anti-HER2 Monoclonal Antibody Trastuzumab Research Articles

Abstract CT175: Safety, tolerability, pharmacokinetics, and antitumor activity of SHR-A1811 in HER2-expressing/mutated advanced solid tumors: A global phase 1, multi-center, first-in-human study

Abstract Background: SHR-A1811 is an ADC comprised of a humanized anti-HER2 monoclonal antibody (trastuzumab), a cleavable linker, and a DNA topoisomerase I inhibitor payload. Here we assessed SHR-A1811 in HER2-expressing/mutated unresectable, advanced, or metastatic solid tumors. Methods: Pts were eligible if they had HER2 positive breast cancer (BC), HER2 positive gastric/GEJ carcinoma, HER2 low-expressing BC, HER2-expressing/mutated NSCLC, or other HER2-expressing/mutated solid tumors, and were refractory or intolerant to standard therapy. SHR-A1811 at doses of 1.0-8.0 mg/kg was given Q3W (IV). The primary endpoints were DLT, safety, and the RP2D. Results: From Sep 7, 2020 to Sep 28, 2022, 250 pts who had undergone a median of 3 prior treatment lines in the metastatic setting received at least one dose of SHR-A1811 in dose escalation, PK expansion, and indication expansion part. As of data cutoff on Sep 28, 2022, 1 pt experienced DLT. Treatment-related adverse events (TRAEs) were reported in 243 (97.2%) pts. Grade ≥3 TRAEs, serious TRAEs, and treatment-related deaths were reported in 131 (52.4%), 31 (12.4%), and 3 (1.2%) pts, respectively. Interstitial lung disease (AESI) was reported in 8 (3.2%) pts. Exposures of SHR-A1811, total antibody, and the payload were generally proportional to dose from 3.2 to 8.0 mg/kg. ORR was 61.6% (154/250, 95% CI 55.3-67.7) in all pts. Objective responses were observed in pts with HER2 positive BC (88/108, ORR 81.5%, 95% CI 72.9-88.3), HER2-low BC (43/77, ORR 55.8%, 95% CI 44.1-67.2), urothelial carcinoma (7/11), colorectal cancer (3/10), gastric/GEJ carcinoma (5/9), biliary tract cancer (5/8), NSCLC (1/3), endometrial cancer (1/2), and H&N cancer (1/1). Subgroup analyses of ORR are shown in Table 1. The 6-month PFS rate was 73.9% in all pts. Conclusions: SHR-A1811 was well-tolerated and showed promising antitumor activity in heavily pretreated advanced solid tumors. Table 1. Subgroup analyses of ORR No. of prior treatment lines in metastatic setting in all pts (N=250) HER2 positive BC (N=108) HER2-low BC (N=77) Other tumor types (N=65) ≤3 81.8% (45/55) 58.7% (27/46) 36.7% (18/49) >3 81.1% (43/53) 51.6% (16/31) 31.3% (5/16) Prior anti-HER2 therapies in pts with BC (N=185)* HER2 positive BC (N=108) HER2-low BC (N=77) All BC (N=185) Any 82.2% (88/107, 73.7-89.0) 68.8% (11/16, 41.3-89.0) 80.5% (99/123, 72.4-87.1) Trastuzumab 81.9% (86/105, 73.2-88.7) 75.0% (9/12, 42.8-94.5) 81.2% (95/117, 72.9-87.8) Pertuzumab 83.0% (39/47, 69.2-92.4) 100% (5/5, 47.8-100) 84.6% (44/52, 71.9-93.1) Pyrotinib 86.9% (53/61, 75.8-94.1) 71.4% (5/7, 29.0-96.3) 85.3% (58/68, 74.6-92.7) Lapatinib 80.0% (28/35, 63.1-91.6) 100% (1/1, 2.5-100) 80.6% (29/36, 64.0-91.8) T-DM1 82.4% (14/17, 56.6-96.2) 100% (3/3, 29.2-100) 85.0% (17/20, 62.1-96.8) Other HER2-ADC (except T-DM1)** 60.0% (9/15, 32.3-83.7) 50.0% (2/4, 6.8-93.2) 57.9% (11/19, 33.5-79.8) ORR in pts with tumor types other than BC (N=65) HER2 IHC3+ or IHC2+/ISH+ (N=36) HER2 IHC2+/ISH- or IHC1+ or unknown (N=29) All other tumor types (N=65) % (n/N) 38.9% (14/36) 31.0% (9/29) 35.4% (23/65) ORR was shown as % (n/N, 95% CI) or % (n/N). *ORR is calculated using the number of subjects previously treated with anti-HER2 cancer therapy in advanced/metastatic setting as denominator; 2-sided 95% CIs are estimated using Clopper-Pearson method. **Includes RC48-ADC, A166, DP303c, MRG002, ARX788, TAA013, DX126-262, PF-06804103, and BAT8001. Citation Format: Herui Yao, Min Yan, Zhongsheng Tong, Xinhong Wu, Min-Hee Ryu, Jee Hyun Kim, John Park, Yahua Zhong, Weiqing Han, Caigang Liu, Mark Voskoboynik, Qun Qin, Jian Zhang, Minal Barve, Ana Acuna-Villaorduna, Vinod Ganju, Seock-Ah Im, Changsheng Ye, Yongmei Yin, Amitesh C. Roy, Li-Yuan Bai, Yung-Chang Lin, Chia-Jui Yen, Hui Li, Ki Young Chung, Shanzhi Gu, Jun Qian, Yuee Teng, Yiding Chen, Yu Shen, Kaijing Zhao, Shangyi Rong, Xiaoyu Zhu, Erwei Song. Safety, tolerability, pharmacokinetics, and antitumor activity of SHR-A1811 in HER2-expressing/mutated advanced solid tumors: A global phase 1, multi-center, first-in-human study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT175.

Pharmacovigilance Analysis of HeartFailure Associated With Anti-HER2 Monotherapies and Combination Regimens for Cancer.

Trastuzumab improves outcomes in patients with HER2-overexpressing malignancies but is associated with decreases in left ventricular ejection fraction. Heart failure (HF) risks from other anti-HER2 therapies are less clear. Using World Health Organization pharmacovigilance data, the authors compared HF odds across anti-HER2 regimens. In VigiBase, 41,976 patients had adverse drug reactions (ADRs) with anti-HER2 monoclonal antibodies (trastuzumab, n=16,900; pertuzumab, n=1,856), antibody-drug conjugates (trastuzumab emtansine [T-DM1], n=3,983; trastuzumab deruxtecan, n=947), and tyrosine kinase inhibitors (afatinib, n=10,424; lapatinib, n=5,704; neratinib, n=1,507; tucatinib, n=655); additionally, 36,052 patients had ADRs with anti-HER2-based combination regimens. Most patients had breast cancer (monotherapies, n=17,281; combinations, n=24,095). Outcomes included comparison of HF odds with each monotherapy relative to trastuzumab, within each therapeutic class, and among combination regimens. Of 16,900 patients with trastuzumab-associated ADRs, 2,034 (12.04%) had HF reports (median time to onset 5.67months; IQR: 2.85-9.32months) compared with 1% to 2% with antibody-drug conjugates. Trastuzumab had higher odds of HF reporting relative to other anti-HER2 therapies collectively in the overall cohort (reporting OR [ROR]: 17.37; 99% CI: 14.30-21.10) and breast cancer subgroup (ROR: 17.10; 99% CI: 13.12-22.27). Pertuzumab/T-DM1 had 3.4 times higher odds of HF reporting than T-DM1 monotherapy; tucatinib/trastuzumab/capecitabine had similar odds as tucatinib. Among metastatic breast cancer regimens, HF odds were highest with trastuzumab/pertuzumab/docetaxel (ROR: 1.42; 99% CI: 1.17-1.72) and lowest with lapatinib/capecitabine (ROR: 0.09; 99% CI: 0.04-0.23). Trastuzumab and pertuzumab/T-DM1 had higher odds of HF reporting than other anti-HER2 therapies. These data provide large-scale, real-world insight into which HER2-targeted regimens would benefit from left ventricular ejection fraction monitoring.

Near-infrared upconversion multimodal nanoparticles for targeted radionuclide therapy of breast cancer lymphatic metastases.

The theranostics of lymph node metastasis has always been one of the major obstacles to defeating breast cancer and an important decisive factor in the prognosis of patients. Herein, we design NaGdF4:Yb,Tm@NaLuF4 upconversion nanoparticles with PEG and anti-HER2 monoclonal antibody (trastuzumab, Herceptin) (NP-mAb), the delivery of NP-mAb through the lymphatic system allows for effective targeting and accumulation in lymphatic metastasis. Combination of radionuclides 68Ga and 177Lu could be chelated by the bisphosphate groups of NP-mAb. The obtained nanoprobe (NP-mAb) and nanonuclear drug (68Ga-NP-mAb or 177Lu-NP-mAb) exhibited excellent stability and show high accumulation and prolong retention in the lymph node metastasis after intratumoral injection into the foot pad by near-infrared fluorescence (NIRF), single-photon emission computed tomography (SPECT) and positron emission tomography (PET) imaging. Utilizing the β-rays released by 177Lu, 177Lu-NP-mAb could not only decrease the incidence of lymph node metastasis, but also significantly decrease the volumes of lymph node metastasis. Additionally, 177Lu-NP-mAb induce no obvious toxicity to treated mice through blood routine, liver and kidney function assay. Therefore, nanoprobe and nanonuclear drug we designed could be acted as excellent theranostics agents for lymph node metastasis, providing potential alternatives diagnose and treatment option for lymph node metastasis.

HER2-Displaying M13 Bacteriophages induce Therapeutic Immunity against Breast Cancer.

Simple SummaryThe high incidence and death rates of breast cancer make the development of new therapies an urgent need. The introduction into the clinic of the anti-HER2 monoclonal antibody trastuzumab considerably improved the overall survival and time-to-disease progression of patients with HER2-positive breast cancer. However, many patients do not benefit from it because of resistance to therapy. Cancer vaccines, by inducing into the patient an anti-cancer specific immunity, might represent an alternative immunotherapeutic approach, but despite promises, so far no anti-HER2 cancer vaccine has been approved for human use. In this study, we propose therapeutic phage-based vaccines, against HER2 and its aggressive isoform Δ16HER2, able to elicit a protective immunity and potentially capable of preventing relapse in HER2-positive breast cancer patients, even in those who develop trastuzumab resistance.The advent of trastuzumab has significantly improved the prognosis of HER2-positive (HER2+) breast cancer patients; nevertheless, drug resistance limits its clinical benefit. Anti-HER2 active immunotherapy represents an attractive alternative strategy, but effective immunization needs to overcome the patient’s immune tolerance against the self-HER2. Phage display technology, taking advantage of phage intrinsic immunogenicity, permits one to generate effective cancer vaccines able to break immune tolerance to self-antigens. In this study, we demonstrate that both preventive and therapeutic vaccination with M13 bacteriophages, displaying the extracellular (EC) and transmembrane (TM) domains of human HER2 or its Δ16HER2 splice variant on their surface (ECTM and Δ16ECTM phages), delayed mammary tumor onset and reduced tumor growth rate and multiplicity in ∆16HER2 transgenic mice, which are tolerant to human ∆16HER2. This antitumor protection correlated with anti-HER2 antibody production. The molecular mechanisms underlying the anticancer effect of vaccine-elicited anti-HER2 antibodies were analyzed in vitro against BT-474 human breast cancer cells, sensitive or resistant to trastuzumab. Immunoglobulins (IgG) purified from immune sera reduced cell viability mainly by impairing ERK phosphorylation and reactivating retinoblastoma protein function in both trastuzumab-sensitive and -resistant BT-474 cells. In conclusion, we demonstrated that phage-based HER2 vaccines impair mammary cancer onset and progression, opening new perspectives for HER2+ breast cancer treatment.

AVB-S6-500, a receptor tyrosine kinase AXL inhibitor, improves the therapeutic efficacy of trastuzumab in uterine serous cancers (278)

<b>Objectives:</b> To determine whether inhibition of <i>GAS6</i>/<i>AXL</i> can improve efficacy to the humanized anti-HER2 monoclonal antibody trastuzumab by decreasing uterine serous cancer cell invasion and cell survival <i>in vitro</i> and tumor burden <i>in vivo</i>. <b>Methods:</b> We treated three uterine serous cancer cell lines (ARK1, ARK2, PUC198) that expressed <i>Her2/neu</i> and <i>AXL</i> receptors with AVB500 (Aravive Biologics, Houston, TX) in combination with Trastuzumab. Clonogenic proliferation assays were performed after three days of treatment, and relative cell viability was calculated using GraphPad Prism. Next, using a Matrigel invasion assay, we examined the invasive capacity of the ARK1 and ARK2 cell lines after 24 hours of treatment with vehicle, AVB500 alone, Trastuzumab alone, or combination Trastuzumab and AVB500. We also performed proximity ligation and IF colocalization assays to show that <i>Her2/neu</i> and <i>AXL</i> receptors form a complex that results in the transphosphorylation and activation of the <i>AXL</i>. We performed <i>in vivo</i> studies using nod-SCID mice injected with 1 x 10⁷ ARK1 and ARK2 cells intraperitoneally, followed by treatment with vehicle control, AVB alone, or Trastuzumab alone or in combination with AVB500 for 35 days and 21 days, respectively. <b>Results:</b> In uterine serous (ARK 2 and PUC 198) cells, there was a significant reduction in cell proliferation when treated with combination Trastuzumab and AVB500 compared to the Trastuzumab-alone group (0.155nm vs 0.202nm, p=0.015; 0.290 vs 0.015, p=0.0044) in ARK 2 and PUC 198 cell lines, respectively. We also observed a reduction in the number of invading tumor cells when cells were treated with combination Trastuzumab and AVB500 versus Trastuzumab alone (3.9 vs 17.7 invading tumor cells/hpf, p=0.0001; 3.2 vs 17.8 invading tumor cells/hpf, p=0.0004) in ARK 1 and ARK 2 cell lines, respectively. Furthermore, the proximity ligation and co-localization assays demonstrated the physical closeness of <i>AXL</i> and <i>HER2</i> receptors in ARK2 cells that could account for the co-regulation of these receptors. Finally, we found that mice treated with Trastuzumab plus AVB -500 developed significantly less tumor burden than mice treated with Trastuzumab alone (0.032 g vs 0.083 g, <i>p</i>= 0.024) in the ARK1 xenograft model and (0.142g vs 0.443 g, <i>p</i>= 0.0015) in the ARK2 xenograft model. <b>Conclusions:</b> The <i>GAS6</i>/<i>AXL</i> inhibitor, AVB-500, potentiated the effect of Trastuzumab to decrease uterine serous cancer cell proliferation and invasion <i>in vitro</i> and tumor burden <i>in vivo</i>. Co-localization of <i>AXL</i> and <i>HER2</i> receptors was seen in both uterine serous cancer cell lines. Our data suggest that the addition of AVB-S6-500 increased the therapeutic efficacy of Trastuzumab therapy in uterine serous cancers. Additional therapeutic and mechanistic experiments are ongoing.

Ultralow Limit Detection of Soluble HER2 Biomarker in Serum with a Fiber-Optic Ball-Tip Resonator Assisted by a Tilted FBG.

An optical-fiber biosensor has been developed for the detection of the breast cancer biomarker soluble human epidermal growth factor receptor-2 (sHER2). The sensor was fabricated by combining a tilted fiber Bragg grating (TFBG) with a ball resonator, allowing us to achieve an excellent sensitivity compared to other optical-fiber-based sensors. The sensor exhibits a resonance comb excited by the TFBG and the spectral profile of the ball resonator. The detection of sHER2 at extremely low concentrations was carried out by tracking the amplitude change of selected resonances. The therapeutic anti-HER2 monoclonal antibody Trastuzumab has been used to functionalize the biosensor with silane surface chemistry. The sensor features a sensitivity of 4034 dB/RIU with a limit of detection (LoD) in buffer and in a 1/10 diluted serum of 151.5 ag/mL and 3.7 pg/mL, respectively. At relatively high protein concentrations (64 ng/mL) binding to sHER (7.36 dB) as compared to control proteins (below 0.7 dB) attested the high specificity of sHER2 detection.

Abstract P4-01-12: Short-term trastuzumab treatment increases oncogenic fitness in HER2 overexpressing breast cancer models

Abstract Overexpression of Human EGF Receptor 2 (HER2) is found in up to 20% of primary invasive breast cancers and is a marker of aggressive metastatic disease and poor prognosis. HER2 is a member of the transmembrane tyrosine kinase receptors family which includes EGFR (HER1), HER3 and HER4. Phosphorylation of tyrosine residues in the cytoplasmic domain upon homo- or heterodimerization results in stimulation of cellular proliferation, migration, angiogenesis, and inhibition of apoptosis. HER2 is deemed a preferred heterodimer partner and presents an excellent target for personalized medicine. Anti-HER2 monoclonal antibody trastuzumab (TZM) has been used in the clinic over the last decades and it is considered as one of the most successful targeted anti-cancer therapies. However, a large fraction of eligible patients displays either primary or acquired resistance to TZM treatment. In this study, we report that even relatively short 24h TZM exposure (TZM-priming) of AU565 HER2-overexpressing breast cancer cells results in rapid and profound alterations in HER receptors’ expression level and signaling, as well as upregulation of markers associated with drug resistance. We provide several lines of evidence to support this claim: Firstly, Western blot analysis of both monolayer AU565 cells incubated in the presence or absence of 20 μg/mL TZM (TZM priming) or human IgG (control), as well as of liquid overlay AU565 spheroids generated from TZM-primed or control cells, consistently shows significant increase of HER3 and pHER2 levels in the TZM-primed samples. Secondly, immunofluorescent analysis strongly indicates that TZM priming substantially upregulates HER3 and pHER2 levels both in 2D and 3D (spheroids) models. Importantly, this process is accompanied by rearrangement of endocytic markers Rab4, CD63, Sorl1 as well as Extra domain B fibronectin, found at the cells’ surface together with HER2 and HER3 upon TZM priming. A similar trend was also found in HER2-overexpressing ovarian cancer cells SKOV-3 subjected to short-term TZM treatment. Furthermore, spheroids made with TZM-primed AU565 cells display significantly faster cell proliferation compared to IgG-primed or untreated control counterparts, as measured via optical coherent tomography imaging and Imaris 3D rendering software. Finally, tumor xenografts implanted in athymic nude mice using TZM-primed AU565 cells display continuous growth in contrast to those generated from untreated cells. These results suggest that short-term TZM treatment may inadvertently increase oncogenic fitness via adaptation to TZM-HER2 cellular binding and subsequent disruption of dimerization and signaling. These results are consistent with reports indicating that regional HER2 expression heterogeneity appears to increase with TZM treatment. Moreover, these observations are especially concerning because radiolabeled TZM probes have been employed in PET imaging in HER2+ cancer patients. In summary, our results suggest that short-term TZM treatment may result in priming and selection for more aggressive cancer phenotype. Citation Format: Alena Rudkouskaya, Cassandra L Roberge, Lauren Elder, Kailie Matteson, David T Corr, Margarida Barroso. Short-term trastuzumab treatment increases oncogenic fitness in HER2 overexpressing breast cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-12.

Abstract P2-13-27: Infusion-related reactions in patients receiving pertuzumab and trastuzumab: A retrospective study

Abstract BACKGROUND: The use of two anti-HER2 monoclonal antibodies (mAbs) (pertuzumab and trastuzumab) combined with taxanes is the standard of care for first line treatment in HER2-positive metastatic breast cancer (mBC). Infusion related reactions (IRR) during first administration of anti-HER2 mAb have been reported. Reintroduction of the causal drugs is often possible, but there is no data to identify patients at highest risk of developing IRR and modalities of safe reintroduction. The aim of this study was to report patients who experienced IRR during anti-HER2 mAbs infusions at Institut Curie Hospitals (ICH) and to describe these reactions. METHODS: All IRR cases must be reported to the ICH pharmacies. We retrospectively inspected the electronic record of patients who experienced an IRR during pertuzumab (Pmab) and trastuzumab (Tmab). We collected patients’ characteristics, grade of IRR and outcomes. RESULTS: From January 2013 to December 2020, a total of 223 patients had at least one Pmab+Tmab infusion as part of their BC treatment. Among them, 28 patients (8%) with anti-HER2 mAb IRR were identified. All patients but one had an HER2-positive BC; the HER2-negative case was a HER3-mutant mBC. Twelve patients (43%) had de novo mBC. Twenty-six patients (93%) received Pmab+Tmab as first line treatment for mBC. Ten patients were previously exposed to Tmab and three to Pmab+Tmab for (neo)adjuvant treatment. At IRR onset, 21 patients (75%) had liver metastasis, with more than five liver metastases for 19 of them. Fifteen patients (55%) had liver enzymes upper normal limit. IRR occurred during first anti-HER2-mAb infusion in 22 patients (79%) despite pre-treatment with antihistaminic and/or glucosteroids for taxanes. The other IRR were reported at the 2nd, 5th, 6th, 8th and &amp;gt; 10th cycle of Pmab+Tmab. Sixteen IRR (57%) were attributed to Pmab infusion, 9 (32%) to Tmab infusion and 3 (11%) to Tmab and/or Pmab. Al IRR were declared to the pharmacovigilance agency. Majority of reactions were mild to moderate (54% of grade 2). Most frequent symptoms associated with IRR were: thrills (68%), hyperthermia (64%); low oxygen saturation (36%), pain (25%), hypotension (21%), cutaneous (18%) and digestive (18%) reaction. Three patients were transferred to ICU. One death was attributed to anti-HER2 mAb IRR (death was attributed to respiratory distress during the first Tmab infusion). Reintroduction off Pmab+Tmab was performed in 18 patients, after pre-treatment with antihistaminic and/or glucosteroids and with vital function monitoring in conventional hospitalization. No recurrence of IRR was observed in 15/18 patients. For the 3 patients with recurrent IRR, symptoms were mild to moderate. Objective response to anti-HER2 treatments was observed in twenty three patients (82%; 8 complete responses; 15 partial responses). Median PFS was 16 months. CONCLUSIONS: Most anti-Her2-mAb IRR occurs during first infusion, are mild to moderate and do not require a transfer in ICU. Our study suggests that patients with high liver burden might be over-represented among those experiencing an IRR. Reintroduction of Pmab+Tmab was safe in most patients, allowing the continuation of the anti-HER2 dual blockade. Citation Format: Delphine Loirat, Juliette Logeart, Pauline Vaflard, Aurélien Noret, Mathilde Saint-Ghislain, Edith Borcoman, Audrey Hurgon, Cyrille Cros, Thomas Genevee, Audrey Bellesoeur, Francesco Ricci, Florence Lerebours, Laurence Escalup, Marie-Paule Sablin, François-Clément Bidard, Paul H. Cottu, Jean-Yves Pierga. Infusion-related reactions in patients receiving pertuzumab and trastuzumab: A retrospective study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-27.

Abstract P5-01-04: Correlation of HER2 receptor expression and in vivo activity of the HER2-targeting therapies trastuzumab deruxtecan (DS-8201a) and T-DM1 activity in a panel of breast XPDX models

Abstract Background: 15-20% of metastatic breast cancers are characterized by overexpression or amplification of human epidermal growth factor receptor 2 (HER2). First-line treatment for HER2-positive metastatic breast cancer includes the anti-HER2 monoclonal antibodies trastuzumab and pertuzumab in combination with chemotherapy. Second-line therapy includes T-DM1, an antibody-drug conjugate (ADC) consisting of trastuzumab conjugated to maytansinoid (DM1). Until recently, patients who progress on T-DM1 were left with few treatment options. Trastuzumab deruxtecan (DS-8201a) is an antibody-drug conjugate (ADC) consisting of an anti-HER2 (human epidermal growth factor receptor 2) antibody linked to a novel topoisomerase I inhibitor payload using a cleavable tetrapeptide-based linker and was recently approved for unresectable or T-DM1-refractory HER2+ breast cancer. In addition, DS-8201a is currently being evaluated as a treatment in breast cancers with low and medium expression of HER2. To better understand the potential for DS-8201a as a treatment in these cancer types, we evaluated this therapy in a panel of ER+/- XPDX models with differential HER2 expression. In addition, we compared DS-8201a activity to T-DM1 and benchmarked efficacy of the topoisomerase I inhibitor irinotecan in all tested models. Methods: One hundred (50ER+/50ER-) breast XPDX models were evaluated in this study. Models were established and characterized for estrogen receptor and HER2 expression by IHC and profiled using WES and RNAseq. For in vivo studies, DS-8201a and T-DM1 were administered IV at 3 mg/kg and irinotecan IP at 100 mg/kg on a weeklyx3 schedule. In vivo study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20 versus control was considered sensitive. Results: Models were grouped by ER positivity (+/-) and model HER2 score and designated as null (0), low (1+), medium (2+) or high (3+); models established from clinically HER2+ patients were also noted. In the ER+ group, HER2 low and medium expressing models accounted for over 80% of the total, while 15% were categorized as high and less than 5% as null. All models with high and 30% with medium HER2 staining were from clinically HER2+ patients, no low or null models were established from breast cancer assigned HER2+ clinically. In the ER- group, HER2 low and medium expressing models accounted for 75% of the total, while 12% were categorized as high and 13% as null. All models with high and 13% with medium HER2 staining were from clinically HER2+ patients; 5% of low or null models were established from breast cancer assigned HER2+ clinically. In vivo, 65% of the ER+ group reported sensitivity to DS-8201a versus 25% to T-DM1; 10% of DS-8201a, and 40% of T-DM1 sensitive models were high expression models and the remaining low and medium with no null models reporting sensitivity to either agent. 35% of tested ER+ models were sensitive to both HER2 therapies and 50% of models sensitive to DS-8201a were also sensitive to irinotecan. In the ER- group, 70% reported sensitivity to DS-8201a versus 25% to T-DM1; 20% of DS-8201a and 25% of T-DM1 sensitive models were high expression models and the remaining low and medium with one null model (ST069) sensitive to both agents. 25% of tested ER- models were sensitive to both HER2 therapies and 40% of models sensitive to DS-8201a were also sensitive to irinotecan. Conclusion: We have compared activity of DS-8201a, T-DM1 and irinotecan in a panel of ER+/- XPDX models and correlated activity based on HER2 expression. This data and panel can be utilized as a valuable tool in better understanding the potential for DS-8201a and other HER2-targeting therapies as a treatment in cancers driven by HER2 expression. Citation Format: Alyssa Simonson, Peter Forofontov, Johnnie R Flores, Kimberly Hernandez, April Cabang, Amy Lang, Gladys Rodriguez, Kyriakos Papadopoulos, Murali Beeram, Arthur Rosenthal, Brittany DeBerry, Lon Smith, Ronald Drengler, Amita Patnaik, Drew Rasco, Luis Rodriguez, Steven Abbate, Scott Ulmer, Michael Wick. Correlation of HER2 receptor expression and in vivo activity of the HER2-targeting therapies trastuzumab deruxtecan (DS-8201a) and T-DM1 activity in a panel of breast XPDX models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-04.

Autophagy and intracellular product degradation genes identified by systems biology analysis reduce aggregation of bispecific antibody in CHO cells

Aggregation of therapeutic bispecific antibodies negatively affects the yield, shelf-life, efficacy and safety of these products. Pairs of stable Chinese hamster ovary (CHO) cell lines produced two difficult-to-express bispecific antibodies with different levels of aggregated product (10–75% aggregate) in a miniaturised bioreactor system. Here, transcriptome analysis was used to interpret the biological causes for the aggregation and to identify strategies to improve product yield and quality. Differential expression- and gene set analysis revealed upregulated proteasomal degradation, unfolded protein response and autophagy processes to be correlated with reduced protein aggregation. Fourteen candidate genes with the potential to reduce aggregation were co-expressed in the stable clones for validation. Of these, HSP90B1, DDIT3, AKT1S1, and ATG16L1, were found to significantly lower aggregation in the stable producers and two (HSP90B1 and DNAJC3) increased titres of the anti-HER2 monoclonal antibody trastuzumab by 50% during transient expression. It is suggested that this approach could be of general use for defining aggregation bottlenecks in CHO cells.

The benefits of trastuzumab in the treatment of HER2+ breast cancer as a function of exposure time.

BackgroundBreast cancer is a heterogeneous disease with overexpression of several receptors, such as human epidermal receptor 2 (HER2), which is a prognostic and predictive biomarker for treatment with the anti-HER2 monoclonal antibody trastuzumab. This study aimed to test the contribution of this regimen in patients with overexpression/amplification of HER2 for periods shorter than the 1-year treatment recommendation.MethodsA retrospective single-centre study involving 155 patients with non-metastatic (stages I–III) invasive ductal HER2+ breast carcinoma, with a median follow-up of 48.9 months after completion of adjuvant therapy, except endocrine therapy.ResultsAbout 60% of patients received trastuzumab therapy for a median time of 365 days. Although the use of trastuzumab for a short period has provided some benefit, analyses of survival with a continuous dependent variable have revealed a minimum time for improved survival. In the multivariate analysis by Cox regression, trastuzumab use duration exceeding 9 weeks resulted in protection against distant metastasis (adjusted HR: 0.307 (0.139–0.678), p = 0.004), disease progression (adjusted hazard ratio (HR) 0.353 (0.175–0.714), p = 0.004) and death (adjusted HR: 0.267 (0.105–0.678), p = 0.005), being superior to multimodal systemic therapy with chemotherapy and to endocrine therapy without trastuzumab, but inferior to almost 1 year of administration of this monoclonal antibody, especially regarding overall survival (adjusted HR: 0.203 (0.069–0.596), p = 0.004).ConclusionDespite showing some benefits, the protective effect derived from a suboptimal time of trastuzumab exposure is inferior to the standard course of 1 year.

Predictors of pathologic complete response to neoadjuvant treatment in HER2-overexpressing breast cancer: a retrospective analysis using real-world data

The human epidermal growth factor receptor 2 (ERBB2, HER2 or HER2/neu) is a transmembrane tyrosine kinase receptor that is overexpressed in approximately 20% of breast cancers. The use of the anti-HER2 monoclonal antibodies Pertuzumab and Trastuzumab in association with chemotherapy has achieved a higher percentage of pathologic complete response (pCR) than conventional chemotherapy. The purpose of our study was to identify factors that could affect the therapeutic response of patients with breast cancer and HER2 overexpression treated with cytotoxic chemotherapy plus double HER2 blockade in neoadjuvant setting at Fundación Arturo López Pérez (FALP). A case–control study was designed to evaluate the effect of clinical and histopathological variables on the response to neoadjuvant therapy. Ninety-four women with non-metastatic breast cancer and HER2 overexpression received neoadjuvant combination chemotherapy with Trastuzumab and Pertuzumab at FALP during the period 2017–2020. Seventy percent of patients achieved pCR, and in the group of hormone receptor negative patients, 89% of patients achieved pCR. Different variables were analysed trying to look for clinicopathological predictors of complete response. This study provides us with real-world data on the efficacy of using this treatment combination in our population of HER2-overexpressing breast cancer patients.

A Semi Rigid Novel Hydroxamate AMPED-Based Ligand for 89Zr PET Imaging.

In this work, we designed, developed, characterized, and investigated a new chelator and its bifunctional derivative for 89Zr labeling and PET-imaging. In a preliminary study, we synthesized two hexadentate chelators named AAZTHAS and AAZTHAG, based on the seven-membered heterocycle AMPED (6-amino-6-methylperhydro-1,4-diazepine) with the aim to increase the rigidity of the 89Zr complex by using N-methyl-N-(hydroxy)succinamide or N-methyl-N-(hydroxy)glutaramide pendant arms attached to the cyclic structure. N-methylhydroxamate groups are the donor groups chosen to efficiently coordinate 89Zr. After in vitro stability tests, we selected the chelator with longer arms, AAZTHAG, as the best complexing agent for 89Zr presenting a stability of 86.4 ± 5.5% in human serum (HS) for at least 72 h. Small animal PET/CT static scans acquired at different time points (up to 24 h) and ex vivo organ distribution studies were then carried out in healthy nude mice (n = 3) to investigate the stability and biodistribution in vivo of this new 89Zr-based complex. High stability in vivo, with low accumulation of free 89Zr in bones and kidneys, was measured. Furthermore, an activated ester functionalized version of AAZTHAG was synthesized to allow the conjugation with biomolecules such as antibodies. The bifunctional chelator was then conjugated to the human anti-HER2 monoclonal antibody Trastuzumab (Tz) as a proof of principle test of conjugation to biologically active molecules. The final 89Zr labeled compound was characterized via radio-HPLC and SDS-PAGE followed by autoradiography, and its stability in different solutions was assessed for at least 4 days.

Inhibition of GAS6/AXL improves efficacy of HER2 inhibitor trastuzumab in uterine serous cancer

<h3>Objectives:</h3> To determine whether inhibition of GAS6/AXL can improve efficacy to the humanized anti-HER2 monoclonal antibody trastuzumab by decreasing uterine serous cancer cell invasion and cell survival <i>in vitro</i> and tumor burden <i>in vivo.</i> <h3>Methods:</h3> We treated AXL-expressing and HER2-expressing uterine serous cancer cell lines (ARK1, ARK2) with trastuzumab, AVB-500, or trastuzumab plus AVB-500. Clonogenic cell survival assays were performed. Matrigel invasion assays were used to assess the invasive capacity of ARK2 cells after 24 hours of treatment with vehicle, AVB-500, trastuzumab, or trastuzumab plus AVB-500. A proximity ligation assay was used to examine colocalization of HER2/neu and AXL. Western blots were performed to assess the effects of the individual drugs and combination on phosphorylation of HER2 and AXL. An intraperitoneal tumor model with 10 million ARK1 cells was treated with vehicle, AVB-500, trastuzumab, or trastuzumab plus AVB-500 for 35 days. The number, size, and volume of tumor nodules were measured. GraphPad Prism was used for statistical analysis. <h3>Results:</h3> We found decreased cell survival by clonogenic assays in ARK1 and ARK2 cells treated with trastuzumab plus AVB-500 compared to trastuzumab alone (relative absorbance 0.155 nm vs 0.202 nm, P=0.015). There was significantly less invasion from treatment with trastuzumab plus AVB-500 than with trastuzumab alone (3.2 vs 17.8 invading tumor cells/hpf, <i>P</i>=0.0004). Western blot showed that cells treated with trastuzumab plus AVB-500 had reduced phospho-AXL compared to cells treated with AVB-500 or trastuzumab alone. Furthermore, the proximity ligation assay demonstrated co-localization of the AXL and HER2 receptors in both ARK1 and ARK2 cells indicating a physical closeness that could account for this co-regulation. We found that mice treated with trastuzumab plus AVB-500 developed significantly less tumor burden than mice treated with trastuzumab alone (0.03205 g vs 0.08316 g, P=0.024), AVB-500 alone (0.0320 g vs 0.1638 g, P<0.0001), and vehicle alone (0.03205 g vs 0.1154 g, P=0.0395). <h3>Conclusions:</h3> The GAS6/AXL inhibitor AVB-500 potentiated the effect of trastuzumab to decrease uterine serous cancer cell proliferation and invasion <i>in vitro</i> and tumor burden <i>in vivo.</i> There was co-localization of AXL and HER2 receptors seen in both uterine serous cancer cell lines.

Association between timely targeted therapy initiation and clinical outcomes in patients with advanced HER2+ gastroesophageal adenocarcinoma.

e16048 Background: Delayed treatment initiation is associated with increased mortality in early-stage cancers. Few studies have examined the impact of timely initiation in advanced cancers. Timely targeted therapy initiation can be especially challenging due to the additional biomarker testing required to identify patients eligible for these treatments. We examined the impact of timely targeted therapy initiation on survival among patients with metastatic HER2+ gastroesophageal adenocarcinoma (GEA). Methods: We performed a retrospective cohort study of patients with metastatic HER2+ GEA treated with first line systemic therapy between 1/2011 and 12/2017 using the nationwide Flatiron Health EHR-derived deidentified database. Timely targeted therapy, defined as initiation of the anti-HER2 monoclonal antibody trastuzumab up to 14 days after first line chemotherapy initiation, was entered as a time-varying exposure on the day of initiation. Patients who initiated trastuzumab after the 14-day cut point or who lacked documentation of having received trastuzumab were classified as having non-timely targeted therapy initiation. Extended Cox regression models were used to compare progression-free (PFS) and overall survival (OS) from the date of first line treatment initiation, adjusted for potential confounders. Pre-planned sensitivity analyses assessed alternative “timeliness” definitions as well as the impact of any trastuzumab administration, regardless of the timing of initiation. Results: We included 459 patients with metastatic HER2+ GEA; 293 (63.8%) received trastuzumab, of whom 223 (76.1%) initiated in a timely manner. The median age was 65.0 years, and patients were predominantly male (82.4%) and non-Hispanic White (78.2%) with an ECOG performance status of 0-1 (85.9%). Relative to non-timely initiation, timely targeted therapy was associated with significantly higher OS (2-year OS 29.6% vs 14.6%; adjusted HR 0.72, 95% CI 0.57-0.91, p = 0.006) and PFS (2-year PFS 8.3% vs 3.9%; adjusted HR 0.78, 95% CI 0.62-0.98, p = 0.030). Results remained similar in sensitivity analyses 1) using alternative “timeliness” definitions (up to 70 days after chemotherapy initiation), and 2) comparing any trastuzumab administration, regardless of the timing of initiation, to no trastuzumab administration (OS - adjusted HR 0.75, 95% CI 0.60-0.95, p = 0.018; PFS - adjusted HR 0.81, 95% CI 0.64-1.02, p = 0.069). Conclusions: OS and PFS were higher among patients with metastatic HER2+ GEA treated with trastuzumab, regardless of the timing of targeted therapy initiation. Although our results provide reassurance to clinicians that delays in targeted therapy may not be detrimental to patient outcomes, efforts should still be made to ensure that all patients with metastatic HER2+ GEA receive first line trastuzumab.

Enhanced Antitumor Effect of Trastuzumab and Duligotuzumab or Ipatasertib Combination in HER-2 Positive Gastric Cancer Cells.

Simple SummaryThe ToGA trial has demonstrated, in HER2-expressing patients, that unresectable and advanced gastric cancer, chemotherapy and trastuzumab in combination increase overall survival, even if it is still unclear why after one year the same patients are non-responsive to trastuzumab treatment. Here, we have demonstrated that in HER2-positive gastric cancer cell lines, the addition of duligotuzumab, targeting HER3 receptor, or ipatasertib, targeting AKT protein, enhances the antitumor effect of trastuzumab in vitro through a full inhibition of the membrane signals, on HER2 and HER3, and of downstream signaling, including AKT, and MAPK pathways. Hence, this study suggests a novel and biomarker-driven therapeutic strategy supporting further evaluation of the anti-tumor efficacy of these combinations in HER2 human gastric cancer patients.The anti-HER2 monoclonal antibody trastuzumab is a key drug for the treatment of HER2-positive gastric cancer (GC); however, its activity is often limited by the onset of resistance and mechanisms of resistance are still poorly understood. Several targeted agents showed synergistic activity by concomitant use with trastuzumab in vitro and are under clinical investigation. The aim of this study was to assess the antitumor activity of duligotuzumab, an anti HER3/EGFR antibody or ipatasertib, an AKT inhibitor, combined with trastuzumab in a panel of HER2-positive human gastric cancer cells (GCC), and the efficacy of such combinations in HER2-resistant cells. We have assessed the efficacy of duligotuzumab or ipatasertib and trastuzumab in combination, analyzing proliferation, migration and apoptosis and downstream intracellular signaling in vitro on human HER2-positive GCC (NCI-N87, OE33, OE19) and in negative HER2 GCC (MKN28). We observed a reduction of proliferation, migration and apoptotic rate in HER2-positive OE33, OE19 and N87 cell lines with the combination of duligotuzumab or ipatasertib plus trastuzumab. In particular, in OE33 and OE19 cell lines, the same combined treatment inhibited the activation of proteins downstream of HER2, HER3, AKT and MAPK pathways. Targeting both HER2 and HER3, or HER2 and AKT, results in an improved antitumor effect on HER2-positive GCC.

Monitoring Receptor Heterodimerization along Intracellular Trafficking Pathways using Anti‐HER2 Therapeutic Antibodies

Overexpression of Human EGF Receptor 2 (HER2) in cancer is a marker of aggressive metastatic disease and poor prognosis. Anti-HER2 monoclonal antibody trastuzumab (TZM) has been successfully used in the clinic over the last decades. However, a large fraction of eligible patients display resistance to this therapy. Deeper investigation of TZM-HER2 binding, internalization and trafficking/degradation in cancer cells may provide some crucial answers. Forster resonance energy transfer Fluorescence lifetime microscopy (FRET-FLIM) offers a unique approach to monitor TZM-HER2 binding via the reduction of donor fluorophore lifetime. Thus, FRET-FLIM provides an efficient method to quantify ligand-receptor binding and internalization in cancer cells with the additional benefit of sensing intracellular microenvironment heterogeneity such as modulation of endocytic trafficking upon drug treatment. In this study, we report quantification of binding of NIR-labeled therapeutic antibodies FRET pairs to their respective receptors, e.g. HER2 and/or HER1, in AU565 breast cancer cells in various combinations, i.e. anti-HER1 cetuximab donor – cetuximab acceptor vs. cetuximab donor - TZM acceptor, to assess the dynamics of HER receptor homo and heterodimerization behavior. We also monitor FRET FLIM events along the intracellular trafficking of these anti-HER2 and anti-HER1 probes. Our data indicate that in the presence of TZM, HER1 as well as HER3 receptors localization is shifted towards plasma membrane. Furthermore, the distribution pattern of fluorescence lifetime during endocytic trafficking differs when comparing “homo” vs “hetero FRET” pairs. These results suggest that HER2 dimerization pattern can switch towards hetero- and oligomerization during TZM treatment which may result in modulation of oncogenic signaling. In parallel, Western blotting and immunofluorescent analysis of AU565 cells strongly indicate that 24h TZM treatment substantially upregulate HER3 and pHER2 levels both in 2D and 3D (spheroids) models. Together, these data imply that in cultured breast cancer models, short-term TZM treatment may inadvertently increase oncogenic fitness probably as a result of adaptation to HER2 - TZM binding.

Abstract PO045: Inhibition of GAS6/AXL improves efficacy of HER2 inhibitor trastuzumab in uterine serous cancer

Abstract Objectives: To determine whether inhibition of GAS6/AXL can improve efficacy to the humanized anti-HER2 monoclonal antibody trastuzumab by decreasing uterine serous cancer cell invasion and cell survival in vitro and tumor burden in vivo. Methods: We treated AXL-expressing and HER2-expressing uterine serous cancer cell lines (ARK1, ARK2) with trastuzumab, AVB-500, or trastuzumab plus AVB-500. Clonogenic cell survival assays were performed. Matrigel invasion assays were used to assess the invasive capacity of ARK2 cells after 24 hours of treatment with vehicle, AVB-500, trastuzumab, or trastuzumab plus AVB-500. A proximity ligation assay was used to examine colocalization of HER2/neu and AXL. Western blots were performed to assess the effects of the individual drugs and combination on phosphorylation of HER2/neu and AXL. An intraperitoneal tumor model with 10 million ARK1 cells was treated with vehicle, AVB-500, trastuzumab, or trastuzumab plus AVB-500 for 35 days. The number, size, and volume of tumor nodules were measured. GraphPad Prism was used for statistical analysis. Results: We found decreased cell survival by clonogenic assays in ARK1 and ARK2 cells treated with trastuzumab plus AVB-500 compared to trastuzumab alone (relative absorbance 0.155 nm vs 0.202 nm, P=0.015). There was significantly less invasion from treatment with trastuzumab plus AVB-500 than with trastuzumab alone (3.2 vs 17.8 invading tumor cells/hpf, P=0.0004). Western blot showed that cells treated with trastuzumab plus AVB-500 had reduced phospho-AXL compared to cells treated with AVB-500 or trastuzumab alone. Furthermore, the proximity ligation assay demonstrated co-localization of the AXL and HER2 receptors in both ARK1 and ARK2 cells indicating a physical closeness that could account for this co-regulation. We found that mice treated with trastuzumab plus AVB-500 developed significantly less tumor burden than mice treated with trastuzumab alone (0.03205 g vs 0.08316 g, P= 0.024), AVB-500 alone (0.0320 g vs 0.1638 g, P&amp;lt;0.0001), and vehicle alone (0.03205 g vs 0.1154 g, P= 0.0395). Conclusions: The GAS6/AXL inhibitor AVB-500 potentiated the effect of trastuzumab to decrease uterine serous cancer cell proliferation and invasion in vitro and tumor burden in vivo likely through AXL and HER2 complexes. Citation Format: Joan Tankou, Michael Toboni, Hollie Noia, Alyssa Oplt, Daniel Wilke, Dineo Khabele, Lindsay Kuroki, Andrea Hagemann, Carolyn McCourt, Premal Thaker, David Mutch, Matthew Powell, Katherine Fuh. Inhibition of GAS6/AXL improves efficacy of HER2 inhibitor trastuzumab in uterine serous cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO045.

Arsenic-induced HER2 promotes proliferation, migration and angiogenesis of bladder epithelial cells via activation of multiple signaling pathways in vitro and in vivo

Arsenic (As) is a known human carcinogen with a hitherto unknown mechanism of action. Dimethylarsinic acid (DMAV) is a methylated metabolite of arsenicals found in most mammals, and long-term exposure to DMAV can lead to bladder cancer in rats. Human epidermal growth factor receptor 2 (HER2) is an oncogenic factor that is overexpressed in bladder cancer, but its role in the initiation and progression of As-induced bladder cancer has not been elucidated. We found that HER2 was up-regulated in human uroepithelial cells treated with arsenite as well as in the bladder tissues of DMAV-exposed rats. HER2 overexpression correlated to increased cell proliferation, epithelial-to-mesenchymal transition (EMT), migration and angiogenesis in vitro. The anti-HER2 monoclonal antibody trastuzumab significantly decreased serum vascular endothelial-derived growth factor (VEGF) levels and that of proliferation-related proteins in the bladder tissues of DMAV-exposed rats. Furthermore, inhibition of HER2, as well as that of the MAPK, AKT and STAT3 pathways, attenuated arsenite-induced proliferation, migration and angiogenesis of human uroepithelial cells, and increased apoptosis rates in vitro. These findings indicate that HER2 mediates the oncogenic effects of As on bladder epithelial cells by activating the MAPK, PI3K/AKT and Src/STAT3 signaling pathways, and is therefore a promising biomarker.

Abstract B60: Isolation of cfDNA and circulating extracellular vesicles allows for biomarker detection in a single aliquot of breast cancer patients’ plasma

Abstract The last few years have witnessed exceptional advances in liquid biopsies based on the isolation and analysis of diverse tumor-derived material circulating in the blood, such as cell-free DNA (cfDNA) and extracellular vesicles (EVs). However, the isolation of different tumor-derived circulating components from a single aliquot of patients’ plasma and the consequent accurate detection of cancer biomarkers is still challenging. By taking advantage of a recently published nickel-based EVs isolation (NBI) protocol (1) that allows for recovery of cfDNA after EVs isolation, we generated a high-sensitivity molecular assay to accurately detect ERBB2 amplification and consequent HER2 overexpression on a limited volume of plasma collected from 20 breast cancer patients (stage I, II, and III) at diagnosis. The amplification of ERBB2 gene and the overexpression of the relevant encoded protein HER2 are currently used in the clinic for breast cancer classification at diagnosis and subsequent treatment decision with the anti-HER2 monoclonal antibody trastuzumab. By performing droplet digital PCR (ddPCR), we were able to detect ERBB2 amplifications in plasma cfDNA from 20 patients selected based on in situ evaluation of tissue biopsies (IHC and FISH). Moreover, we set up an antibody-based affinity reaction to detect HER2 protein on the surface of the isolated EVs that confirmed HER2 overexpression on a subset of patients. Most importantly, we also succeeded in absolute quantification of HER2 transcripts enclosed within EVs by performing ddPCR in samples of patients showing a range of circulating tumor-derived material. The yields and detection performance of this novel methodologic assay were tested on a cohort of healthy individuals (n=20) and on a cohort of triple-negative (and therefore HER2-negative) breast cancer patients (TNBC; n=20; stage: I, II, III). Overall, here we report a pilot study on a novel multimodal noninvasive method for breast cancer-specific biomarker detection from a minimal amount of plasma (1,5mL) integrating cfDNA-derived information with EVs-derived RNA and proteins analysis. This proof of concept may ultimately translate into relevant clinical applications for disease diagnosis as well as for therapy selection and monitoring of disease progression. Reference: 1. Notarangelo M et al. Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse extracellular vesicles from blood. EBioMedicine 2019;43:114–26. Citation Format: Vera Mugoni, Caterina Nardella, Orsetta Quaini, Yari Ciani, Michela Notarangelo, Antonella Ferro, Orazio Caffo, Vito D'Agostino, Francesca Demichelis. Isolation of cfDNA and circulating extracellular vesicles allows for biomarker detection in a single aliquot of breast cancer patients’ plasma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B60.