from both primary and metastatic HER2-positive breast cancer will enhance our understanding of underlying biological processes.MethodsHere, we used WGS and RNA sequencing data of 700 metastatic breast tumors, of which 68 being HER2+, to search for specific genomic features of HER2+ disease and therapy resistance. Furthermore, we integrated results with transcriptomic data to associate tumors exhibiting a HER2+-specific gene expression profile with ERBB2 mutation status, prior therapy and relevant gene expression signatures.ResultsOverall genomic profiles of primary and metastatic HER2+ breast cancers were similar, and no specific acquired genomics traits connected to prior anti-HER2 treatment were observed. However, specific genomic features were predictive of progression-free survival on post-biopsy anti-HER2 treatment. Furthermore, a HER2-driven expression profile grouped HER2-amplified tumors with ERBB2-mutated cases and cases without HER2 alterations. The latter were reported as ER positive in primary disease, but the metastatic biopsy showed low ESR1 expression and upregulation of the MAPK pathway, suggesting transformation to ER independence.ConclusionsIn summary, although the quantity of variants increased throughout HER2-positive breast cancer progression, the genomic composition remained largely consistent, thus yielding no new major processes beside those already operational in primary disease. Our results suggest that integrated genomic and transcriptomic analyses may be key in establishing therapeutic options.

Detecting Human Epidermal Growth Factor Receptor 2 (HER2) Amplification: Proof of Concept of an Alternative Approach.

There are multiple genes that are co-amplified along with human epidermal growth factor receptor 2 (HER2) in chromosome 17. GRB7 and PGAP3 are two such genes. We hypothesize that the protein products of these genes may serve as immunohistochemistry (IHC) markers for detecting HER2 amplification in breast cancer. Tissue sections from one hundred and thirty-five primary breast carcinoma cases were subjected to immunohistochemical staining for antibodies against HER2, GRB7, and PGAP3 and graded on a scale of 1 to 3. Both membranous staining and cytoplasmic staining were assessed for GRB7 and PGAP3. For equivocal HER2 IHC positivity, fluorescent in situ hybridization was performed to get the final HER2 status. IHC staining for GRB7 and PGAP 3 was a moderate to strong predictorfor HER2 status (area under the curve (AUC) of 0.768, 0.868,0.754, and 0.790 for GRB7 membranous staining, GRB7 cytoplasmic staining, PGAP3 membranous staining, and PGAP3 cytoplasmic staining respectively). A combination of GRB7 cytoplasmic and PGAP3 membranous staining resulted in an AUC of 0.905 (95% CI0.855-0.954), while a combination of GRB7 and PGAP3 cytoplasmic staining resulted in an AUC of 0.902 (95% CI 0.851-0.953). The point estimates for the AUC of GRB7 and combined GRB7 and PGAP3 in predicting the AUC suggest a strong predictive ability of these markers to predict HER2. With further refinement in technique, cytoplasmic staining and membranous IHC staining for GRB7 and PGAP3 have potential to serve as surrogate markers for HER2 status. The strategy of using protein products of co-amplified genes of HER2 is likely to be successful in technical validation.

BSBM-15 SYSTEMIC AND LOCAL IMMUNE RESPONSES FOLLOWING STEREOTACTIC RADIOSURGERY TO BRAIN METASTASES FROM HER2-AMPLIFIED BREAST CANCER

Abstract BACKGROUND Brain metastases (BMs) afflict 30-50% of HER2-amplified breast cancer (HER2-BC) patients. Stereotactic radiosurgery (SRS) is a highly effective focal treatment for BMs. Extracranial radiotherapy can promote anti-tumor immune responses that synergize with immunotherapy, but whether this applies to the brain microenvironment remains unclear. Here, we examined blood and BM samples from HER2-BC patients in the TROG 16.02 translational sub-study for immunological effects of SRS (ACTRN12616001265460). METHODS Blood samples from 10 patients taken pre- and 7-14 days post-SRS to intact HER2-BC BMs or post-resection BM cavities were analyzed by mass and flow cytometry for immune cell and cytokine modulation. One patient received pre-operative SRS for a BM that recurred 6 months after resection, followed by re-resection of the BM 7 days post-SRS. Tumors from pre- and post-SRS timepoints in this patient were analyzed by bulk RNAseq and immunohistochemistry. RESULTS Monocytes, CD8+ T effector memory and regulatory T cells were enriched in blood post-SRS, while conventional dendritic cells, CD4+ T early and CD4+ TEMRA cells were diminished. Stem cell factor (SCF) concentration, a growth factor important for monocyte production, was elevated in plasma post-SRS. In the tumor, SRS upregulated gene signatures for monocyte/macrophage processes (including SCF gene transcript levels), antigen presentation, and T cell activation. Cell type deconvolution from RNAseq suggested an SRS-induced loss of metastatic tumor cells and enrichment of macrophages and CD4+ T cells. Immunohistochemistry corroborated the influx of CD68+ macrophages and CD3+ T cells into tumor and brain regions post-SRS, with spatial co-localization evident between the cell types. Furthermore, the pre-SRS immune-excluded phenotype in tumor regions was no longer observed post-SRS. CONCLUSION Systemic and local immunological changes in this homogenous patient cohort receiving SRS to HER2-BC BMs suggest a radiation-induced adaptive immune response intracranially, involving the monocyte-macrophage lineage as antigen presenting cells, that should be further explored.

Improved concordance of challenging human epidermal growth factor receptor 2 dual in-situ hybridisation cases with the use of a digital image analysis algorithm in breast cancer.

Accurate assessment of human epidermal growth factor receptor 2 (HER2) expression by HER2 immunohistochemistry and in-situ hybridisation (ISH) is critical for the management of patients with breast cancer. The revised 2018 ASCO/CAP guidelines define 5 groups based on HER2 expression and copy number. Manual pathologist quantification by light microscopy of equivocal and less common HER2 ISH groups (groups 2-4) can be challenging, and there are no data on interobserver variability in reporting of these cases. We sought to determine whether a digital algorithm could improve interobserver variability in the assessment of difficult HER2 ISH cases. HER2 ISH was evaluated in a cohort enriched for less common HER2 patterns using standard light microscopy versus analysis of whole slide images using the Roche uPath HER2 dual ISH image analysis algorithm. Standard microscopy demonstrated significant interobserver variability with a Fleiss's kappa value of 0.471 (fair-moderate agreement) improving to 0.666 (moderate-good) with the use of the algorithm. For HER2 group designation (groups 1-5), there waspoor-moderate reliability between pathologists bymicroscopy [intraclass correlation coefficient (ICC) = 0.526], improving to moderate-good agreement (ICC = 0.763) with the use of the algorithm. In subgroup analysis, the algorithm improved concordance particularly in groups 2, 4 and 5. Time to enumerate cases was also significantly reduced. This work demonstrates the potential of a digital image analysis algorithm to improve the concordance of pathologist HER2 amplification status reporting in less common HER2 groups. This has the potential to improve therapy selection and outcomes for patients with HER2-low and borderline HER2-amplified breast cancers.

HER2 in situ hybridization testing in breast cancer: Applying algorithm-assisted assessment to reduce interobserver variability in difficult cases.

e13561 Background: Accurate assessment of HER2 expression by HER2 immunohistochemistry and in-situ hybridization (ISH) is critical for the management of patients with breast cancer. The revised 2018 ASCO/CAP guidelines defined 5 subgroups based on HER2 expression and copy number. Few reports have investigated interobserver variability when applying the revised guidelines. Digital algorithms may have a role in assisting pathologists in assessing cases with equivocal and uncommon patterns of HER2 amplification. This study was designed to evaluate interobserver variability in the gold standard manual assessment of HER2 ISH, particularly in less common HER2 ISH groups. Subsequently, we evaluated whether a new digital image analysis algorithm (Roche uPath HER2 Dual ISH image analysis) could reduce interobserver variability and potentially improve patient selection for targeted anti-HER2 therapy. Methods: Of 2982 breast cancer cases, we derived an enriched cohort of 99 patient cases with HER2 IHC 2+ or 3+ and selective ISH results (groups 2-4, evidence of HER2 heterogeneity, borderline negative group 5 and borderline positive group 1). As per guidelines, a minimum of 20 tumour cells per slide were manually and independently counted by 4 anatomical pathologists. After a washout period and second round of de-identification, the slides were scanned (DP200; Roche) and re-scored using the digital algorithm. Results: Pathologist reporting of patient HER2 status (HER2 positive vs. HER2 negative) using manual microscopy demonstrated significant interobserver variability, with a Fleiss’s kappa value of 0.471 (95% CI: 0.378-0.564; P= 1.24E-21) indicating fair-moderate agreement. Interobserver variability in manual scoring of HER2 ISH at the level of HER2 group designation (groups 1-5) showed poor-moderate reliability between pathologists, with an intraclass correlation coefficient (ICC) of 0.526 (95% CI: 0.407-0.641; P= 9.28E-21). Repeat analysis of the cohort using algorithm-assisted quantification resulted in moderate-substantial concordance in HER2 patient status reporting, with a Fleiss’s kappa value of 0.666 (95% CI: 0.572-0.759; P= 1.70E-40). Algorithm-assisted quantification resulted in moderate-good agreement of HER2 group designation, with an ICC of 0.763 (95% CI: 0.683-0.832; P= 8.76E-48). In sub-group analysis, use of the algorithm improved concordance particularly in groups 2,4 and 5. Time to report cases was also significantly reduced with the use of the algorithm. Conclusions: Our study demonstrates the potential to improve the concordance of HER2 amplification status reporting in less common HER2 subgroups using a digital algorithm and potentially improve therapy selection and outcomes for patients with HER2-low and borderline HER2-amplified breast cancers.

Dissecting sources of variability in patient response to targeted therapy: anti-HER2 therapies as a case study

Background and purposeDespite their use to treat cancers with specific genetic aberrations, targeted therapies elicit heterogeneous responses. Sources of variability are critical to targeted therapy drug development, yet there exists no method to discern their relative contribution to response heterogeneity. Experimental approachWe use HER2-amplified breast cancer and two agents, neratinib and lapatinib, to develop a platform for dissecting sources of variability in patient response. The platform comprises four components: pharmacokinetics, tumor burden and growth kinetics, clonal composition, and sensitivity to treatment. Pharmacokinetics are simulated using population models to capture variable systemic exposure. Tumor burden and growth kinetics are derived from clinical data comprising over 800,000 women. The fraction of sensitive and resistant tumor cells is informed by HER2 immunohistochemistry. Growth rate-corrected drug potency is used to predict response. We integrate these factors and simulate clinical outcomes for virtual patients. The relative contributions of these factors to response heterogeneity arecompared. Key resultsThe platform was verified with clinical data, including response rate and progression-free survival (PFS). For both neratinib and lapatinib, the growth rate of resistant clones influenced PFS to a higher degree than systemic drug exposure. Variability in exposure at labeled doses did not significantly influence response. Sensitivity to drug strongly influenced responses to neratinib. Variability in patient HER2 immunohistochemistry scores influenced responses to lapatinib. Exploratory twice daily dosing improved PFS for neratinib but not lapatinib. Conclusion and implicationsThe platform can dissect sources of variability in response to target therapy, which may facilitate decision-making during drug development.

Outcome comparison of pyrotinib with current standard of care in the second/third line setting in advanced non-small cell lung cancer patients with HER2 mutation.

Outcome comparison of pyrotinib with current standard of care in the second/third line setting in advanced non-small cell lung cancer patients with HER2 mutation.

Abstract P4-07-28: Prognostic Impact of Delaying Multimodality Treatment in HER2 Positive Breast Cancer

Abstract Objective: Multimodality treatment in breast cancer is a key to the improved survival outcomes. The effects of delays in multimodality treatment in HER2 amplified breast cancer were studied. Patients and Methods: Of the patients with primary HER2 amplified breast cancer who were treated between 2009 and 2018 in a single institution, 1,075 patients met the inclusion criteria. The patterns of the multimodality treatments and the prognostic effects of treatment delays were studied. Kaplan-Meier method was used to estimate the relapse free survival (RFS) and overall survival (OS). Hazard ratio (HR), their 95% confidence interval (CI) and p value were computed using the Cox proportional-hazards model adjusting for 11 covariates including age, ethnicity, clinical T stage, clinical N stage, ER, PR, Ki67, LVI, histologic grade, surgery type (mastectomy versus lumpectomy), neoadjuvant versus adjuvant chemotherapy. The Harrell’s C statistics were used to determine the Cox model accuracy. Adjusted multivariate analyses of recurrence/death and death were computed using Cox proportional-hazards model. Delays in treatment were defined as starting treatment beyond 60 days after diagnosis or after the completion of the leading treatment. Results: Of the patients included, 49% received neoadjuvant chemotherapy/HER2 target treatment and surgery, 43% had adjuvant treatments and surgery and 8% had surgery only without chemotherapy. Timely commencement of treatment in the 3 groups were 85.7%, 72.1% and 78.4% respectively. The 5-year RFS was 88.7% and OS was 98.2%. Patients who received neoadjuvant and adjuvant chemotherapy were combined, and six treatment groups with delays in various treatments were compared: no delay in chemotherapy/target treatment and surgery (1); surgery delay (2); chemotherapy delay (3); delays in both treatments (4) and surgery only groups with (5) or without delays (6). Concordance statistics showed that the covariate adjusted Cox proportional-hazards model had a better accuracy than the unadjusted. Compared to those without delays, patients with both chemotherapy and surgery delayed had worse recurrence/death (adjusted HR = 4.11, 95% CI: 1.39-12.5, p= 0.0161). Delays in either surgery or chemotherapy also increased recurrence/death, although to a lesser magnitude. The adjusted death in surgery delay, chemotherapy delay and delays in both had HR 2.91; 2.22; and 2.29 respectively when compared to the group without delays in either treatment although not significant at p < 0.05. Adjusted recurrence/death and death were similar between neoadjuvant and adjuvant groups (HR 0.99 & HR 0.85 respectively). The group that received surgery only without chemotherapy was associated with worse recurrence/death (adjusted HR=1.78, 95% CI: 0.19 – 16.9) Conclusion: Delays in any treatment adversely impacted recurrence and death and chemotherapy/target treatment is a critical component in treating patients with operable HER2 positive breast cancer. Citation Format: Helena Chang, Jeffrey Gornbein, Sin Yee Lim. Prognostic Impact of Delaying Multimodality Treatment in HER2 Positive Breast Cancer [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 P4-07-28.

Abstract P2-01-06: Real-world outcome and cost analysis of the addition of pertuzumab to neoadjuvant therapy in localized HER2 positive breast cancer: a single center experience

Abstract BACKGROUND: Breast cancer is the most common cancer in woman and can be classified based on the expression of hormonal receptor as well as the human epidermal growth factor receptor 2 (HER2). HER2 amplification is associated with an aggressive clinical course and higher recurrence rates following curative intend surgery. For non-metastatic T2 or node-positive HER2-positive disease neoadjuvant treatment is favored. The National Comprehensive Cancer Network (NCCN) guidelines have endorsed the use of dual HER2 blockage with trastuzumab and pertuzumab combined with chemotherapy in the neoadjuvant setting. However, pertuzumab isn’t frequently reimbursed in public health care systems for this indication. Patients who don’t achieve a pathological complete response (pCR) at surgery are eligible for adjuvant T-DM1, adding significant side effects on patients and cost on the healthcare system. METHODS: We conducted a retrospective analysis of patients receiving anti-HER2 therapy in the neoadjuvant setting at the Jewish General hospital between 2015 and 2021. After 2019, pertuzumab was routinely added to standard neoadjuvant therapy enabling us to compare patients treated with or without dual-HER2 blockade. Our primary endpoint is the percentage of pCR at surgery. Secondary objectives are to estimate and compare the cost of anti-HER2 targeted therapy in the perioperative setting and side effect burden on patients. Statistical analyses were done using fisher exact test with statistical significance defined p value < 0.05 in a one-sided test. Drug cost was calculated using publicly available resources. RESULTS: We identified 83 patient who underwent neoadjuvant chemotherapy for HER2 amplified breast cancer. 44 patients received only trastuzumab has anti-HER2 therapy and 39 patients were treated with dual HER2 blockade containing pertuzumab. The addition of pertuzumab was associated with improved the pCR rate (67% vs. 27%; p = 0.0016). The increased pCR rate was observed in hormone-receptor positive and negative tumors. We also described a non-statistically significant trend in reduction in the requirement for axillary dissection with the use of pertuzumab (28% vs. 39%; P=0.2208). The increased in pCR rate with pertuzumab reduced the number of patients eligible for adjuvant T-DM1. If all patients with residual disease had received adjuvant T-DM1, the cost of neoadjuvant pertuzumab would be neutral, with a mean anti-HER2 drug cost of 65 150 CA$ in the pertuzumab-trastuzumab group and 66 116 CA$ in the trastuzumab group. CONCLUSION: Our real-world analysis confirmed that neoadjuvant chemotherapy with dual HER2-blockade was well tolerated and associated with increased the pCR rate compared to regimens containing trastuzumab only. This measure is neutral on drug cost by reducing the amount of patients eligible for adjuvant T-DM1. Further research is warranted to estimate the overall health-care utilization costs of neoadjuvant pertuzumab-trastuzumab in settings where adjuvant T-DM1 is available. Table 3: Pathologic complete response (pCR) and type of surgery in patients who received dual HER2 blockade with neoadjuvant pertuzumab-trastuzumab plus chemotherapy and in patients receiving trastuzumab only with chemotherapy at Jewish General Hospital between 2015 and 2021. ** p < 0,01, ns non-statistically significant Citation Format: Francois Panet, Matt Young, Stephanie Wong, Alice Dragomir, April A. N. Rose, Lawrence Panasci. Real-world outcome and cost analysis of the addition of pertuzumab to neoadjuvant therapy in localized HER2 positive breast cancer: a single center experience [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 P2-01-06.

Abstract P6-12-06: Neratinib vs. Trastuzumab plus Ribociclib in ERBB2-positive breast cancer: Preclinical mechanistic efficacy study

Abstract Background: Despite the wide-ranging clinical success of human epidermal growth factor receptor 2 (HER2)-directed therapies, many HER2-positive breast cancer patients eventually progress because of the development of primary or acquired resistance. PIK3CA is found often mutated in breast cancer (>30%, TCGA dataset) and responsible for HER2-directed treatment failure. Purpose: Inhibition of pan-tyrosine kinases of HER-receptors along with the blockade of estrogen receptor (ER) prevents the activation of downstream effectors and crosstalk between HER2 and ER, leading to tumor cell death. Similarly, the combined inhibition of HER2-receptor signaling with cell cycle arrest leads to efficient tumor cell apoptosis. Here, we evaluate the mechanistic efficacy and comparability of neratinib (N) (an irreversible pan-ERBB tyrosine kinase inhibitor) in combination with fulvestrant (F) against ribociclib (R) plus trastuzumab (T) in HER2-amplified breast cancer cells. Methods: HER2+ breast cancer cell lines with Rb- wild-type- BT474 (ER+), SKBR3 (ER-), and MDA-MB453 (ER-, PIK3CA mutated [H1047R]) were used for the study. Cells were treated with N+F (F added in ER+ cell line only) or R or T as a single-agent or combination and assessed for real-time proliferation, 3D ON-TOP assay, changes in mitochondrial potential, and apoptosis. Cells treated with R and/or T were additionally examined for cell cycle arrest and changes in CDK4 mRNA transcription. Baseline CCND1 mRNA transcripts were quantified by RT-qPCR. Immunohistochemistry was used to assess baseline BCL2 expression in HER2+ tumor microarrays (TMA). Western blot was used to evaluate the effects on key downstream signaling proteins in response to the above treatment or combination. Results: High CCND1 expression was found in HER2+ cell lines that show promise for CDK4/6 inhibition. High BCL2 expression was found in HER2+ TMA that confirmed the natural resistance to programmed cell death. BT474 and MDA-MB453 were highly sensitive to N (+F), and high growth inhibition was evident at lower doses (< 20 nM). SKBR3 was comparatively less sensitive to N monotherapy and required dosing >160 nM to induce marked cell death. BT474 and MDA-MB453 had pronounced cytostatic responses to R or T+R, while a moderate response was observed in SKBR3. Substantial reduction in CDK4 transcription was noted following R or R+T treatment in MDA-MB453/BT474 but not in SKBR3. Apoptosis assays confirmed enhanced cell death with the R+T combination in BT474 and MDA-MB453 but not in SKBR3. Inhibition of key downstream oncogenic signaling (p-AKT/p-S6RP/p-ERK) was more evident with N compared to R, T, or R+T combination. Attenuated p-Rb expression (Ser 780 & Ser 807/811) was detected in all cell lines from R administration, indicating interruption in cell cycle progression. Conclusion: Mechanistically, N+F was superior to T+R in terms of inhibition of HER2 and its downstream signaling in the HER2+/ER+ BC model. N+F induced significantly more apoptosis and inhibited cell proliferation compared to T+R. Additionally, N monotherapy was highly effective in HER2-amplified/ER-negative breast cancer cells with PIK3CA mutation. Citation Format: Nischal Koirala, Jennifer Aske, Xiaoqian Lin, Nandini Dey, Pradip De. Neratinib vs. Trastuzumab plus Ribociclib in ERBB2-positive breast cancer: Preclinical mechanistic efficacy study [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 P6-12-06.

Abstract OT1-14-01: Efficiency and safety of palbociclib in patients with locally advanced or metastatic breast cancer: a muti-center real world study

Abstract Endocrine therapy is the main treatment for premenopausal women with HR+/HER2- breast cancer. Palbociclib is an oral CDK4/6 inhibitor which preclinical evidence that ER+ and HER2-amplified breast cancer cell lines are most sensitive to CDK4/6 inhibition of proliferation and is used in vitro in combination with endocrine therapy showed better tumor suppressive effect. To evaluate the efficacy and safety of palbociclib in patients with locally advanced or metastatic breast cancer, we enrolled females with hormone receptor-positive breast cancer who treat with Palbociclib from July 2018 to March 2022. The study has so far included 267 patients in 4 centers. 32.83% patients were treated in first-line, 31.32% and 38.85% patients in second and third lines. 30.34% patients had hepatic metastasis. 35.47% patients were sensitive to previous endocrine therapy; 18.23% patients had primary resistance to endocrine therapy, while 46.31% patients had secondary resistance to endocrine therapy. The median PFS was 12.67 months (95% CI 11.51-13.92), and Median overall survival (OS) was not reached. Among all patients, the overall response rate (ORR) was 25.84%, and the disease control rate (DCR) was 78.62%. The main adverse events related to treatment were neutropenia (91.38%), white blood cell decreased (90.09%), anemia (43.78%), and thrombocytopenia (37.93%). The most common grade 3/4 adverse event was neutropenia (55.61%). 11.99% of patients had dose reductions. More patients still needed for further analyze. Citation Format: Yongmei Yin, Xiang huang, Wei Li, Xinyu Wu, Yijia Hua, Chunxiao Sun. Efficiency and safety of palbociclib in patients with locally advanced or metastatic breast cancer: a muti-center real world study [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 OT1-14-01.

Prognostic role of immune environment in luminal B early breast cancer.

The importance of the immune microenvironment in triple negative and HER2-amplified breast cancer (BC) is well-established; less is known about the immune environment in luminal breast cancers. We aimed to assess for the impact of immune biomarkers on BC outcome in a group of luminal B patients with archived tissue and annotated clinical information. Patients with early breast cancer (EBC) treated in a single institution over a 14-year period, with prospectively collected data were included. Luminal B EBC patients were identified and defined into three cohorts: A: grade 2 or 3, ER & PR positive, HER2-negative; B: Any grade, ER positive, PR and HER2-negative (Ki67 ≥ 14% in cohorts A & B); and C: Any grade, ER or PR positive, HER2-positive. Within each cohort, patients with a relapsed BC event (R) were compared on a 1:1 basis with a control patient (C) who remained disease-free, balanced for key characteristics in an effort to balance the contribution of each clinical group to outcome. Archival breast, involved and uninvolved axillary nodes were assessed by immunohistochemistry for biomarkers identifying effector and suppressor immune cells, and compared between R and C. In total, 120 patients were included (80, 22, and 18 patients in cohorts A, B, and C, respectively). R were 1.5 years older (p=0.016), with all other characteristics being balanced. Overall, there were no statistically significant differences in immune biomarkers in breast or nodal tissue of R and C. However, there was a trend toward higher levels of TILs in breast tumors of C, while GAL-9 was consistently expressed on lymphocytes and tumor cells in all breast and nodes of C and was absent from all tissues of R. These trends in checkpoint molecule expression deserve further research.

Interobserver Variation in the Assessment of Immunohistochemistry Expression Levels in HER2-Negative Breast Cancer: Can We Improve the Identification of Low Levels of HER2 Expression by Adjusting the Criteria? An International Interobserver Study

The classification of human epidermal growth factor receptor 2 (HER2) expression is optimized to detect HER2-amplified breast cancer (BC). However, novel HER2-targeting agents are also effective for BCs with low levels of HER2. This raises the question whether the current guidelines for HER2 testing are sufficiently reproducible to identify HER2-low BC. The aim of this multicenter international study was to assess the interobserver agreement of specific HER2 immunohistochemistry scores in cases with negative HER2 results (0, 1+, or 2+/in situ hybridization negative) according to the current American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. Furthermore, we evaluated whether the agreement improved by redefining immunohistochemistry (IHC) scoring criteria or by adding fluorescent in situ hybridization (FISH).We conducted a 2-round study of 105 nonamplified BCs. During the first assessment, 16 pathologists used the latest version of the ASCO/CAP guidelines. After a consensus meeting, the same pathologists scored the same digital slides using modified IHC scoring criteria based on the 2007 ASCO/CAP guidelines, and an extra “ultralow” category was added.Overall, the interobserver agreement was limited (4.7% of cases with 100% agreement) in the first round, but this was improved by clustering IHC categories. In the second round, the highest reproducibility was observed when comparing IHC 0 with the ultralow/1+/2+ grouped cluster (74.3% of cases with 100% agreement). The FISH results were not statistically different between HER2-0 and HER2-low cases, regardless of the IHC criteria used.In conclusion, our study suggests that the modified 2007 ASCO/CAP criteria were more reproducible in distinguishing HER2-0 from HER2-low cases than the 2018 ASCO/CAP criteria. However, the reproducibility was still moderate, which was not improved by adding FISH. This could lead to a suboptimal selection of patients eligible for novel HER2-targeting agents. If the threshold between HER2 IHC 0 and 1+ is to be clinically actionable, there is a need for clearer, more reproducible IHC definitions, training, and/or development of more accurate methods to detect this subtle difference in protein expression levels.