Survival In HER2 Research Articles

DUSP6 inhibition overcomes neuregulin/HER3-driven therapy tolerance in HER2+ breast cancer

Despite clinical benefits of tyrosine kinase inhibitors (TKIs) in cancer, most tumors can reactivate proliferation under TKI therapy. Here we present transcriptional profiling of HER2+ breast cancer cells transitioning from dormant drug tolerant cells to re-proliferating cells under continuous HER2 inhibitor (HER2i) therapy. Focusing on phosphatases, expression of dual-specificity phosphatase DUSP6 was found inhibited in dormant cells, but strongly induced upon regrowth. DUSP6 expression also selectively associated with poor patient survival in HER2+ breast cancers. DUSP6 overexpression conferred apoptosis resistance, whereas its pharmacological blockade prevented therapy tolerance development under HER2i therapy. DUSP6 targeting also synergized with clinically used HER2i combination therapies. Mechanistically DUSP6 is a positive regulator of HER3 expression, and its impact on HER2i tolerance was mediated by neuregulin-HER3 axis. In vivo, genetic targeting of DUSP6 reduced tumor growth in brain metastasis model, whereas its pharmacological targeting induced synthetic lethal therapeutic effect in combination with HER2i. Collectively this work demonstrates that DUSP6 drives escape from HER2i-induced dormancy, and that DUSP6 is a druggable target to overcome HER3-driven TKI resistance.

Abstract 7660: L-Arginine metabolism is of prognostic relevance in women with breast cancer, but varies considerably between subtypes in vitro - A bedside-to-bench translational approach

Abstract Availability of the semi-essential amino acid L-arginine (L-arg) is limiting for proliferation in highly proliferative tissues. L-Arg is also a substrate for nitric oxide synthases (NOS) and arginases (ARG) 1 and 2. Methylation of L-arg residues within proteins by protein L-arg methyltransferases (PRMTs) leads to asymmetric (ADMA) and symmetric dimethylarginine (SDMA). Protein L-arg methylation is an important post-translational mechanism to regulate gene expression and cell cycle control. We measured L-arg and its metabolites in a prospective cohort of patients with primary breast cancer (BC). We further measured these metabolites and expression of genes involved in L-arg metabolic pathways in cell lines representing intrinsic BC subtypes. Plasma L-arg-related metabolite concentrations from 243 women with primary BC were associated with total mortality and disease recurrence during a median follow-up of 88 (IQR, 82-93) months. MCF-7, BT-474, SK-BR-3, and MDA-MB-468 cells representing ER-positive, HER2-positive, and triple negative BC were cultured in standard conditions and compared to MCF-12A normal breast epithelial cells. The plasma (in vivo) and intracellular concentrations (in vitro) of L-arg, L-ornithine (L-orn), L-citrulline (L-cit), ADMA, and SDMA were measured by UPLC-MS/MS. mRNA expression was quantified by qRT-PCR and protein expression was analyzed by Western Blot. In vivo, the plasma concentrations of all L-arg metabolites differed significantly between BC patients and healthy controls. L-Arg and ADMA were significantly higher in patients with recurrent disease and in those who died during follow up. ADMA was significantly associated with total mortality and recurrent disease in Luminal A patients; low L-orn/L-arg ratio was associated with survival in HER2+ BC patients, and low L-cit was associated with survival in triple negative BC. With the exception of MCF-7 cells, DDAH1 and DDAH2 mRNA concentrations in vitro were significantly higher in BC cells than in MCF-12A cells (DDAH1: 32-44 fold, DDAH2: 1.7-4.2 fold; p<0.05). These differences were mirrored by DDAH1 and DDAH2 protein concentrations. By contrast, MCF-7 cells were characterized by low DDAH1 and DDAH2, but high PRMT4 and PRMT6 mRNA expression and high L-arg content. BT-474 cells showed high ARG2 expression and high L-arg and L-orn concentrations, and MDA-MB-468 cells had the highest L-cit/L-arg ratio. In conclusion, regulation of L-arg metabolic pathways shows a complex and differential pattern between intrinsic BC subtypes, which is mirrored in representative cell lines in vitro. ADMA is a prognostic biomarker in Luminal A breast cancer patients; its metabolizing enzyme, DDAH, is highly overexpressed in BC cells in vitro. Thus, fingerprinting of L-arg metabolism may offer novel personalized treatment options within intrinsic BC subtypes. Citation Format: Juliane Hannemann, Leticia Oliveira Ferrer, Antonia Röglin, Fiona Kleinsang, Volkmar Müller, Isabell Witzel, Rainer Böger. L-Arginine metabolism is of prognostic relevance in women with breast cancer, but varies considerably between subtypes in vitro - A bedside-to-bench translational approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7660.

Comprehensive Clinical Characterization of Decade-Long Survivors of Metastatic Breast Cancer.

Elucidating the clinical features of metastatic breast cancer (MBC) patients with an exceptionally favorable prognosis may offer insights to improve the survival of more typical patients. We collected comprehensive real-world data on clinicopathologic characteristics, treatments, and outcomes of 110 consecutive MBC patients who survived for over ten years from the clinical data warehouse of Samsung Medical Center. The cohort included 54 hormone receptor (HR)-positive/HER2-negative (HR+/HER2-), 21 HR+/HER2+, 16 HR-/HER2+, and 14 triple-negative breast cancer (TNBC) patients. The median age at MBC diagnosis was 48.5 years. Approximately 70% of patients initially had a single-organ metastasis. The most common site of metastasis was the lung (46.4%), followed by distant lymph nodes (37.3%). During a median follow-up of 14.6 years, the median duration of systemic therapy was 11, 8.4, 7.3, and 0.8 years in the HR+/HER2-, HR+/HER2+, HR-/HER2+, and TNBC subgroups, respectively. Seven HER2+ and ten TNBC patients received systemic treatment for less than two years and remained treatment-free for most of the follow-up period, suggesting a potential chance of cure. The TNBC subtype (p < 0.001) and local treatment with curative intent within 1 year of MBC diagnosis (p = 0.002) were significantly associated with long-term treatment-free survival. The survival of HER2+ MBC and TNBC patients, but not that of HR+/HER2- patients, plateaued approximately 13 years after MBC diagnosis. A small subset of patients with HER2+ MBC and metastatic TNBC may be curable with multimodality therapy. Prospective studies integrating clinical and genomic data may identify unique clinicogenomic features of MBC patients who can achieve durable disease control without prolonged chemotherapy.

Role of plasma circulating HER2/EFTUD2 ratio in predicting pathological complete response (pCR) after neoadjuvant dual anti-HER2 therapy in HER2-positive breast cancer (HER2+ BC).

e12592 Background: Achieving pCR following neoadjuvant chemo and anti-HER2 therapy is associated with a significantly better survival in HER2+ BC. There is currently a lack of a robust biomarker to predict pCR in HER2+ BC. Using EFTUD2 as chromosome 17 reference probe, circulating HER2/EFTUD2 plasma DNA copy number ratio (H-ratio) detected by droplet digital PCR (ddPCR) was shown to have high concordance with the tumor HER2 status. We aim to evaluate whether the change in H-ratio is associated with response to neoadjuvant dual anti-HER2 therapy. Methods: We prospectively recruited patients with HER2+ BC who received neoadjuvant taxane, trastuzumab and pertuzumab followed by radical surgery from May 2019 to April 2022. Serial plasma samples (n = 49) were collected at pre-treatment (Tpre), at 4th cycle (Tmid) and at completion of neoadjuvant treatment (Tpost). H-ratio was determined in each plasma sample using droplet ddPCR (QX200 ddPCR system, Bio-rad). H-ratio responders were defined as patients having a declining Tpost H-ratio, whereas patients having a rising or persistent Tpost H-ratio were defined as H-ratio non-responders. The relationship between pCR, H-ratio and various clinicopathological characteristics were evaluated by Spearman’s correlation, Fisher’s exact test and Wilcoxon signed-rank test. Results: Eighteen clinical stage II or III HER2+ BC patients with a median age of 56 years old (range 33 - 71) were included. The median H-ratio at Tpre, Tmid and Tpost were 1.27, 1.12 and 1.10, respectively. Higher Tpre H-ratio was significantly associated with larger tumor size (p = 0.004) and higher tumor grade (G1-2 vs G3; median H-ratio 1.15 vs 1.78; p = 0.024). Six patients (33.3%) were H-ratio responders. Ten (55.6%) of 18 patients achieved pCR after completion of neoadjuvant treatment. The pCR rate in H-ratio responders was significantly higher than the H-ratio non-responders (100% vs. 36.4%; p = 0.017). In predicting pCR, H-ratio response outperformed hormonal receptor (HR) negativity (HR- vs HR+; 72.7% vs 28.6%; p = 0.088) or high tumor grade (G1-2 vs G3; 66.7% vs 33.3%; p = 0.201). There was no association between pCR and Tpre/Tmid/Tpost H-ratio. With a median follow up of 22.8 months (range 8.2 - 41.8), there were no relapse or death. Conclusions: A high pre-treatment plasma circulating HER2/EFTUD2 ratio is associated with large tumor size and high tumor grade in HER2+ BC. The decline of HER2/EFTUD2 ratio after neoadjuvant dual anti-HER2 therapy predicts pCR, and may serve as a potential biomarker for treatment de-escalation.

Is HER2 expression related to CDKi efficacy in HER2-negative luminal metastatic breast cancer?

e13073 Background: Cyclin kinase inhibitors (CDKi) are the standard of care in the first line of luminal (Lum) metastatic breast cancer (MBC). HER2 overexpression is a classic risk factor in BC, however, the use of targeted therapy has changed the prognosis of this disease, even with anti-HER2 efficacy data in cases of low HER2 expression. We wonder if the level of HER2 expression can be a predictor of efficacy in treatment with CDKi. Methods: Descriptive, observational, retrospective and analytical study of patients with MBC treated with CDKi, in the period 2018-2022. Descriptive and analytical statistical analysis (IBM SPSS Statistics 22) using logistic regression and Kaplan-Meier with long-rank test to analyze the relationship between CKi efficacy and HER2 expression (negative = 0+; HER2 low = 1+ and 2 + with SISH negative). Results: We analyzed 152 patients (p) with Lum MBC under treatment with CDKi (median 58 years), in first and successive lines. HER2 expression: 31% 46p (0+), 54% 81p (1+) and 14% 21p (2+ and SISH negative). 41% were treated with palbociclib, 34% with ribociclib, and 26% with abemaciclib. Radiological response (RR) to CDKi was achieved by 34.1% (CRR 2.4%, PRR 31.7), and the rest stable disease (SD) or progressive disease (PD): SD 42% and PD 22%. In the overall OR, there was no evidence of a risk association between the response to CDKi and the level of HER2 expression. We obtained similar results in the analysis stratified by type of CDKi. In the multivariate study, for the variables CDKi type and HER2 expression, the logistic regression model was not statistically significant either. Regarding the Kaplan-Meier analysis, we compared the median PFS and OS using the Long-rank test, based on the expression of HER2. In the complete series we did not find statistically significant differences between the curves. In the palbociclib subgroup the curves split in favor of HER2 0+ vs. HER2 low: 44 months 95%CI (7.6-80) vs 16 months 95%CI (7.9-24); p = 0.01. Regarding OS, we found no significant differences. With ribociclib, the curves separate in favor of HER2 low versus HER2 0+: 33 months 95%CI (7.2-18) vs. 13 months 95%CI (23-42); p = 0.03. In OS we did not find significant differences either. In the subgroup with abemaciclib, we observed a trend towards greater survival in HER2 0+ patients, but with no significant difference between the PFS or OS curves. Conclusions: Based on our study, the expression of HER2 could be considered a predictor of efficacy depending on the CDKi used. We assume greater survival with palbociclib in HER2 negative patients (0+) and with ribociclib in HER2 low patients. We consider it essential to develop studies with a larger sample size and clinical trials that allow us to demonstrate the hypothesis of our work with scientific evidence.

Phase I study of adoptive T cell therapy following HER2-pulsed dendritic cell vaccine and pepinemab/trastuzumab in patients with metastatic HER2-positive breast cancer (MBC).

TPS1113 Background: Despite major improvement of overall survival of HER2+ MBC with effective HER2 targeted therapies, many patients experience significant toxicities and develop progressive disease during treatment. Therefore, new and more effective therapeutic options are needed. This novel approach will evaluate whether the combination of three immunotherapies in addition to trastuzumab: dendritic cell (DC) vaccination, anti-SEMA4D blocking antibody (pepinemab) and CD4+ T cell adoptive transfer can lead to improved outcomes for patients with MBC refractory to HER2-targeted agents. BC have been considered as immunologically cold which is attributed to immune evasion and suppression of host effector immune cells homing into tumor bed. Progressive loss of Th1 immunity against HER2 oncodriver correlates with poor prognosis. HER2 peptide pulsed type I dendritic cells (HER2-DC1) restored anti-HER2 CD4+ Th1 immune response and improved pathologic complete response (pCR) in HER2+ BC. Antibodies to SEMA4D have been shown to modulate the TME by increasing effector cell infiltration and reducing immunosuppression. In preclinical studies, treatment with anti-SEMA4D and HER2-DC1 in mice bearing established HER2+ tumors improved DC homing, expansion of CD4+ T cells, and complete tumor regression, compared to treatment with anti-SEMA4D or HER2-DC1 alone. Further, subsequent expansion and adoptive transfer of CD4+ T cells induced synergistic anti-tumor activity by activating CD8+ T mediated cytotoxicity. Pepinemab was well-tolerated and showed signs of anti-tumor activity in in immunotherapy-resistant and PD-L1 negative/low non-small cell lung cancer patients when combined with checkpoint inhibitor (avelumab). Methods: This open label Phase 1 study is enrolling up to 28 patients with HER2+ MBC. Patients will be treated with 6 weekly injections of dendritic cell (DC1) vaccines in combination with trastuzumab and pepinemab. We hypothesize these therapies may elicit CD4+ HER2-specific T cell responses. HER2-specific T cells will be expanded ex vivo and subsequently infused to patients following lymphodepletion with cyclophosphamide. Trastuzumab and pepinemab will be given as maintenance in addition to booster DC1 vaccines. Patients (ECOG 0,1) must have had disease progression while on trastuzumab for the treatment of HER2+ MBC and received no more than 3 lines of chemotherapy in the setting of metastatic disease. Dose escalation will consist of 3-6 patients each with increasing amounts of transferred CD4+ T cells, followed by dose expansion of 10 patients at the MTD. The primary objective is safety and tolerability; secondary objectives will include evaluation of T cell immunity and immune subsets, efficacy, PK/PD/ADA of pepinemab, and biomarker assessments. Clinical trial information: NCT05378464 .

Abstract 2032: Intratumoral tumor infiltrating lymphocytes is associated with cell proliferation and better survival but not with response to chemotherapy

Abstract Introduction: Abundance of tumor infiltrated lymphocytes (TILs) in the peripheral region of tumor assessed pathologically is associated with improved response to chemotherapy and better prognosis in Her2+ and triple-negative breast cancer (TNBC). Since recent progress in bioinformatics has allowed for computational deconvolution of bulk tumor, we investigated the clinical relevance of intratumoral TILs in primary breast cancer and response to neoadjuvant chemotherapy (NAC). Methods: A total of 4139 bulk tumor RNA sequencing data were analyzed for the association of high intratumoral TILs with clinicopathologic factors and biology in primary breast cancer cohorts of TCGA and SCAN-B. Total of 1719 bulk samples from three other cohorts (GSE163882, GSE25066, and GSE194040) were utilized to investigate the association between pathologic complete response rate (pCR) to neoadjuvant chemotherapy and intratumoral TILs. Results: Among several deconvolution algorithms, the sum of xCell lymphocytes was chosen to estimate intratumoral TIL since it correlated the strongest with multiple signals that represent lymphocytes. The cohorts were divided into high or low TIL tumor by median cut-off, and number of TILs in high-TIL ER+/Her2- was roughly the same as that of low-TIL TNBC. As expected, high TILs were associated with better disease specific survival in Her2+ and TNBC subtypes consistently in TCGA and SCAN-B cohorts. Immune-related gene sets were uniformly enriched to high TIL tumors regardless of subtype, and the difference in cytolytic activity by TILs were the largest in TNBC. Macrophages, dendritic cells, lymphatic and blood endothelial cells were infiltrated in TIL high tumors. Thus, estimated intra-tumor TILS showed similar immune-related characteristics with pathology-assessed TILs. However, ER+/Her2- was most strongly correlated with cell proliferation biologically, pathologically, and molecular biologically. High TIL was significantly associated with higher mutation related scores only in ER+HER2-. Unexpectedly, intratumoral TILs and all the types of lymphocytes showed low AUC for predicting pCR to NAC (AUC &amp;lt; 0.6). Gene expression-based scores generated by LASSO model showed low AUC in all the cohorts neither (AUC &amp;lt; 0.65). Conclusion: Transcriptomic signature of Intratumoral TILs is associated with cell proliferation in ER+HER2- breast cancer and better survival in HER2+ breast cancer and TNBC but is not as consistently associated with response to neoadjuvant chemotherapy as pathologically assessed TILs. Citation Format: Rongrong Wu, Masanori Oshi, Mariko Asaoka, Masayuki Nagahashi, Li Yan, Yasuo Miyoshi, Takashi Ishikawa, Kazuaki Takabe. Intratumoral tumor infiltrating lymphocytes is associated with cell proliferation and better survival but not with response to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2032.

Abstract P3-06-01: Unleashing NK- and CD8 T cells by combining monalizumab (anti-NKG2A) and trastuzumab for metastatic HER2+ breast cancer: first results MIMOSA trial

Abstract Background Although treatment options and survival of HER2+ metastatic breast cancer (MBC) patients have greatly improved, the majority of MBC-patients still die of this disease. The relatively high levels of TILs observed in this BC subtype provide a rationale for immunomodulatory strategies, however, PD1-blockade has only shown modest response rates in this setting. While PD-1 blockade mainly acts on T cells, monalizumab targets the inhibitory immune checkpoint NKG2A which interacts with HLA-E on tumor cells, thereby unleashing NK- as well as CD8 T cells. We hypothesize that monalizumab can promote antibody-dependent cell-mediated cytotoxicity (ADCC) which is critical for trastuzumab efficacy. Clinical activity was shown in patients with head and neck cancer when monalizumab was combined with cetuximab (anti-EGFR). Here, we present the first results of the MIMOSA-trial investigating the efficacy of the novel combination monalizumab and trastuzumab in patients with HER2+ MBC. Methods In the phase II MIMOSA-trial (NTC04307329), HER2+ MBC patients were treated biweekly with 4mg/kg trastuzumab and 750mg monalizumab. Key eligibility criteria were progressive disease despite anti-HER2 therapy, had received a minimum of one and a maximum of three lines of palliative chemotherapy, had measurable disease according to RECIST1.1, and a serum LDH-level below 500U/L. Primary endpoint was objective response rate according to RECIST1.1. Secondary endpoints included clinical benefit rate (complete response CR, partial response PR or stable disease SD for at least 6 months) according to RECIST1.1, progression-free survival, overall survival and safety. Dose-limiting toxicities were continuously monitored throughout the trial and evaluated using a pre-defined Pocock-type boundary rule. Following a Simon’s two-stage design, 11 patients were included in stage I of the trial. If two or more responders were observed, further exploration is warranted in stage II. Results Between January 2021 and April 2022, eleven women of which ten are currently evaluable were enrolled in the trial. Patients received a median of two lines of prior treatment for MBC, of which 6 out of 11 patients were treated with trastuzumab emtansine (T-DM1). The majority of patients had hormone receptor positive BC (72% of the patients) and had low levels of tumor-infiltrating lymphocytes (TILs) with a median of 1% (ranging from 1% to 20%). Patients received a median of four cycles of trastuzumab and monalizumab. Treatment was well tolerated with no dose-limiting toxicities. No objective responses were observed in the first ten out of eleven evaluable patients. Therefore, MIMOSA stage I did not meet its primary endpoint, leading to discontinuation of the trial. Conclusions The novel combination of trastuzumab and monalizumab did not induce objective responses in heavily pre-treated HER2+ MBC patients. Citation Format: Veerle Geurts, Leonie Voorwerk, Karolina Sikorska, Roberto Salgado, Koen van de Vijver, Marloes van Dongen, Inge Kemper, Ingrid A. Mandjes, Martine Heuver-mes, John Haanen, Gabe S. Sonke, Hugo Horlings, Marleen Kok. Unleashing NK- and CD8 T cells by combining monalizumab (anti-NKG2A) and trastuzumab for metastatic HER2+ breast cancer: first results MIMOSA trial [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 P3-06-01.

Abstract HER2-04: HER2-04 Prevalence of HER2-low among Metastatic Breast Cancer Patients and Their Outcomes Compared to HER2 IHC 0

Abstract Prevalence of HER2-low among Metastatic Breast Cancer Patients and Their Outcomes Compared to HER2 IHC 0 Background: Amplification and/or overexpression of the HER2 gene is detected in approximately 15% to 20% of breast cancer (BC) patients. HER2-targeted therapies improve survival in HER2+ BC. Current HER2 diagnostic tests and thresholds were designed and optimized to predict benefit from HER2-directed therapies. The DESTINY-Breast04 trial demonstrated notable efficacy of HER2 antibody-drug conjugate trastuzumab deruxtecan, in patients whose tumors are not conventionally HER2+, but defined as HER2-low (IHC 1 or 2+ without HER2 amplification). A better understanding of the HER2-low phenotype is therefore of critical importance. Methods: Eligible pts were adults with metastatic HER2 IHC (0,1+,2+/FISH negative) BC seen at MD Anderson between 2006 and 2019. Primary biopsy of the breast was used to test biomarkers. HER2 low was defined as IHC 1+ or 2+ and FISH-negative. HER2-negative was defined as HER2 IHC 0. Multi-covariate logistic regression models were used to evaluate effect of clinical factors on HER2 low status and pCR. Overall survival (OS) was defined as time from diagnosis date until death. Disease free survival (DFS) was defined as time from definitive surgery date until the first local/distant recurrence or death from any cause. Patients alive without events (death for OS, recurrence/death for DFS) were censored at last follow-up date. OS and DFS times were estimated by Kaplan-Meier method. Multivariate Cox proportional hazards regression models were applied to assess effect of covariates of interest on OS and DFS. Results: A total of 3053 early stage and 1203 de novo female BC patients were included in the analysis. The prevalence of HER2 low for early stage patients was 59.3% (1811/3053) and for de novo metastatic disease 59.3% (713/1203). In early stage patients, white race, higher nuclear grade and positive ER status were significantly associated with HER2 low status in multicovariate logistic regression. Univariately, pCR rate was significantly associated with negative ER (10.2% in negative vs 2.7% in positive), negative PR (8.6% vs 2%) and negative lymphatic vascular invasion (9.2% vs 2%). Multicovariate logistic regression showed ER (p=0.0124), PR (p=0.0439) status and lymphatic vascular invasion (p&amp;lt; 0.0001) were significantly associated with pCR status. With median follow-up of 8.5 years, median OS time was 5.3 years (95% CI: [4.9, 5.4]), 5.4 years and 4.8 years in HER2 low and HER2 0 groups, respectively. In multicovariate Cox regression, HER2 low was significantly associated with longer OS (HR=0.87, p=0.008), adjusted for age, race, stage, nuclear grade, lymphatic-vascular invasion and ER/PR status. In patients who received neoadjuvant chemotherapy, adjusted for stage, nuclear grade, ER and PR status and pCR, HER2 low was significantly associated with a longer OS time (HR=0.87, p=0.04). Median DFS time was 1.9 years (95% CI: (1.8, 2.0)). Age, stage, histology, nuclear grade, and ER and PR status were significantly associated with DFS by multicovariate Cox regression. Among de novo cases, higher nuclear grade (HR [II vs I]=1.838, p =0.008; HR [III vs I]=1.856, p=0.007) and positive ER status (OR=1.933, p &amp;lt;.0001) were associated with high percentage of HER2 low by multicovariate logistic regression. Median OS time was 3.2 years (95% CI: (3.0, 3.5)). By multicovariate Cox regression, race, histology, nuclear grade, ER and PR status, and HER2 low status (HR=0.834, p=0.0260) were significantly associated with OS time. Conclusions: In metastatic BC pts, HER2 low status was significantly associated with a longer OS and DFS when compared to HER2 0. Nuclear grade and ER positivity was significantly associated with HER2 low status. Biomarkers on recurrence tumor will be presented. This real world data helps to establish the prevalence of HER2 low and their outcomes for this selective cohort. Citation Format: Akshara Singareeka Raghavendra, Diane Liu, Jason Mouabbi, Debu Tripathy. HER2-04 Prevalence of HER2-low among Metastatic Breast Cancer Patients and Their Outcomes Compared to HER2 IHC 0 [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 HER2-04.

Abstract OT1-10-02: Trial of Neratinib Plus Capecitabine in Subjects With HER2-Negative Metastatic Breast Cancer With Brain Metastases and Abnormally Active HER2 Signaling

Abstract Background: Development of brain metastasis (BM) portends poor prognosis in patients (pts) with metastatic breast cancer (MBC) mainly due to lack of systemic therapy with strong activity in CNS. While survival in HER2+ BC BMs has improved due to development of various HER2 therapies with activity against BM, outcome for HER2- BC BMs remains dismal. A novel assay (CELsignia) which measures underlying HER2 signal (HS) of live cancer cells in response to HER2 agonists &amp; antagonists showed that about 25% of BC deemed HER2- by standard methods have underlying abnormal HS. Preclinical data showed various HER2 therapies inhibit tumor growth of such HER2- BC with abnormal HS. Among tested HER2 inhibitors with known CNS activity, neratinib had the lowest IC50. We propose a trial which identifies HER2- BC BM pts with abnormal HS &amp; assesses activity of neratinib based therapy. Design: This is a phase II single arm study with 2 steps- step 0 (biopsy) &amp; step 1 (treatment). Pts eligible for Step 0 will be registered as “Immediate Treatment (IT)” (intent to register to Step 1 immediately) or “Future Treatment” [registration can be delayed up to 28 weeks (wks)] &amp; undergo biopsy of most accessible extra CNS (eCNS) tumor. Tumor will be sent for CELsignia testing (2 wks turn around). Additional biopsies may be obtained for standard of care (SOC). IT pts can be treated with a brief course of capecitabine 1gm/m2 BID for 1 wk followed by 1 wk off while waiting for CELsigia result. Pts with BC having abnormal HS will be screened &amp; registered to step 1. Enrolled pts will receive neratinib 240mg daily (with SOC antidiarrheal prophylaxis) + capecitabine 750mg/m2 BID for 2 wks in every 3 wks cycle (C). Tumor assessments will be done prior to C3, 5 &amp; 7 then every 12 wks with MRI brain, CT chest abdomen pelvis &amp; bone scan using RECIST1.1 (eCNS disease) and a modified BM RANO criteria (&amp;gt;5mm, measurable disease, CNS). In isolated CNS progression, investigators have option of treatment beyond progression after SOC local treatment of progressing BMs. Key Eligibility Criteria: Step 0: Inclusion Criteria (IC): histologically confirmed HER2- BC (primary or metastatic); has radiological evidence of BM; prior treatment with CDK4/6 inhibitor + endocrine therapy required for hormone receptor+ BC, no prior specific treatment required for triple negative BC; radiological evidence of eCNS measurable disease (RECIST1.1) accessible for biopsy. Exclusion Criteria (EC): prior use of capecitabine in metastatic setting; known/suspected leptomeningeal disease. Step 1: IC: Abnormal HS; New or progressing BM that is measurable (&amp;gt;5mm); minimum washout periods in wks: last chemotherapy 2, hormonal therapy 1 except fulvestrant 4, targeted therapies 3, eCNS radiation 1, any investigational treatment 4. ECOG performance status 0-2; adequate end organ functions. EC: whole brain radiation in last 3 months (m). Endpoints: Co-primary: Overall survival (OS) and CNS progression free survival (CNS PFS). Key Secondary: Objective response rate, clinical benefit rate and duration of response (CNS using BM RANO, eCNS using RECIST1.1), eCNS PFS, various feasibility endpoints. Safety. Statistical methods: To detect 70% improvement in this trial in CNS PFS and OS compared to historical control (BEACON trial BM subset) 2.7m vs. 4.6m and 4.8m vs. 8.2 m with 80% power and 1-sided 5% significance level, a sample size of 22 is required. About 88 patients will undergo biopsy to enroll 22 pts in step 1. One futility assessment is planned when the first 12 pts have been treated for at least 6 wks or have failed (died or progressed) prior to 6 wks. If 9 or more in the first 12 have died or progressed by 6 wks of treatment, the study will close for futility. PFS and OS will be analyzed using Kaplan-Meier survival functions. Contact: Ajay Dhakal MBBS, ajay_dhakal@urmc.rochester.edu NCT#: NCT04965064 Funding: Celcuity, Puma Biotechnology Status: IRB approved. Anticipated activation Aug 2022, Accrual duration: 2 years. Seeking participating sites. Citation Format: Ajay Dhakal, Ruth O’Regan, Carla Falkson, David Hicks, Douglas Hawkins, Bradley Turner, Nimish Mohile. Trial of Neratinib Plus Capecitabine in Subjects With HER2-Negative Metastatic Breast Cancer With Brain Metastases and Abnormally Active HER2 Signaling [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 OT1-10-02.

Suppression of Platelet-Derived Growth Factor Receptor-Alpha Overcomes Resistance to Trastuzumab through STAT3-Dependent IL-6 Reduction in HER2-Positive Breast Cancer Cells

Platelet-derived growth factor receptor (PDGFR) plays an essential role in the proliferation and invasion of malignant cancer cells. However, the functional role of PDGFR alpha (PDGFRA) in HER2-positive (HER2+) breast cancer has not been fully clarified yet. Thus, the objective of this study was to investigate the clinical significance of PDGFRA and the therapeutic potential of PDGFR inhibitors as part of an effort to overcome trastuzumab (TRZ) resistance. Aberrant PDGFRA expression is closely associated with decreased survival in HER2+ breast cancers. Therefore, we established BT474 trastuzumab-sensitive (TRZ_S) and trastuzumab-resistant (TRZ_R) cells to investigate the association between PDGFR signaling and TRZ resistance. We found that PDGFRA was significantly upregulated in the BT474 TRZ_R cells. In addition, IL-6 expression, which was also found to be upregulated in the TRZ_R cells, was induced by PDGFC, a ligand of PDGFR. Next, we investigated the effects of ponatinib and sunitinib, PDGFR inhibitors, on the BT474 TRZ_R and HCC1954 (TRZ-resistant cell line) cells. These inhibitors decreased cell viability and migration in a dose-dependent manner. Additionally, IL-6 expression was decreased by ponatinib in both the BT474 TRZ_R and HCC1954 cells. In contrast, IL-6 was not suppressed by TRZ, implying that the PDGFRA/STAT3/IL-6 axis is associated with resistance to TRZ. In addition, we found that STAT3 and ERK phosphorylation were increased in the BT474 TRZ_R cells. IL-6 expression was suppressed by a STAT3 inhibitor, indicating that IL-6 expression is modulated downstream of STAT3. Taken together, these results suggest that PDGFRA could serve as a therapeutic target to overcome TRZ resistance.

Dishevelled 2 regulates cancer cell proliferation and T cell mediated immunity in HER2-positive breast cancer

BackgroundDishevelled paralogs (DVL1, 2, 3) are key mediators of Wnt pathway playing a role in constitutive oncogenic signaling influencing the tumor microenvironment. While previous studies showed correlation of β-catenin with T cell gene expression, little is known about the role of DVL2 in modulating tumor immunity. This study aimed to uncover the novel interaction between DVL2 and HER2-positive (HER2+) breast cancer (BC) in regulating tumor immunity and disease progression.MethodsDVL2 loss of function studies were performed with or without a clinically approved HER2 inhibitor, Neratinib in two different HER2+ BC cell lines. We analyzed RNA (RT-qPCR) and protein (western blot) expression of classic Wnt markers and performed cell proliferation and cell cycle analyses by live cell imaging and flow cytometry, respectively. A pilot study in 24 HER2+ BC patients was performed to dissect the role of DVL2 in tumor immunity. Retrospective chart review on patient records and banked tissue histology were performed. Data were analyzed in SPSS (version 25) and GraphPad Prism (version 7) at a significance p < 0.05.ResultsDVL2 regulates the transcription of immune modulatory genes involved in antigen presentation and T cell maintenance. DVL2 loss of function down regulated mRNA expression of Wnt target genes involved in cell proliferation, migration, invasion in HER2+ BC cell lines (±Neratinib). Similarly, live cell proliferation and cell cycle analyses reveal that DVL2 knockdown (±Neratinib) resulted in reduced proliferation, higher growth arrest (G1), limited mitosis (G2/M) compared to non-targeted control in one of the two cell lines used. Analyses on patient tissues who received neoadjuvant chemotherapy (n = 14) further demonstrate that higher DVL2 expression at baseline biopsy pose a significant negative correlation with % CD8α levels (r = − 0.67, p < 0.05) while have a positive correlation with NLR (r = 0.58, p < 0.05), where high NLR denotes worse cancer prognosis. These results from our pilot study reveal interesting roles of DVL2 proteins in regulating tumor immune microenvironment and clinical predictors of survival in HER2+ BC.ConclusionOur study demonstrates potential immune regulatory role of DVL2 proteins in HER2+ BC. More in-depth mechanistic studies of DVL paralogs and their influence on anti-tumor immunity may provide insight into DVLs as potential therapeutic targets benefiting BC patients.

CTTN Overexpression Confers Cancer Stem Cell-like Properties and Trastuzumab Resistance via DKK-1/WNT Signaling in HER2 Positive Breast Cancer

Despite the therapeutic success of trastuzumab, HER2 positive (HER2+) breast cancer patients continue to face significant difficulties due to innate or acquired drug resistance. In this study we explored the potential role of CTTN in inducing trastuzumab resistance of HER2+ breast cancers. Genetic changes of CTTN and survival of HER2+ breast cancer patients were analyzed in multiple breast cancer patient cohorts (METABRIC, TCGA, Kaplan-Meier (KM) plotter, and Hanyang University cohort). The effect of CTTN on cancer stem cell activity was assessed using the tumorsphere formation, ALDEFLUOR assay, and by in vivo xenograft experiments. CTTN-induced trastuzumab resistance was assessed by the sulforhodamine B (SRB) assay, colony formation assays, and in vivo xenograft model. RNA-seq analysis was used to clarify the mechanism of trastuzumab resistance conferred by CTTN. Survival analysis indicated that CTTN overexpression is related to a poor prognosis in HER2+ breast cancers (OS, p = 0.05 in the Hanyang University cohort; OS, p = 0.0014 in KM plotter; OS, p = 0.008 and DFS, p = 0.010 in METABRIC). CTTN overexpression-induced cancer stem cell-like characteristics in experiments of tumorsphere formation, ALDEFLUOR assays, and in vivo limiting dilution assays. CTTN overexpression resulted in trastuzumab resistance in SRB, colony formation assays, and in vivo xenograft models. Mechanistically, the mRNA and protein levels of DKK-1, a Wnt antagonist, were downregulated by CTTN. Treatment of the β-catenin/TCF inhibitor reversed CTTN-induced cancer stem cell-like properties in vitro. Combination treatment with trastuzumab and β-catenin/TCF inhibitor overcame trastuzumab resistance conferred by CTTN overexpression in in vitro colony formation assays. CTTN activates DKK-1/Wnt/β-catenin signaling to induce trastuzumab resistance. We propose that CTTN is a novel biomarker indicating a poor prognosis and a possible therapeutic target for overcoming trastuzumab resistance.

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

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

HER2-positive invasive lobular carcinoma: a rare breast cancer which may not necessarily require anti-HER2 therapy. A population-based study.

HER2-positive (HER2 +) invasive lobular breast cancer (ILC) is rare and poorly characterised. In particular, patient outcomes compared to those associated with HER2 + invasive ductal cancer (IDC) and HER2-negative (HER2-) ILC, as well as the benefits of anti-HER2 therapy, are not well established. We analysed the data from the Côte d'Or Registry of Breast and Gynaecological Cancers (France) for all patients diagnosed with early-stage HER2 + ILC (62 cases), HER2 + IDC (833 cases) and HER2- ILC (685 cases) between 1998 and 2015 to compare overall and disease-free survival (OS and DFS) between these groups in correlation with anti-HER2 therapy. ILCs were associated with older age, larger tumours, lower histological grades, higher hormonal receptor positivity rates and multifocality, and more common endocrine therapy. OS and DFS between the three groups did not differ. We found that anti-HER2 therapy was associated with a survival benefit in patients with HER2 + IDC. In contrast, the survival of HER2 + ILC patients was not improved by anti-HER2 treatment, remaining close to that of HER2- ILC patients. HER2 + ILC seems not to be associated with better outcomes than HER2 + IDC but may not differ from HER2- ILC in terms of survival.

A gene expression signature in HER2+ breast cancer patients related to neoadjuvant chemotherapy resistance, overall survival, and disease-free survival.

To identify prognostic markers for treatment and survival in HER2+ patients. Patients treated with neoadjuvant chemotherapy were assigned to sensitive and resistant groups based on their treatment response. Differentially expressed genes (DEGs) were identified using RNA-seq analysis. KEGG pathway, gene ontology, and interactome analyses were performed for all DEGs. An enrichment analysis Gene set enrichment analysis was performed. All DEGs were analyzed for overall (OS) and disease-free survival (DFS). A total of 94 DEGs were related to treatment resistance. Survival analysis showed that 12 genes (ATF6B, DHRS13, DIRAS1, ERAL1, GRIN2B, L1CAM, IRX3, PRTFDC1, PBX2, S100B, SLC9A3R2, and TNXB) were good predictors of disease-free survival, and eight genes (GNG4, IL22RA2, MICA, S100B, SERPINF2, HLA-A, DIRAS1, and TNXB) were good predictors of overall survival (OS). We highlighted a molecular expression signature that can differentiate the treatment response, overall survival, and DFS of patients with HER2+ breast cancer.

166P Adjuvant trastuzumab plus pertuzumab (TP) versus trastuzumab (T) alone in patients achieving pathologic complete response after chemotherapy with TP

The APHINITY trial showed that an addition of 1 year pertuzumab (P) to standard chemotherapy with trastuzumab (T) improves invasive disease-free survival in HER2+ breast cancer patient with node-positive. Meanwhile, in patients with pathologic complete response (pCR) after neoadjuvant chemotherapy (NACT) with TP, it lacks of evidence whether an addition of P might be omitted in adjuvant treatment. We compared survival outcome according to the type of adjuvant anti-HER2 therapy in patients with pCR after NACT with TP.

Efficacy and Safety of Pyrotinib in Human Epidermal Growth Factor Receptor 2-Positive Advanced Breast Cancer: A Multicenter, Retrospective, Real-World Study.

PurposePyrotinib, a novel human epidermal growth factor receptor 2 (HER2)-targeted tyrosine kinase inhibitor (TKI), has led to remarkable survival outcomes in HER2-positive advanced breast cancer (ABC) in clinical trials and was approved for second-line standards of treatment for HER2+ ABC in China. However, the clinical trials could not fully reflect reality of clinical practice, and predictive factors were still lacking. This study aimed to assess the actual efficacy and safety of pyrotinib in HER2+ ABC in real-world setting.Patients and MethodsIn this multicenter, retrospective, observational real-world study, we analyzed 171 patients with HER2+ ABC, who received pyrotinib-based treatment from November 2017 to November 2020. The primary end point was progression-free survival (PFS). Secondary end points included overall survival (OS), objective response rate (ORR), clinical benefit rate (CBR) and safety.ResultsUp to November 30, 2021, the median PFS (mPFS) was 12.0 months for all patients. One hundred and sixty-two patients (94.7%) with measurable lesions had been included in efficacy assessment. The ORR and CBR were 45.1% and 81.5%, respectively. A significantly longer PFS was reported in patients who received pyrotinib as first-line treatment, had the ECOG-PS of 0–1, as well as those who were lapatinib-naive. In addition, multivariable analysis indicated that ECOG-PS of 2–4, positive hormone receptor (HR) status, and presence of visceral metastasis were independent negative predictors of PFS. As far as we know, this study first reported the survival outcome of pyrotinib cross-line treatment, with a mPFS of 5.0 months. All grades of adverse events (AEs) occurred in 171 patients (100%), and the most common AE was diarrhea (86.5%).ConclusionThis study further demonstrated the outstanding efficacy and safety of pyrotinib and reported the potential predictors of survival in HER2+ ABC.

Abstract 3423: A novel FcγRIIB-blocking antibody to enhance FcγR-dependent antitumor immunity with anti-HER2 therapy

Abstract Therapeutic antibodies have improved survival of both hematologic and solid cancer patients, inducing long-lasting responses and even cures. Clinically successful antibodies exert antitumor activity by targeting tumor cells directly (e.g. anti-HER2 mAb in HER2+ breast cancer, anti-CD20 mAb in B cell cancer) or by targeting and activating immune cells that seek and destroy cancer cells in the tumor microenvironment (immune checkpoint blocking antibodies; ICB). Still, many patients fail to respond or acquire resistance to these therapies. Understanding mechanisms and overcoming resistance to distinct classes of antibody drugs, is therefore clearly warranted and has the potential to further improve cancer outcomes. The inhibitory Fc gamma receptor (FcγR) IIB imbues resistance to cancer immunotherapy by several mechanisms, acting both on tumor and immune effector cells. We previously developed an FcγRIIB blocking antibody (BI-1206) which is currently being explored in clinical trials (NCT03571568, NCT04219254, and NCT02933320). BI-1206 was found to have cytolytic activity against malignant B cells and the ability to block rituximab internalization from tumor cells, as well as enhance rituximab therapeutic activity in mice humanized for CD20 and FcγRIIB or bearing relapsed/refractory CLL in vivo. Emerging data demonstrate that FcγRs modulate the therapeutic activity of many therapeutic antibodies among them HER2 targeting antibodies such as Trastuzumab. Trastuzumab alone or in combination with chemotherapy significantly improves overall survival of HER2+ breast cancer patients. However, many patients remain uncured and develop Trastuzumab resistance resulting in relapse of the disease. Means of improving anti-HER2 therapy and overcoming resistance are therefore highly desirable. We report the generation of a fully human FcγRIIB-blocking antibody (BI-1607) engineered to eliminate Fc-mediated FcγR binding and function (Fc-null anti-FcγRIIB). Using a mechanism-of-action-matched surrogate antibody, we demonstrate that Fc-null anti-FcγRIIB enhances therapeutic efficacy, both in tumor growth and overall survival of anti-HER2 in the syngeneic immune competent TUBO mouse model. This enhanced therapeutic efficacy is associated with increased tumor influx of myeloid and NK-cells. Collectively our results provide proof-of-concept for Fc-null anti-FcγRIIB to enhance anti-HER2 in the treatment of cancer. Citation Format: Linda Mårtensson, Robert Oldham, Marie Borggren, Mathilda Kovacek, Therese Blidberg, Ulla-Carin Tornberg, Ingrid Karlsson, Steve Beers, Mark Cragg, Ingrid Teige, Björn Frendeus. A novel FcγRIIB-blocking antibody to enhance FcγR-dependent antitumor immunity with anti-HER2 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3423.

Multifocal and pathologically-confirmed brain metastasis complete response to trastuzumab deruxtecan.

Antibody–drug conjugates have transformed the treatment of HER2+ breast and other cancers. Unfortunately, the CNS remains a sanctuary site for many such patients in part due to poor macromolecule penetration across the blood–brain tumor barrier. Trastuzumab deruxtecan (T-DXd), a high-payload antibody–drug conjugate, was recently found to improve progression-free survival in HER2+ breast cancer patients versus prior-generation trastuzumab emtansine, prompting us to evaluate CNS activity in a woman with brain-only metastatic disease. T-DXd achieved complete response despite heavy pretreatment. Three persistent, previously-irradiated lesions were biopsy-proven to represent treatment effect. Subsequent recurrence occurred upon treatment holiday; partial response was observed with rechallenge. This case suggests T-DXd is active in HER2+ breast cancer brain metastases and supports further prospective evaluation.