Non-amplified Metastatic Breast Cancer Research Articles

Abstract 1618: Highly populated phylogenetic reconstruction in patients with hormone receptor positive - HER2 non-amplified metastatic breast cancer

Abstract Background: Tumor progression still needs to be further elucidated in hormone receptor positive (HR+) breast cancer, the most common subtype of breast cancer. Here, leveraging the extensive sampling approach of our postmortem rapid tissue donation program UPTIDER (NCT04531696), we used phylogenic reconstruction to analyze disease progression. Patients and Methods: Thirteen patients with HR+/HER2 non-amplified primary breast cancer, recruited in UPTIDER between 12/2020 and 05/2022, were considered. Shallow whole-genome sequencing was performed on 495 solid samples including a matched germline DNA and archived primary tumor sample for each patient. Log2 coverage ratios were computed with CNVkit and profiles with median absolute pairwise deviation < 0.35 were co-segmented per patient using the copynumber R package. Purity and ploidy were assessed by ABSOLUTE and manually reviewed. Samples with purity > 30% were kept for downstream analysis. Phylogenetic reconstruction was performed using MEDICC2 for each patient. Metrics derived from the trees were: (i) tree length, (ii) trunk length, (iii) trunk ratio as trunk length/tree length*100. Overall survival (OS) was defined as time between primary diagnosis and death. Comparison was done using Wilcoxon test, time to event analysis using cox regression. Endocrine resistance was defined as per the ESMO guidelines (PMID:34678411). Results: 81% (399/495) of the samples passed QC. Phylogenies included a median of 28 samples per patient (range: 9-59) and 10/13 phylogenies included primary samples at diagnosis. Multi-seeding from the primary tumor was observed in 7 patients, suspected in 1, and a single seeding was observed in 2 patients. Median OS was 8.0 years. Higher trunk ratios tended to associate with poorer OS (Hazard ratio: 1.08 per 1% increase, 95% confidence interval: 0.99-1.16, p=0.06). In organs where multiple metastatic lesions were sampled (n= 73 organs, median of 3 samples/organ, range: 2-18 and median of 5 organs per patient, range: 1-10) seeding from at least 2 organs was detected in 48% (35/73) of the organs. Phylogenies from patients with secondary endocrine resistance (7 patients) tended to have a shorter trunk compared to patients with primary endocrine resistance (5 patients, median of 18 and 48 alterations respectively, p=0.15). Conclusion: Branching evolution from the primary tumor was more frequently observed than linear evolution. Phylogenies revealed extensive tumor heterogeneity, questioning treatment decisions based on a single biopsy. Additionally, evaluation of multiple metastatic samples per organ showed seeding from multiple organs in 48% of organs evaluated, indicating greater disease heterogeneity than previously thought. Phylogenetic differences were also noted according to the type of endocrine resistance (primary versus secondary). Citation Format: Francois Richard, Tatjana Geukens, Maxim De Schepper, Amena Mahdami, Karen Van Baelen, Marion Maetens, Gitte Zels, Ha-Linh Nguyen, Anirudh Pabba, Sophoa Leduc, Edoardo Isnaldi, Josephine Van Cauwenberge, Kristien Borremans, Hava Izci, Imane Bachir, Sigrid Hatse, Peter Vermeulen, Evy Vanderheyden, Bram Boeckx, Diether Lambrechts, Ann Smeets, Ines Nevelsteen, Kevin Punie, Patrick Neven, Hans Wildiers, Wouter Van Den Bogaert, Jonas Demeulemeester, Elia Biganzoli, Giuseppe Floris, Christine Desmedt. Highly populated phylogenetic reconstruction in patients with hormone receptor positive - HER2 non-amplified metastatic breast cancer [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 1618.

Selected Articles from This Issue

Neratinib is an irreversible pan-HER inhibitor approved for patients with HER2 amplified breast cancer. MutHER trial evaluated the antitumor activity of neratinib and neratinib in combination with fulvestrant for HER2 non-amplified metastatic breast cancer harboring activating mutations in HER2, a population otherwise not eligible for HER2-targeted therapy. The study demonstrated the antitumor effect of neratinib, especially in invasive lobular breast cancer. Interestingly, responses were observed with the addition of trastuzumab to neratinib upon disease progression, suggesting dual HER2 blockade may be important in targeting HER2 mutation. Further investigation of trastuzumab in combination with neratinib is therefore warranted.Fusions involving the NTRK1/2/3 genes have been identified in many tumor types, several of which often metastasize to the CNS. Entrectinib is a potent TRK inhibitor that was specifically designed to penetrate and remain in the CNS. Demetri and colleagues present the latest results of an integrated analysis of three phase I/II trials: entrectinib continued to induce deep and durable systemic and intracranial responses across tumor types and was well tolerated. Entrectinib could address the need for a CNS-active treatment in NTRK fusion-positive solid tumors; therefore, encouraging broader NTRK fusion testing would improve patient access to effective, personalized treatments.Concurrent radiation therapy (RT) with cetuximab, an anti-EGFR monoclonal antibody (mAb), is a standard treatment for locally advanced head and neck squamous cell carcinoma (HNSCC); however, control in high-risk disease is suboptimal. CTLA-4+ T regulatory cells (Treg) dampen cellular immunity and correlate negatively with clinical outcomes. Ferris and colleagues conducted a phase I trial to evaluate the safety and preliminary efficacy of the CTLA-4 targeting mAb, ipilimumab, with standard, fixed cetuximab-RT. They identified the recommended phase II dose and observed acceptable PFS and OS without cytotoxic chemotherapy. High expression of coinhibitory receptors PD1/LAG3/CD39 on baseline tumor infiltrating Treg was marginally associated with worse PFS. This is the first trial to combine CTLA-4 targeting with definitive cetuximab-RT in HNSCC, integrating immune checkpoint inhibitors into curative-intent RT platforms.Otani and colleagues used immunofluorescence staining and RNA-seq to uncover a significant induction of MDSC cell populations into brains of tumor-bearing mice treated with oHSV. This was mediated by NOTCH induction. In human patients, NOTCH signaling also correlated with a higher myeloid cell infiltration. The tumor infiltrating myeloid cells further spread NOTCH signaling in tumors and also created a CCL-2 feed-forward loop to induce more myeloid cells into the tumors. Increased CCL2 was also observed in serum of GBM patients treated with oHSV. Blockade of NOTCH signaling rescued these immunosuppressive effects and enhanced antitumor immunity.

Disease characterization in liquid biopsy from HER2-mutated, non-amplified metastatic breast cancer patients treated with neratinib

Metastatic breast cancer (mBC) patients have a high risk of progression and face poor prognosis overall, with about one third (34%) surviving five years or more. In rare instances (2–4% of cases) patients with mBC have ERBB2 (HER2) activating mutations but are ERBB2 non-amplified. Neratinib is a potent, irreversible inhibitor that binds HER2 and inhibits downstream signaling. We used the previously validated high-definition single cell assay (HDSCA) workflow to investigate the clinical significance of the liquid biopsy in ERBB2 mutant, non-amplified, post-menopausal mBC patients starting neratinib and fulvestrant combination therapy. Characterization with a comprehensive liquid biopsy methodology (HDSCA) included genomic analysis of both the cell-free DNA (cfDNA) and single circulating tumor cells (CTCs) to monitor tumor evolution and identify potential mutational variants unique to the patient’s clinical response. A limited series of five sequentially enrolled patients presented here were from the MutHER (https://www.clinicaltrials.gov, NCT01670877) or SUMMIT (https://www.clinicaltrials.gov, NCT01953926) trials. Patients had an average of 5.4 lines of therapy before enrollment, variable hormone receptor status, and ERBB2 mutations at diagnosis and during treatment. CTC enumeration alone was not sufficient to predict clinical response. Treatment pressure was shown to lead to an observable change in CTC morphology and genomic instability (GI), suggesting these parameters may inform prognosis. Single cell copy number alteration (CNA) analysis indicated that the persistence or development of a clonal population of CTCs during treatment was associated with a worse response. Hierarchical clustering analysis of the single cells across all patients and timepoints identified distinct aberrant regions shared among patients, comprised of 26 genes that are similarly affected and may be related to drug resistance. Additionally, the genomic analysis of the cfDNA, identified new mutations in ERBB2, PIK3CA, and TP53 that arose likely due to treatment pressure in a patient with poor response, further providing insights on the dynamics of the cancer genome over the course of therapy. The data presented in this small cohort study demonstrates the feasibility of real-time molecular profiling of the cellular and acellular fractions of the liquid biopsy using the HDSCA methodology. Additional studies are necessary to determine the potential use of morphometric and genomic analysis as a prognostic tool to advance personalized oncology.

Abstract CT026: A phase II trial of neratinib (NER) or NER plus fulvestrant (FUL) (N+F) in HER2 mutant, non-amplified (HER2mut) metastatic breast cancer (MBC): Part II of MutHER

Abstract Introduction: The irreversible pan-HER inhibitor NER showed modest single agent activity for HER2mut MBC in Part I of MutHER trial. In Part II, we hypothesized that (1) N+F would improve activity in estrogen receptor positive (ER+) HER2mut MBC due to ER-HER2 crosstalk and (2) dual HER2 blockade by adding trastuzumab at disease progression (PD) could overcome resistance. Methods: Pts with ER+HER2mut MBC were enrolled to 2 cohorts (FUL treated or naive) to receive N+F with diarrhea prophylaxis. ER- pts received NER in an exploratory ER- cohort. Trastuzumab was added at PD if approved by insurance. Simon's Minimax 2-stage phase II design with the primary endpoint of clinical benefit rate (CBR: rates of complete/partial response [CR/PR] plus stable disease [SD] >24 weeks [wks]), with anticipated vs null hypothesis being CBR of 55% vs 35% (FUL treated) or 65% vs 40% (FUL naïve) with 80% power, 1 sided 0.05 alpha, was used. Secondary endpoints included progression free survival (PFS) and adverse events (AEs). Serial blood samples were analyzed for circulating tumor DNA (ctDNA) by Guardant360 for concomitant mutations, HER2mut variant allele frequency (VAF) dynamics, and resistance mechanisms. Results: Between Sep. 2015 and Oct. 2020, 40 pts with HER2mut MBC were enrolled, completing the 1st stage of each ER+ cohort. 35 pts (21 FUL treated, 10 FUL naïve, 4 ER-) were evaluable for response, with median age 63 (35-82) years, 3 (0-12) prior MBC regimen, lobular BC in 13 (37%) and visceral mets in 32 (91%) pts. 21 (68%) ER+ pts had prior CDK4/6 inhibitor. All but 1 pt has come off study due to PD. Table 1 shows the efficacy by cohort. Further enrollment is closed per protocol. Adding trastuzumab at PD induced CB in 4 (3 PR, 1 SD≥24 wks) of 5 pts (1 ER-, 4 ER+), with PFS 28 (95% CI 18~NA) wks. Common AEs across cohorts were diarrhea (G3 21%) and fatigue (G3 5%). No G4 AEs. ctDNA HER2mut was detected in 72% (23/32) baseline (BL) samples tested. In pts with paired samples, HER2mut VAF decreased at C1D15/C2D1 from BL in 75% (15/20) and rose in 89% (16/18) at PD. Acquired HER2mut, including the T798I gatekeeper mutation, were detected in 2 pts at PD. Mutations in TP53 (53%), PIK3CA (43%), and CDH1 (35%) were common, but none significantly associated with PFS in all or ER+ pts. Conclusions: NER, or N+F, is active for HER2mut MBC with good tolerability. Adding trastuzumab at PD induced further response, supporting dual HER2 blockade for HER2mut MBC. Table 1.EfficacyCohortFUL treatedFUL naïveER-Best Response, n evaluablen = 21n = 10n = 4CR, n100PR, n431SD (≥ 24 wks), n300SD (< 24 wks), n1030PD, n343CBR, n with CB/total n evaluable, % (95% CI)8 of 20*, 40% (19~64%)3 of 10, 30% (7~65%)1 of 4, 25% (0.6~81%)mPFS (95% CI), wks, ITT (n)24 (16~31) wks, (n = 24)20 (8~NA) wks, (n = 11)8.5 (8~NA) wks, (n = 5)*20 of 21 pts are evaluable for CBR in the FUL treated Cohort as 1 pt had SD as best response and treatment is still ongoing. ITT (intent to treat) population is used for mPFS estimate. Citation Format: Cynthia X. Ma, Jingqin Luo, Rachel A. Freedman, Timothy Pluard, Julie Nangia, Janice Lu, Frances Valdez-Albini, Melody Cobleigh, Jason Jones, Nancy U. Lin, Eric Winer, P. Kelly Marcom, Shana Thomas, Jill Anderson, Brittney Haas, Kimberly M. Hamann, Richard Bryce, Alshad S. Lalani, Lisa Carey, Matthew Goetz, Feng Gao, Gretchen Kimmick, Mark Pegram, Matthew J. Ellis, Ron Bose. A phase II trial of neratinib (NER) or NER plus fulvestrant (FUL) (N+F) in HER2 mutant, non-amplified (HER2mut) metastatic breast cancer (MBC): Part II of MutHER [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT026.

Abstract PS4-13: Irreversible inhibition of HER2 activating mutations with neratinib enhances the pre-clinical efficacy of trastuzumab emtansine and trastuzumab deruxtecan

Abstract Background: HER2 activating mutations occur in 2-5% of metastatic breast cancer (MBC) patients, and three phase II or basket clinical trials have shown that the irreversible pan-HER tyrosine kinase inhibitor, neratinib, has good single agent efficacy for HER2 mutated MBC patients. Current trials are combining neratinib with other targeted therapies to increase response rate and progression free survival for these patients. Methods: We established patient derived xenografts (PDX) and organoids from two patients with HER2 mutated, non-amplified MBC and used them to test neratinib with the antibody drug conjugates (ADC’s), trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd), both in 3D culture and in vivo. Real time, in vivo uptake of these ADC’s was visualized with a near infrared fluorophore. Results: PDX lines WHIM51 and WHIM64 were established from ER+, HER2 non-amplified MBC patients that had HER2 activating mutations. WHIM51 has HER2 exon 20 insertion mutation at amino acid 776 (ERBB2 A775_G776insYVMA) and WHIM64 has a HER2 L869R missense mutation, both of which are located in the HER2 tyrosine kinase domain. Both of these HER2 mutations have been previously characterized and are known activating mutations. Organoids were established from both PDX’s and were grown in 3D culture. Drug combination testing of neratinib with T-DM1 in 3D culture showed strong synergy and the mechanism was explored. We demonstrate that neratinib and other irreversible HER2 inhibitors increase the endocytic uptake of T-DM1, but this effect does not occur with the reversible HER2 inhibitors, tucatinib and lapatinib. Real time, in vivo uptake of T-DM1 was measured by labeling the ADC with a near infrared fluorophore and we observed statistically significant increase in T-DM1 uptake with neratinib pre-treatment. Combining neratinib with T-DM1 increased apoptosis at day 3 post-treatment and enhanced tumor shrinkage. With the FDA approval of T-DXd at the end of 2019, we hypothesized that this same mechanism may apply to neratinib combined with T-DXd. We have tested both the combinations of neratinib + T-DXd and neratinib + T-DM1 in vivo in both HER2 mutant PDX’s and observed statistically significant tumor regression with the neratinib + ADC combinations as compared to either T-DXd or T-DM1 on its own. Conclusions: Neratinib increases the endocytosis of trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd), thereby increasing tumor cell kill and causing greater tumor regression in HER2 mutated MBC. These data provide preclinical justification for trials of neratinib plus HER2 ADCs including T-DXd or T-DM1 in HER2 mutant or HER2+ MBC. Further, this mechanism of neratinib stimulated HER2 endocytosis may also apply to HER2 low MBC. Citation Format: Ron Bose, Shunqiang Li, Tina M. Primeau, Maureen K. Highkin, Ashley R. Tipton, Nagalaxmi Vemalapally, Xuefeng Gao, Gail Sudlow, Irmina Diala, Yu Tao, Jingqin Luo, Ian Hagemann, Chieh-Yu Lin, Richard P. Bryce, Alshad S. Lalani, Samuel Achilefu, Cynthia X. Ma. Irreversible inhibition of HER2 activating mutations with neratinib enhances the pre-clinical efficacy of trastuzumab emtansine and trastuzumab deruxtecan [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-13.

Metronomic treatment of vinorelbine with oral capecitabine is tolerable in the randomized Phase 2 study XeNa including patients with HER2 non-amplified metastatic breast cancer

Background Metronomic treatment is hypothesized to be less toxic and more effective as compared to standard maximal tolerable dosing treatment in metastatic cancer disease. Material and methods We tested the metronomic treatment principle with vinorelbine in a randomized phase 2 setting combined with standard capecitabine treatment in the XeNa trial with Clinical Trials.gov identifier number: NCT0141771. 120 patients with disseminated HER2 non-amplified breast cancer were included. Randomization was between Arm A: vinorelbine 60 mg/m2 day 1 + day 8 in the first cycle followed by 80 mg/m2 day 1 + day 8 in the following cycles or Arm B: vinorelbine 50 mg three times a week. Capecitabine 1000 mg/m2 twice a day for days 1–14 was administered in both arms. Results The treatment was generally well-tolerated. The response rate (RR) was 24% (arm A) versus 29% (arm B) (p = .67). The clinical benefit rate (CBR) 46.8% (arm A) versus 51.7% (arm B) (p = .72). We found a median progression-free survival (PFS) of 7.1 months (95% confidence interval [CI] 3.9–10.3) in arm A and 6.3 months (95% CI 4.1–8.5) in arm B (p = .25) whereas median overall survival (OS) was 23.3 months (95% CI 20.2–26.4) in arm A and 22.3 months (95% CI 14.3–30.3) in arm B (p = .76). Conclusions We confirmed that the combination of vinorelbine and capecitabine was well tolerated. Metronomic treatment can be used with acceptable adverse events (AEs), but we did not find significant difference in the effect compared to the standard treatment.

Clinical activity of MCLA-128 (zenocutuzumab) in combination with endocrine therapy (ET) in ER+/HER2-low, non-amplified metastatic breast cancer (MBC) patients (pts) with ET-resistant disease who had progressed on a CDK4/6 inhibitor (CDK4/6i).

1037 Background: MCLA-128 (zenocutuzumab) is an ADCC-enhanced humanized bispecific antibody targeting HER2 and HER3 and potently blocking HER3-ligand induced receptor dimerization. Upregulation of Her2:Her 3 pathway is a means of resistance to ET in HR+ breast cancer, indicating a potential role for MCLA-128. In preclinical studies, the combination of MCLA-128 with ET in breast cancer xenografts outperformed single drug treatments. The current study explores the use of MCLA-128 to rescue pts with ET-resistant MBC who have progressed on a CDK4/6i. Methods: This phase II, open-label trial planned for up to 40 evaluable women with HR+, HER2 low (IHC 1+/IHC 2+ with negative FISH) MBC, who had progressed on a CDK4/6i and up to 3 lines of ET, who had received ≤ 2 chemotherapy regimens in the metastatic setting. Pts received MCLA-128 (750 mg, 2h IV, flat dose) q3w combined with last ET on which the pt had previously progressed immediately prior to study entry. Disease control rate (DCR; RECIST 1.1, per investigator), best overall response (BOR), overall response rate (ORR), safety, and PK, are evaluated. Data cut off was 14Nov2019. Results: 48 pts were treated, all of whom had progressed on a CDK4/6i. Pts had received a median 2 prior ET lines (range 1-5) and 1 line (range 1-3) of chemotherapy. Pts had a median number of 3 metastatic sites (range 1-6) and 42 (88%) had visceral involvement. Among 42 pts evaluable for efficacy, DCR was 45% (90% CI 32-59) with 2 pts having unconfirmed PR and 19 pts SD as BOR. Common related AEs (all grades; G3-4) were asthenia/fatigue (27%; 2%), diarrhea (25%; 0), nausea (21%; 0). No clinically significant LVEF decline was seen. At the end of cycle 1, mean trough level of MCLA-128 was 15.5 µg/mL, and mean terminal half-live was 102 h (n = 19-21). Data on the primary endpoint, clinical benefit rate at 24 weeks, and biomarkers will be provided. Conclusions: The addition of MCLA-128 to the last line of ET showed clinical activity after ET+CDK4/6i failure and a favorable safety profile. Clinical trial information: NCT03321981 .

Outcome of Everolimus-Based Therapy in Hormone-Receptor-Positive Metastatic Breast Cancer Patients After Progression on Palbociclib.

Background:Despite the approval of mTOR inhibitor everolimus and CDK4/6 inhibitors in the management of hormone-receptor-positive HER2 non-amplified metastatic breast cancer (HR+ HER2-MBC), the optimal sequence of therapy is unclear. There are no clinical data on efficacy of everolimus in HR+ HER2-MBC after cancer progresses on CDK4/6 inhibitors.Objective:The objective of this study is to find the efficacy of everolimus in HR+ HER2-MBC after they progress on a CDK4/6 inhibitor palbociclib.Methods:This is a retrospective, 2-institute review of HR+ HER2-MBC from Jan 2015 to March 2018 treated with everolimus after progression on palbociclib. Primary end point was median progression-free survival (PFS), secondary end points objective response rate (ORR), clinical benefit ratio (CBR), and overall survival (OS).Results:Out of 41 women with median age 61 years (33, 87) enrolled, 66% had received adjuvant systemic therapy, 61% had visceral disease, and 95% had prior nonsteroidal aromatase inhibitors. About 83% women had 3 or more chemotherapy or hormonal therapies prior to everolimus. Kaplan-Meier estimates showed a median PFS of 4.2 months (95% confidence interval [CI]: 3.2-6.2). The median OS was 18.7 months (95% CI 9.5 to not reached). Objective response rate and CBR were both 17.1%.Conclusion:Everolimus was associated with modest PFS and ORR in HR+ HER2-MBCs postprogression on palbociclib.

Abstract 4527: Patient-derived organoids and xenografts identify neratinib plus HER2 antibody drug conjugate as a synergistic drug combination for HER2 mutated, nonamplified metastatic breast cancer

Abstract HER2 activating mutations are a novel, druggable genomic alteration in metastatic breast cancer (MBC). These HER2 mutations are predominantly found in HER2 gene amplification negative, hormone receptor positive breast cancers. We have previously demonstrated that HER2 mutations can be potently inhibited by the second generation, irreversible pan-HER tyrosine kinase inhibitor, neratinib (Bose et al., Cancer Discovery 2013). Further, we performed a phase II clinical trial to treat HER2 mutated MBC and we found that neratinib monotherapy produced a clinical benefit rate of 31% and progression free survival (PFS) of 16 weeks in a heavily pre-treated patient population (Ma et al., Clin. Can. Res. 2017). A second clinical trial, the SUMMIT trial (Hyman et al., Nature 2018), similarly showed a response rate of 32% and median PFS of 3.5 months for neratinib monotherapy for HER2 mutated, metastatic breast cancer. The objective of the current study is to explore novel combination strategies to improve the efficacy of neratinib in HER2 mutated breast cancer. In order to accelerate progress on testing multiple drug combinations, we developed organoids from two patient-derived xenografts (PDX’s) of HER2 mutated, ER positive metastatic breast cancer from our institution. We found that the ex vivoculture of these patient-derived organoids provides a platform to rapidly perform drug screens and drug combination testing on a scale that cannot be matched by other existing experimental platforms for patient-derived samples. The drug sensitivity of these organoids cultured ex vivo recapitulates the data previously obtained with transfected cell lines and in vivo experiments using PDX’s. Further, multiple drug combinations can be tested on these organoids in just two weeks, which is much shorter than the four to six months required for the corresponding slow-growing ER positive, breast cancer PDX’s that they are derived from. Strong, single agent activity was seen with neratinib, the HER2 antibody drug conjugate (ADC) ado-trastuzumab emtansine (T-DM1), and the chemotherapy drug vinorelbine. Therefore, we tested combinations of neratinib plus the HER2 ADC and neratinib plus vinorelbine on these patient derived organoids. Neratinib plus HER2 ADC showed a strong drug synergy in both HER2 mutated organoids, as judged by the Loewe model of drug synergy. Prior publications suggest that the mechanism of action of neratinib in this combination is by increasing HER2 ubiquitylation and endocytic degradation, which will increase the uptake of the ADC that binds to HER2. We are now performing 384 well drug screens with these HER2 mutated, ER positive metastatic breast cancer organoids, and the results of the screens will be shown in our presentation. Citation Format: Shunqiang Li, Highkin Maureen, Tina M. Primeau, Stephanie L. Pratt, Irmina Diala, Richard E. Cutler, Grace Mann, Alshad S. Lalani, Cynthia X. Ma, Ron Bose. Patient-derived organoids and xenografts identify neratinib plus HER2 antibody drug conjugate as a synergistic drug combination for HER2 mutated, nonamplified metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4527.

Abstract 4459: Morphologic and genomic characterization of circulating tumor cells in patients with ERBB2 mutant HER2 non-amplified metastatic breast cancer treated with neratinib

Abstract Metastatic breast cancer patients have a high risk of progression and face poor prognosis overall, with about one third (34%) surviving 5 years or more. In rare instances (2-4% of cases) patients with metastatic breast cancer have ERBB2 (HER2) activating mutations, but are HER2 negative (non-amplified. Neratinib is a potent, irreversible inhibitor that binds HER2 and inhibits downstream signaling. We used the previously validated high-definition single cell analysis (HD-SCA) workflow to investigate the clinical significance of circulating tumor cells (HD-CTCs) in HER2-mutant, HER2 non-amplified, post-menopausal metastatic breast cancer patients starting neratinib and fulvestrant combinational therapy. Genomic analysis of the cell-free DNA (cfDNA) and single cell HD-CTCs was conducted to monitor tumor evolution and identify potential mutational variants unique to the patient’s clinical response. A subset of 5 patients presented here were from the MutHER (NCT01670877) and/or SUMMIT (NCT01953926) trials. Patients had an average of 5.4 lines of therapy before enrollment, variable hormone receptor status, and ERBB2 mutations at diagnosis and during treatment. HD-CTC enumeration alone was not sufficient to predict clinical response. Interestingly, treatment pressure was shown to lead to an observable change in HD-CTC morphology and genomic instability, suggesting these parameters may inform prognosis. Single cell copy number variation (CNV) analysis indicated that the persistence or development of a clonal population of HD-CTCs during treatment was associated with a worse response. Hierarchical clustering analysis of the single cells across all patients and timepoints identified distinct aberrant regions shared among patients, comprised of 26 genes that are similarly affected and may be related to drug resistance. Additionally at the cfDNA level, we identified new mutations in ERBB2, PIK3CA, and TP53 that arose due to treatment pressure in a patient with poor response, further informing us on the dynamics of the cancer genome over the course of therapy. The data presented in this small cohort study demonstrates the feasibility of real-time molecular profiling of the cellular and acellular fractions of the liquid biopsy using the HD-SCA platform. Additional insight is warranted to determine the potential use of morphometric and genomic analysis as a prognostic tool to advance personalized oncology. Citation Format: Stephanie Nicole Shishido, Rahul Masson, Lisa Welter, Liya Xu, Anishka D'Souza, Darcy Spicer, James Hicks, Janice Lu, Peter Kuhn. Morphologic and genomic characterization of circulating tumor cells in patients with ERBB2 mutant HER2 non-amplified metastatic breast cancer treated with neratinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4459.

A phase Ib dose allocation study of oral administration of lucitanib given in combination with fulvestrant in patients with estrogen receptor-positive and FGFR1-amplified or non-amplified metastatic breast cancer

PurposeThe primary objective of this multicentric dose allocation and dose expansion study was to determine the MTD and the DLTs of the lucitanib (a tyrosine kinase inhibitor of the FGFR/VEGFR/PDFGR pathways)/fulvestrant combination.MethodsPostmenopausal women with ER+/HER2− mBC, who have relapsed during or after treatment with fulvestrant, were eligible. The study had a dose allocation part to assess the tolerability of the combination followed by a dose expansion part.ResultsEighteen patients with ER+, mBC were enrolled; median age was 66 years, 50% had a PS: 0 and all had received previous endocrine treatment. The study was prematurely terminated after 18 patients (15 in part 1 and 3 in part 2) based on preclinical experiments that failed to confirm the hypothesis that addition of lucitanib would reverse sensitivity to endocrine treatments. Based on data of global lucitanib development, it was decided to stop the dose allocation at 12.5 mg and to start the dose expansion part at 10 mg/day. The most common grade ≥ 3 toxicities (> 10% of patients) were hypertension (78%) and asthenia (22%). All patients required at ≥ 1 interruption, 13 patients (72%) required ≥ 1 dose reduction. Three patients (72%) withdrew from the study for AEs (at 10 mg). Three patients achieved a confirmed PR (10 mg n = 1; 12.5 mg n = 2).ConclusionAlthough the combination is feasible it requires close monitoring of the patients for the management of adverse events. Further investigation is required to better understand the potential role of FGFR inhibition in reversing resistance to endocrine treatment.

Emerging data and future directions for CDK4/6 inhibitor treatment of patients with hormone receptor positive HER2-non-amplified metastatic breast cancer.

Cyclin-dependent kinase (CDK4/6) inhibitors in combination with endocrine therapy are currently the optimal first line treatment for hormone receptor (HR) positive, human epidermal growth factor receptor 2 (HER2) non-amplified metastatic breast cancer (MBC). However, not all patients benefit from this treatment and all patients will inevitably progress. Identifying therapeutic strategies in this setting is therefore of immediate clinical importance. We present an overview of the mechanisms of resistance to CDK4/6 inhibitors and review potential biomarkers that may guide therapy selection. We also discuss the use of CDK4/6 inhibitors in the context of non-HR-positive/HER2-non-amplified breast cancer and in combination with therapies other than endocrine therapy.

Outcome of everolimus based therapy in hormone receptor positive metastatic breast cancer patients after progression on palbociclib combination.

1064Background: BOLERO 2 trial showed improved progression free survival (PFS) with everolimus (EV) + exemestane combination over exemestane alone in hormone receptor positive, HER2 non-amplified m...

Efficacy of Palbociclib Combinations in Hormone Receptor–Positive Metastatic Breast Cancer Patients After Prior Everolimus Treatment

PurposeOutcome data on hormone receptor positive (HR+), human epidermal growth factor receptor 2 (HER2) nonamplified (HER2−) metastatic breast cancer (MBC) treated with palbociclib after treatment with everolimus are lacking. The PALOMA-3 trial, showing benefit of palbociclib plus fulvestrant compared to fulvestrant alone in HR+HER2− MBC after progression while receiving endocrine therapy excluded women previously treated with everolimus. The objective of this study was to examine outcomes of HR+HER2− MBC with prior exposure to everolimus while receiving palbociclib-based therapy. Patients and MethodsA retrospective, single-institute review was conducted of HR+HER2− MBC from January 2014 to November 2016 in patients treated with palbociclib after prior treatment with everolimus. Progression-free survival (PFS) was defined as the time from initiation of palbociclib to the date of progression as determined by the treating physician based on radiologic, biochemical, and/or clinical criteria. Response rates were determined on the basis of available radiologic data. Objective response rate (ORR) was defined as the rate of any complete or partial responses; clinical benefit rate (CBR) was the rate of complete response, partial response, or stable disease for at least 24 weeks. ResultsTwenty-three patients with a mean (range) age of 68 (42-81) years were identified. Kaplan-Meier estimate showed median PFS of 2.9 months (95% confidence interval, 2.1-4.2); ORR was 0 of 23 and CBR was 4 (17.4%) of 23. In the PALOMA-3 trial, median PFS, ORR, and CBR of palbociclib cohort were 9.5 months (95% confidence interval, 9.2-11.0), 19%, and 67%, respectively. ConclusionThere is a limited clinical activity of palbociclib combinations after progression with everolimus combination therapy. Further studies are necessary to confirm these findings.

Circulating tumor cells and genomic profiling of patients with ERBB2 mutant, HER2 non-amplified metastatic breast cancer treated with neratinib.

e12554 Background: ERBB2 mutations in the absence of gene amplification are rare, with an incidence of 2-4%. Neratinib is a HER2/EGFR tyrosine kinase inhibitor being evaluated for use in ERBB2 mutated breast cancer. Neratinib has been found to have clinical activity on heavily pre-treated ERBB2 mutant breast cancer patients. We are evaluating the response and genomic profiles of 3 postmenopausal patients with metastatic ERBB2 mutant/non-amplified breast cancer receiving neratinib and fulvestrant NCT01953926, NCT01670877. Methods: Samples were collected at different points during treatment and CTCs were identified. Other representative cells were tracked but not classified as CTCs. CD45-CK+ cells with cytoplasmic and/or nuclear apoptosis were defined as CTC-Apoptotic. CD45- cells expressing little to no CK but otherwise meeting morphological criteria for CTCs are CTC-LowCK. CD45-CK+ cells with small nuclear size are CTC-SmallCK. Single CTCs will be analyzed using whole genome copy number variation to determine chromosomal alterations. Results: The patients had an average of 4.7 lines of therapy for metastatic disease before neratinib. Two had stable disease and one had progression. The patient who progressed had a rise in the number of CTCs from 37 to 52 cells/ml and drop in apoptotic cells from 5 to 0 cells/ml. Conclusions: The high CTC count of the patient who progressed may suggest more aggressive disease. The drop in the apoptotic cell count may correlate with a failure to respond to therapy. Samples have been sent for copy number variation profiling. The goal is to identify any genomic amplifications or deletions associated with clinical response and progression after targeted therapy. We hope to demonstrate the timeframe of tumor evolution in response to therapy and provide a framework for the use of fluid biopsies to monitor disease progression. [Table: see text]

Phase II trial of neratinib for HER2 mutated, non-amplified metastatic breast cancer (HER2mut MBC).

516Background: HER2 mutation occurs in ~2% of breast cancer (BC). Preclinical models suggested neratinib, an irreversible HER2/EGFR tyrosine kinase inhibitor, could be an effective treatment. This trial was conducted to assess the clinical benefit rate (CBR), defined as complete response (CR), partial response (PR) or stable disease (SD) ≥ 6 months (mo.), for neratinib in patients (pts) with HER2mutMBC (NCT01670877). Methods: This single arm phase II trial employed the Simon’s Optimal 2-stage design (1st stage: n=10, 2nd stage: n=19) to detect CBR of 20% versus the null hypothesis of 5% (80% power, 1-sided alpha 0.05). The primary endpoint is met if ≥ 4 CB is observed. Eligibility required the presence of somatic HER2mut and absent HER2 amplification. Activating HER2mut by in vitro study was required in the 2nd stage. Pts received neratinib 240 mg PO daily (28-day cycle) with prophylactic loperamide. Response was assessed every 2 cycles by RECIST 1.1. Enrollment was stopped early due to a protocol amendme...

Abstract OT1-03-03: FINESSE - An open, 3-cohort, phase II trial testing oral administration of lucitanib in patients with FGFR1-amplified or non-amplIfied oestrogeN rEceptor poSitive metaStatic breast cancEr

Abstract Background: Lucitanib is a potent, oral inhibitor of the tyrosine kinase activity of Fibroblast Growth Factor Receptors 1-3 (FGFR1-3), Vascular Endothelial Growth Factor Receptors 1-3 (VEGFR1-3) and Platelet-Derived Growth Factor Receptors α/β (PDGFRα/β). FGF aberrancy, as defined by amplification of either FGFR1, or 11q (containing FGF ligands 3, 4, CCND1, and 19), or both, is a hallmark genomic alteration that can be observed in up to 25% of patients with breast cancer. In a phase I clinical trial of lucitanib at daily doses of 5 to 20 mg, heavily pretreated patients with advanced breast cancer patients and FGF aberrancy experienced an objective response rate (ORR) of 50% and a median progression-free survival (PFS) over 9 months (Soria et al, 2014). This compelling clinical activity has led to the initiation of a global clinical development program for lucitanib in breast cancer. Trial design: this is a phase II trial testing the efficacy of lucitanib at the dose of 15 mg daily in patients with ER+/HER2- metastatic breast cancer who have received at least one first-line systemic anticancer therapy in the metastatic setting. After informed consent, metastatic tissue (fresh biopsy or archival) is centrally evaluated by FISH for FGFR1- and/or 11q- amplification. Based on FISH results, patients are allocated to cohort 1 (FGFR1-amp), cohort 2 (11q-amp) or cohort 3 (neither). Patients with dual amplification are allocated to cohort 1. The primary objective is to evaluate the ORR of single agent lucitanib in the three cohorts. Secondary objectives include clinical benefit rate, PFS, safety and pharmacokinetics in addition to exploratory biomarker analyses. A Simon two-stage design will be used for each of the cohorts to test the null hypothesis that the ORR is 5% or less versus 20% using a one-sided test with 5% level of significance and 90% power. In each cohort separately, an initial 21 patients with measurable disease at baseline will be assessed at the end of stage 1. If at least 2 patients respond per the pre-specified criteria, this cohort will accrue additional 20 patients. The null hypothesis will be rejected if there are at least 5 responders among all 41 patients. Eligibility Criteria: ER+/HER2- metastatic breast cancer who have received at least a first line of systemic anticancer therapy and no more than 2 line of chemotherapy with or without targeted therapy in the metastatic setting and have ECOG performance status ≤ 2. Patients with uncontrolled hypertension and at risk of developing hypertension related complications are not eligible. Conclusion: FINESSE is a phase II trial testing lucitanib, a multikinase inhibitor, in three selected populations in order to investigate the ORR in FGFR1 or 11q amplified or non-amplified populations and to explore the role of FGFR1 or 11q amplifications through correlative translational analyses. As of May 21st 2015, 40 patients have been enrolled, 19 of them in the FGFR1-amplified arm. Citation Format: Andre F, Daly F, Azim Jr HA, Agrapart V, Fumagalli D, Gingras I, Guitart M, Lange A, Turner NC, Pierrat M-J, Loibl S, Poirot C, Curigliano G, Loi S, Pallis A, Piccart M, Cortes J. FINESSE - An open, 3-cohort, phase II trial testing oral administration of lucitanib in patients with FGFR1-amplified or non-amplIfied oestrogeN rEceptor poSitive metaStatic breast cancEr. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT1-03-03.

Abstract PD5-05: Neratinib for ERBB2 mutant, HER2 non-amplified, metastatic breast cancer: Preliminary analysis from a multicenter, open-label, multi-histology phase II basket trial

Abstract Background: Somatic ERBB2 (HER2) mutations occur in approximately 2% of patients with breast cancer and are found in a predominantly mutually exclusive manner with ERBB2 amplification. These mutations result in increased signaling and oncogenic transformation. Neratinib, a pan-ERBB irreversible tyrosine kinase inhibitor, potently inhibits growth of ERBB2 mutant tumor cell lines and xenografts. An ongoing signal-seeking phase II 'basket' study is evaluating neratinib in patients with multiple histologies harboring ERBB2 mutations (NCT01953926). Novel mutations identified in enrolled patients were characterized for biologic activity in a variety of in vitro model systems. A preliminary analysis of the HER2 non-amplified metastatic breast cancer cohort is presented. Methods: Patients with ERBB2 mutant metastatic breast cancer documented by local testing methods received single-agent oral neratinib 240 mg once daily until progression or intolerable toxicity. High-dose loperamide prophylaxis was mandatory during cycle 1. The primary endpoint was the objective response rate at 8 weeks, defined using anatomic (RECIST 1.1) and/or metabolic (PET Response Criteria) assessments. Secondary endpoints were best overall response rate, clinical benefit rate, progression-free survival, duration of response, and safety. Results: 17 patients with metastatic breast cancer were enrolled and received neratinib (13 patients are evaluable for efficacy to date). Patients had a median of 3 prior anticancer regimens. Other baseline characteristics were: median age 59 years; bone involvement 71%; visceral disease 82%. Tumor characteristics were: ductal/lobular 76%/24%; ERBB2 mutation single nucleotide variants/indels 82%/18%; HER2 amplified/non-amplified 0%/100%; hormone receptor positive/negative 82%/18%. Five patients (39%) had an objective response at 8 weeks (95% CI 14–68%). In the patients who responded, ERBB2 mutations were: 1 complete response (L755S); 4 partial responses (L755S, V777L, V777L, and L869R). The most common all-grade adverse events (in ≥15% of patients) across all cohorts (n=93) were: diarrhea (62%), fatigue (28%), nausea (36%), vomiting (30%), anemia (15%), and constipation (29%). The most common grade 3/4 adverse event was diarrhea (14%, all grade 3). Updated efficacy results, centralized genomic analyses on archival tumor samples, and in vitro characterization of novel ERBB2 mutants will be presented. Conclusions: Single-agent neratinib shows encouraging signs of clinical activity in patients with heavily pretreated, ERBB2 mutant, HER2 non-amplified metastatic breast cancer. The breast cancer cohort demonstrated sufficient activity to meet the study's pre-specified efficacy requirements according to a Simon's two-stage design, and suggests that a confirmatory trial of neratinib for targeting ERBB2 driver mutations in metastatic breast cancer is warranted. Safety was acceptable and diarrhea was manageable with loperamide prophylaxis. Citation Format: Hyman DM, Piha-Paul SA, Rodón J, Saura C, Puzanov I, Shapiro GI, Loi S, Joensuu H, Hanrahan AJ, Modi S, Lalani AS, Xu F, Garza SJ, Cutler RE, Bryce R, Meric-Bernstam F, Baselga J, Solit DB. Neratinib for ERBB2 mutant, HER2 non-amplified, metastatic breast cancer: Preliminary analysis from a multicenter, open-label, multi-histology phase II basket trial. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD5-05.

Abstract OT1-1-03: Phase Ib dose allocation study of oral administration of lucitanib given in combination with fulvestrant in patients with estrogen receptor-positive and FGFR1-amplified or non-amplified metastatic breast cancer (INES)

Abstract Background: Lucitanib is a potent, oral inhibitor of the tyrosine kinase activity of fibroblast growth factor receptors 1-3 (FGFR1-3), vascular endothelial growth factor receptors 1-3 (VEGFR1-3) and platelet-derived growth factor receptors α/β (PDGFRα/β). FGF aberrancy, as defined as FGFR1-or 11q- amplification, is a hallmark genomic alteration in breast cancer, observed at a frequency of up to 25% of patients and is strongly associated with luminal B type. Breast cancer patients with measurable disease and FGF aberrancy treated in the ongoing Phase 1/2 clinical trial of lucitanib monotherapy experienced an overall response rate of 50% (6 out of 12 patients). FGFR1-knock down was shown to decrease cell proliferation and reverse resistance to endocrine therapy in a FGFR1-amplified breast cancer cell line, hence supporting the idea of combining lucitanib with an endocrine agent such as fulvestrant, at the time of resistance. This has led to this Phase Ib study of lucitanib in combination with fulvestrant in metastatic breast cancer. Trial design: INES is a multicenter, open-label, 2 -part study to assess the tolerability of lucitanib in terms of Maximum Tolerated Dose (MTD) and Dose-Limiting Toxicities (DLTs) when administered with fulvestrant. A Continual Reassessment Method (CRM) will be used for the 1st part. A minimum of 3 patients will be enrolled at the initial dose level of 10 mg daily in combination with fulvestrant. Additional doses of 12.5 mg and 15 mg of lucitanib will be tested with the option of deescalating to 7.5 mg in case of DLTs. A minimum of 9 patients will be included at the MTD. In the 2nd part, 2 cohorts will be opened: fourteen FGF+ patients (FGFR1-or 11q- amplification), and fourteen non-amplified patients. All patients will receive fulvestrant 500 mg monthly and lucitanib at the recommended dose (RD) until unacceptable toxicity according to the investigator, disease progression or patient withdrawal. The main objective is to identify the Phase II RD when lucitanib is combined with fulvestrant. Secondary objectives include determination the Pharmacokinetic (PK) profile of lucitanib and metabolites; Measurement of tumour response; Description of the pharmacodynamic (PD) profile of lucitanib and investigation of any potential exposure dose-response relationships for safety, efficacy and PD. Eligibility Criteria: Patients with estrogen receptor-positive, HER2 negative, breast cancer after progression or recurrence on prior therapy including fulvestrant. Patients should have ECOG performance status 0 or 1. Patients with uncontrolled hypertension are not eligible. For part 2, the presence of a metastatic site for biopsy to assess the presence of FGFR1- and/or 11q- amplification, which will be analysed centrally using FISH, is required. Conclusion: INES is a phase Ib trial testing lucitanib in combination with fulvestrant in order to select the RD for phase II and seek preliminary efficacy signal in FGFR1- or 11q- amplified or non- amplified patients. As of June 2014, 3 patients have been enrolled in the 10 mg dose escalation cohort. Citation Format: Mario Campone, Thomas Bachelot, Fabrice André, Chadi Saba, Valérie Agrapart, Marie-Jeanne Pierrat, Frédéric Dubois, Thibault Chesnel, Camille Poirot. Phase Ib dose allocation study of oral administration of lucitanib given in combination with fulvestrant in patients with estrogen receptor-positive and FGFR1-amplified or non-amplified metastatic breast cancer (INES) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT1-1-03.

FINESSE: An open, three-cohort, phase II trial testing oral administration of lucitanib in patients with FGFR1-amplified or nonamplified estrogen receptor-positive metastatic breast cancer.

TPS1134 Background: FGF aberrancy is observed in approximately 25% of breast cancer (BC) patients. Preclinical studies demonstrated that targeting FGFR could lead to antitumor effects. Lucitanib is...