Abstract B028: Functional precision medicine in challenging breast cancer subtypes: Developing preclinical models for tailored therapy in complex and rare histologies

Purpose: Metaplastic breast cancer (MpBC) is a highly chemoresistant and aggressive breast cancer variant with limited therapeutic options. Most MpBCs harbor a triple-negative breast cancer (TNBC) phenotype, yet have a worse prognosis compared to TNBC. We and others discovered two key molecular alterations in MpBC associated with poor overall survival: 1) hyperactivation of the phosphoinositide 3-kinase (PI3K) signaling pathway and 2) enhanced inducible nitric oxide synthase (iNOS) signaling. Nitric oxide produced from iNOS is able to activate various oncogenic pathways, such as PI3K signaling. In MpBC, both PI3K and NOS signaling pathways may synergistically work to enhance chemoresistance. We propose to evaluate whether pan-NOS inhibitor NG-monomethyl-l-arginine (L-NMMA) augments the efficacy of alpha isoform-specific PI3K inhibitor alpelisib in MpBC in vitro and in vivo models. Methods: MpBC cell lines (Hs578T, BT549, SUM159) and MpBC Patient-Derived Xenograft (PDX) models were used in this study. Cell viability was determined with SRB Assay and the combination index was evaluated using Chou-Talalay Method. Flow cytometry was conducted to evaluate cellular apoptosis and cell cycle distribution analysis. In vivo efficacy of NOS and PI3K inhibition was assessed using MpBC PDX models. Transcriptional analysis of PDX tumors treated with vehicle control, single-agent (L-NMMA or alpelisib), or combination therapy (L-NMMA+alpelisib) was conducted to reveal top enriched pathways. Immunoblotting and immunofluorescence analyses of treated MpBC cell lines and PDX tissues were performed. Results: Combination of L-NMMA and alpelisib synergistically reduced cellular proliferation (CI<1), enhanced apoptosis, depleted nucleotide pools, and arrested MpBC cell lines in G2/M phase of cell cycle relative to single-agent treatment. Furthermore, NOS+PI3K inhibition significantly suppressed tumor volume in MpBC PDX models with PIK3CA mutations, although less significantly in MpBC PIK3CA-wild type PDX models, augmented efficacy of taxane chemotherapy, and improved overall survival. Transcriptional analysis of MpBC PDX tumors treated with combination therapy versus vehicle control revealed that epithelial-to-mesenchymal transition, DNA repair, and E2F targets were among the top enriched pathways, suggesting their plausible involvement in the mechanism of response. Immunoblotting/immunofluorescence analyses of cell lines/PDX models confirmed that combination therapy enhances DNA damage, reduces expression of homologous recombination-mediated DNA repair proteins, and induces mesenchymal-to-epithelial transition, rendering MpBC cells potentially more chemosensitive. Conclusion: Combined NOS and PI3K inhibition is a novel therapeutic strategy that may improve the efficacy of chemotherapy in patients with MpBC. Citation Format: Tejaswini P. Reddy, Akshjot Puri, Liliana Guzman-Rojas, Bijan Mahboubi, Wei Qian, Jianying Zhou, Baek Kim, Stacy Moulder, Helen Piwnica-Worms, Roberto Rosato, Jenny C. Chang. NOS inhibition augments PI3K inhibitor-induced DNA damage and mesenchymal-to-epithelial transition in metaplastic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2658.

Background: Patient-derived xenografts (PDX) and organoids (PDO) have become important translational model systems for cancer research, especially for drug responses prediction. Since PDX models have limitation as low success rate and takes long time, PDO is emerging as a new technique. Here, we compared the drug responses in PDX and PDO models using tissues obtained from treatment-refractory breast cancer patients. Methods: Tumor tissues from breast cancer patients were implanted into the mammary fat pads of immunodeficient mice. Tumor size of mice were measured 3 times a week using digital caliper. When xenograft tumors reached 200mm3 in size, drug treatment was started. Drugs selection was based on gene expression patterns, the presence of available drugs, and clinical treatment history. The organoids were established from PDX tumor pieces. Organoids were seeded and cultured in 96-well plates (2000 cells per well) for drugs testing. We treated with a single or combination drugs in PDX and PDO models. For interpretation of drug sensitivity results of PDO or PDX, we referred to IC50 database of 2D cell lines or results from references. Results: We compared the drugs response efficacy in five cases with paired PDX and PDO models; 1 hormone receptor (HR) positive+; HER2 negative-, 1 HR+;HER2+ and 3 TNBC (triple-negative breast cancer) subtypes. In HR+;HER2- case, drug test results between PDX and PDO partially matched in single treated group. In HR+;HER2+, the results of combination treated groups were partially matched in PDX and PDO model. The results for two TNBC samples matched in single or combination treated groups. Especially, tumor size or cell viability of one TNBC case showed significant differences between control and sorafenib/everolimus combination treated groups. The other case of TNBC type had partially matched in PDX and PDO model. We will analyze the consistency for the genomic profiles of tumors in patients, PDX, and PDO models. Conclusion: We have compared the various drugs responses through the successful establishment of PDO and PDX from the different breast cancer subtypes. Although the results are not perfectly matched, it showed that these models have potential to assist the chemotherapy strategies for each patient and predict outcome of treated patient's prognosis. In the future, we will be focused on explaining why the results of drug response between PDX and PDO were inconsistent. (This study was supported by National Cancer Center, Korea, 1710450, and 1810101) Citation Format: Jin-Sun Ryu, You-sun Noh, Bo-Ra Kim, Yun-Hee Kim, A-Ra Jeon, Sung Hoon Sim, In Hae Park, Eun Gyeong Lee, Eun Sook Lee, Keun Seok Lee, Sun-Young Kong. Comparison of drug responses using patient-derived xenograft (PDX) and patient-derived organoid (PDO) models from treatment-refractory breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1667.

BACKGROUND: Patient-derived xenograft (PDX) models of breast cancer replicate the diverse histologic and molecular features of patient tumors and provide a renewable source of human tumor tissue. However, collection of tissue by core needle biopsy is problematic due to patient discomfort, bleeding risk and the limited number of passes a patient can tolerate. Several studies have catalogued the maintenance of molecular features of patient tumors in PDX models of breast cancer. METHODS: To support the neoadjuvant molecular diagnostic and drug development program in triple negative breast cancer (TNBC), a pilot study was conducted to determine if fine needle aspiration (FNA) could be used for building PDX models. Subsequently, PDX models are being established in alignment with ongoing clinical trials at MDACC. The molecular evolution of patient's tumors, matched with PDXs engrafted from their tumors, is under study throughout the neoadjuvant treatment of TNBC using RNA sequencing, whole-exome sequencing, deep sequencing of cancer genes, and histologic analyses. RESULTS: To date, 20 established PDX models have been developed and stable PDX models continue to be generated at a rate of 2-3 per month. Several of these models are derived from serial FNAs derived from patients throughout neoadjuvant treatment. These models retain histologic and molecular features of the original patient tumors. Serial patient biopsies, matched with PDX models, have enabled measurement of the mutational and transcriptomic evolution in vivo of TNBC undergoing neoadjuvant treatment. We have standardized the use of FNAs to generate PDX models both pre- and post-neoadjuvant therapy in the following ongoing neoadjuvant clinical trials: 1. MDACC 2014-0185 (PI Stacy Moulder, 360 patients), 'ARTEMIS: A Randomized TNBC-Enrolling trial to confirm Molecular profiling Improves Survival' 2. MDACC 2014-0045 (PI Jennifer Litton, 20+ patients), 'A pilot study of BMN673 as a neoadjuvant study in patients with a diagnosis of invasive breast cancer and a deleterious BRCA mutation' CONCLUSION: We demonstrated that PDX models from tissue collected by FNA recapitulate the biology and clinical course of the patient's tumor. Sequencing analyses revealed that neoadjuvant chemotherapy and PDX engraftment enrich for cancer gene mutations. We observe association of the rate of successful PDX engraftment with clinical parameters such as the patient's residual cancer burden (RCB) status at the time of surgery (upon completion of neoadjuvant treatment). In addition, we observe that PDX models derived from serial patient biopsies throughout treatment are more resistant to chemotherapy treatment. These models recapitulate the variety of chemotherapy responses observed in patients with TNBC and serve as powerful tools for preclinical biomarker and discovery studies. Citation Format: Echeverria GV, Cai S, Tu Y, McCoy A, Lau R, Redwood A, Rauch G, Adrada B, Candelaria R, Santiago L, Thompson A, Litton J, Moulder S, Symmans F, Chang JT, Piwnica-Worms H. A molecularly annotated collection of breast cancer patient-derived xenograft models aligned with ongoing clinical trials built from fine needle aspiration samples throughout neoadjuvant treatment [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-05-01.

<div>Abstract<p>Taxanes are standard therapy in clinical practice for metastatic breast cancer; however, primary or acquired chemoresistance are a common cause of mortality. Breast cancer patient-derived xenografts (PDX) are powerful tools for the study of cancer biology and drug treatment response. Specific DNA methylation patterns have been associated to different breast cancer subtypes but its association with chemoresistance remains unstudied. Aiming to elucidate docetaxel resistance mechanisms, we performed genome-wide DNA methylation in breast cancer PDX models, including luminal and triple-negative breast cancer (TNBC) models sensitive to docetaxel, their matched models after emergence of chemoresistance and residual disease after short-term docetaxel treatment. We found that DNA methylation profiles from breast cancer PDX models maintain the subtype-specific methylation patterns of clinical samples. Two main DNA methylation clusters were found in TNBC PDX and remain stable during the emergence of docetaxel resistance; however, some genes/pathways were differentially methylated according to docetaxel response. A DNA methylation signature of resistance able to segregate TNBC based on chemotherapy response was identified. Transcriptomic profiling of selected sensitive/resistant pairs and integrative analysis with methylation data demonstrated correlation between some differentially methylated and expressed genes in docetaxel-resistant TNBC PDX models. Multiple gene expression changes were found after the emergence of docetaxel resistance in TNBC. DNA methylation and transcriptional changes identified between docetaxel-sensitive and -resistant TNBC PDX models or residual disease may have predictive value for chemotherapy response in TNBC.</p>Implications:<p>Subtype-specific DNA methylation patterns are maintained in breast cancer PDX models. While no global methylation changes were found, we uncovered differentially DNA methylated and expressed genes/pathways associated with the emergence of docetaxel resistance in TNBC.</p></div>

Poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPi) have recently emerged as therapeutic options for patients with homologous recombination-deficient (HRD) breast or ovarian cancer, two heterogeneous diseases associated with high mortality rates. As shown in several clinical studies, patient response to PARPi is invariably followed by eventual mid-long term resistance and progression under treatment. The molecular processes contributing to PARPi-resistance are at present under-explored. Therefore, a huge effort is being made to better understand how to overcome resistance and to identify ad-hoc combinations with other targeted therapies to improve tumor response and extend progression-free survival. We have previously published the PARPi-response profile of a panel of 40 breast cancer (BC) patient-derived xenografts (PDXs). The models tested showed heterogeneous response to PARPi, and only partial association with the genomic status of BRCA genes, the only currently acknowledged clinical marker to select patients that can benefit from PARPi administration. Although these models are ideal preclinical tools for the evaluation of drug combinations to improve tumor response to PARPi as single agent, the use of these models for early preclinical evaluation of combination efficacy is not straightforward in reason of the limited throughput and of the ethical issue with respect of the 3Rs. The use of cellular models is still considered as a standard early preclinical test to evaluate drug response before moving to in vivo assays. However, the main concern when using in vitro models is the representativeness of the results obtained when transposed to in vivo models. In this study, we have setup an in vitro assay that recapitulates the response to PARPi observed in vivo in our BC PDXs, with particular focus on the models that show resistance to PARPi. To this aim we generated 9 cellular models from 9 BC PDX models: HBCx-2, HBCx-3, HBCx-6, HBCx-8, HBCx-9, HBCx-17, HBCx-19, HBCx-39 and T174. Two cellular models (HBCx-3, and HBCx-19) were established from ER+ BC PDX and 7 from ER- BC PDXs and two models, HBCx-8 and HBCx-17, harbor BRCA1 and BRCA2 mutation, respectively. Seven out of 9 models are resistant to PARPi in vivo, with HBCx-6 PDX showing partial tumor regression and HBCx-17 PDX showing tumor stabilization upon treatment. Several different 2D-culture experimental conditions, namely different cell growth conditions, drug concentrations, duration of cell exposure to drugs, time points and readouts, have been tested to evaluate response to olaparib. The results showed that a 2D colony assay is the best experimental strategy to faithfully evaluate tumor cell sensitivity, minimizing the false positive results when compared to the in vivo data.. This cell panel will be used to identify combination of PARPi with a library of FDA-approved targeted therapy to identify the treatments to be moved forward in vivo. Citation Format: Olivier Déas, Romain Rousseau, Sophie Banis, Kathleen Flosseau, Enora Le Ven, Jean-Gabriel Judde, stefano cairo. Establishment of an in vitro assay that phenocopies tumor response to PARP inhibitors in vivo [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 3810.

We aimed to identify the factors affecting the successful tumor engraftment in breast cancer patient-derived xenograft (PDX) models. Further, we investigated the prognostic significance and the functional importance of the PDX engraftment-related genes in triple-negative breast cancers (TNBC). The clinico-pathologic features of 81 breast cancer patients whose tissues were used for PDX transplantation were analyzed to identify the factors affecting the PDX engraftment. A gene signature associated with the PDX engraftment was discovered and its clinical importance was tested in a publicly available dataset and in vitro assays. Nineteen out of 81 (23.4%) transplanted tumors were successfully engrafted into the PDX models. The engraftment rate was highest in TNBC when compared to other subtypes (p=0.001) and in recurrent or chemotherapy-resistant tumors compared to newly diagnosed primary tumors (p=0.024). PDX tumors originated from the TNBC cases showed more rapid tumor growth in mice. Gene expression profiling showed that down-regulation of genes involved in the tumor-immune interaction was significantly associated with the successful PDX engraftment. The engraftment gene signature was associated with worse survival outcome when tested in publicly available mRNA datasets of TNBC cases. Among the engraftment-related genes, PHLDA2, TKT, and P4HA2 showed high expression in triple-negative breast cancer cell lines, and siRNA-based gene silencing resulted in reduced cell invasion and proliferation in vitro. Our results show that the PDX engraftment may reflect the aggressive phenotype in breast cancer. Genes associated with the PDX engraftment may provide a novel prognostic biomarker and therapeutic targets in TNBC.

Background: Cyclin/cyclin-dependent kinase (CDK) complexes regulate the activity of RB1 and E2F to drive cell cycle progression while ensuring fidelity of DNA replication. The interaction between cyclins and a critical subset of their substrates, including RB1 and E2F, is mediated by a conserved short linear RxL motif. RxL peptides have been shown to inhibit substrate phosphorylation by CDK2/cyclin A (AACR 2022 poster #5379). Previously, we have shown that macrocycles that selectively inhibit RxL-mediated binding to cyclins A and B (RxL inhibitors) lead to growth inhibitory activity in multiple cancer cell lines, and result in tumor regression in small cell lung cancer (SCLC) and ovarian cancer xenograft models (AACR 2023 poster #1560). Here, we test the antitumor activity of CID-016 in patient-derived xenograft (PDX) models from breast cancer. Methods: Bioinformatic analyses using RNA sequencing (RNAseq) and whole exome sequencing (WES) data from 39 breast cancer cell lines was employed to identify biomarker profiles that associate with sensitivity to CID-016. The antiproliferative activity of CID-016 was assessed by GI50 after 4-8 days of treatment. Hallmark pathway scores were calculated by Gene Set Variation Analysis (GSVA) method using MSigDb Hallmark collection from cell lines and existing PDX models. Based on the calculated hallmark pathway score, triple negative breast cancer (TNBC) PDX models were selected to test the anti-tumor activity of CID-016. In vivo, NMRI-Foxn1nu/nu mice were implanted with tumor fragments in the lower flank and treated with vehicle or CID-016 oral formulation of 50-100 mg/kg BID or 100 mg/kg QD, respectively for at least 28 days. Tumor volume and body weight were measured biweekly. Pharmacodynamic (PD) analyses will be conducted after treatment with CID-016. Results: Treatment of the breast cancer cell line panel with CID-016 demonstrated greater potency by GI50 in TNBC compared to other breast cancer cell lines (p=0.009). Gene expression levels of CCNB1, CCNA2, CCNE1 as well as target hallmark scores for “E2F targets”, “G2M checkpoint” and “Mitotic spindle” were significantly higher in TNBC vs other breast cancer cell lines. Oral treatment with CID-016 resulted in tumor regression in at least one triple-negative breast cancer PDX model and was well tolerated with no significant body weight changes. In vivo PD analyses will be assessed after 5 days of treatment with CID-016. Conclusions: The selective cyclin A/B RxL inhibitor CID-016 exhibits potent antitumor activity in TNBC preclinical models, consistent with the proposed mechanism of RB1/E2F pathway dysregulation. Given their compelling characteristics, orally bioavailable RxL inhibitors are advancing as the only first-in-class orally bioavailable dual inhibitors of cyclins A and B into clinical development. Citation Format: Mariana Paes Dias, Li-Fen Liu, Bernard Levin, Cristina Molina, Marta Guzmán, Olga Rodríguez, Evelyn W. Wang, Violeta Serra. Novel orally bioavailable macrocycles that target cyclin A and B elicit antitumor activity in breast cancer patient-derived xenograft models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C04.

Compared with other breast cancer subtypes, triple-negative breast cancer (TNBC) is associated with relatively poor outcomes due to its metastatic propensity, frequent failure to respond to chemotherapy, and lack of alternative, targeted treatment options, despite decades of major research efforts. Our studies sought to identify promising targeted therapeutic candidates for TNBC through in vitro screening of 1,363 drugs in patient-derived xenograft (PDX) models. Using this approach, we generated a dataset that can be used to assess and compare responses of various breast cancer PDXs to many different drugs. Through a series of further drug screening assays and two-drug combination testing, we identified that the combination of afatinib (epidermal growth factor receptor (EGFR) inhibitor) and YM155 (inhibitor of baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5; survivin) expression) is synergistically cytotoxic across multiple models of basal-like TNBC and reduces PDX mammary tumor growth in vivo. We found that YM155 reduces EGFR expression in TNBC cells, shedding light on its potential mechanism of synergism with afatinib. Both EGFR and BIRC5 are highly expressed in basal-like PDXs, cell lines, and patients, and high expression of both genes reduces metastasis-free survival, suggesting that co-targeting of these proteins holds promise for potential clinical success in TNBC.

Triple-negative breast cancers (TNBCs) constitute approximately 12% of all breast cancer cases and are approximately twice as prevalent in African-American populations. Louisiana has a high proportion of African-American residents (32.3% in 2017), and New Orleans has among the highest incidences of TNBC in the country. Louisiana patients also have a high incidence of co-morbidities that affect breast cancer biology and outcomes, including type 2 diabetes and obesity. TNBCs have an aggressive clinical presentation due to high rates of metastasis, recurrence and chemoresistance. There are currently no clinically approved targeted therapies for TNBC; cytotoxic chemotherapy is the first-line treatment for TNBC, and recurrent, chemoresistant cancers are usually fatal. TNBCs are molecularly heterogeneous, consisting of at least four molecular subgroups, and immunologically heterogeneous. Both molecular and immunologic properties are associated with clinical outcomes and are seriously understudied in patients under-represented in biomedical research. Patient-derived xenografts (PDXs), as well as patient-derived organoids (PDO), are currently the best model for translational oncology therapeutic research because they accurately recapitulate the complex architecture and heterogenous genetic and molecular composition of solid cancers. To date, the majority of TNBC research has been based on Caucasian patients, although incidence rates of TNBC are higher in African-American cohorts. Our collaborative team aims to overcome this obstacle by establishing and characterizing TNBC PDX models that represent this understudied cohort. We currently have ten TNBC PDX models representing different patient ethnicities, responsiveness to chemotherapies, as well as different TNBC molecular subtypes and metastatic behavior. We dissect and evaluate the various individual components (tumor cell biology, stroma, immune, extracellular matrix) of TNBC tumors. We utilize these models in vivo, ex vivo and in vitro to examine how unique kinases and targeted inhibitors affect the distinct tumor characteristics. In addition to in vivo treatment studies, we generated cell lines and PDOs and we utilize novel techniques such as tissue decellularization to examine extracellular matrix components. We also analyze mechanistically relevant transcript (qRT-PCR) and protein (Western blot, immunohistochemistry) expression patterns that are unique to each PDX model to evaluate the effects of targeted therapies. We work with surrounding laboratories in the greater New Orleans area (Tulane, LSU, Xavier) that are also focused on therapeutic discovery of TNBC in a collaborative effort to provide translational models for their projects. Our aim is to leverage novel PDX models from understudied patients with a range of clinical and molecular presentations to guide the selection of therapeutically targetable pathways and therapeutic agents in specific molecular subtypes of TNBC. Citation Format: Margarite D. Matossian, Steven Elliott, Hope E. Burks, Maryl Wright, Rachel A. Sabol, Van T. Hoang, Deniz A. Ucar, Alex Alfortish, Jovanny Zabaleta, Fokhrul Hossain, Tiffany Chang, Henri Wathieu, Nicholas Pashos, Bruce Bunnell, Krzysztof Moroz, Arnold Zea, Adam Riker, Steven D. Jones, Elizabeth C. Martin, Lucio Miele, Bridgette M. Collins-Burow, Matthew E. Burow. Applications of patient-derived triple-negative breast cancer xenografts that represent understudied patients in Louisiana in targeted therapeutic research [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C110.

Breast cancer (BC) is marked by significant genetic, morphological and clinical heterogeneity. To capture this heterogeneity and unravel the molecular mechanisms driving tumor progression and drug resistance, we established a comprehensive patient-derived xenograft (PDX) biobank, focusing particularly on luminal (estrogen receptor, ER+) and young premenopausal patients, for whom PDX models are currently scarce. Across all BC subtypes, our efforts resulted in an overall success rate of 17% (26 established PDX lines out of 151 total attempts), specifically 15% in luminal, 12% in human epidermal growth factor receptor 2 positive (HER2+) and 35% in triple negative BC. These PDX mirrored morphologic and genetic features of BC from which they originated, serving as a reliable tool to investigate drug resistance and test therapeutic strategies. We focused on understanding resistance to CDK4/6 inhibitors (CDK4/6i), which are crucial in the treatment of patients with advanced luminal BC. Treating a sensitive luminal BC PDX with the CDK4/6i palbociclib revealed that, despite initial tumor shrinkage, some tumors might eventually regrow under drug treatment. RNA sequencing, followed by gene set enrichment analyses, unveiled that these PDXs have become refractory to CDK4/6i, both at biological and molecular levels, displaying significant enrichment in proliferation pathways, such as MTORC1, E2F and MYC. Using organoids derived from these PDX (PDxO), we observed that acquisition of CDK4/6i resistance conferred cross-resistance to endocrine therapy and that targeting MTORC1 was a successful strategy to overcome CDK4/6i resistance. Considered together, these results indicate that our PDX models may serve as robust tools to elucidate the molecular basis of BC disease progression and, by providing the possibility to simultaneously test different therapies on the same tumor, to surmount treatment resistance. While this approach is of course not feasible in the clinic, its exploitation in PDX may expedite the identification and development of more successful therapies for patients with advanced luminal BC. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A patient-derived xenograft (PDX) model is an in vivo animal model which provides biological and genomic profiles similar to a primary tumor. The characterization of factors that influence the establishment of PDX is crucial. Furthermore, PDX models can provide a platform for chemosensitivity tests to evaluate the effectiveness of a target agent before applying it in clinical trials. We implanted 83 cases of breast cancer into NOD.Cg-Prkdcscid Il2rgtm1Sug/Jic mice, to develop PDX models. Clinicopathological factors of primary tumors were reviewed to identify the factors affecting engraftment success rates. After the establishment of PDX models, we performed olaparib and carboplatin chemosensitivity tests. We used PDX models from triple-negative breast cancer (TNBC) with neoadjuvant chemotherapy and/or germline BRCA1 mutations in chemosensitivity tests. The univariate analyses (p<0.05) showed factors which were significantly associated with successful engraftment of PDX models include poor histologic grade, presence of BRCA mutation, aggressive diseases, and death. Factors which were independently associated with successful engraftment of PDX models on multivariate analyses include poor histologic grade and aggressive diseases status. In chemosensitivity tests, a PDX model with the BRCA1 L1780P mutation showed partial response to olaparib and complete response to carboplatin. Successful engraftment of PDX models was significantly associated with aggressive diseases. Patients who have aggressive diseases status, large tumors, and poor histologic grade are ideal candidates for developing successful PDX models. Chemosensitivity tests using the PDX models provide additional information about alternative treatment strategies for residual TNBC after neoadjuvant chemotherapy.

BACKGROUND Metaplastic breast cancer (MpBC) is an extremely rare, therapeutically recalcitrant and aggressive variant of triple negative breast cancer (TNBC). We have previously shown molecular alterations in inducible nitric oxide (iNOS) signaling in MpBC is associated with worse overall survival. Preclinical models have shown pan-NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) reduces tumor growth and epithelial to mesenchymal transition in mesenchymal cell lines. We have also previously shown in TNBC patients treated with LNMMA and docetaxel, the non-responders have a higher expression of M2 macrophage vs the responders have decreased pro-tumor N2 neutrophils at the end of therapy. Here, we report the results of L-NMMA plus taxane in a cohort of MpBC patients. METHODS We conducted a phase 1B/2 trial, with L-NMMA (starting dose of 7.5 mg/kg which was escalated to recommended phase 2 dose of 20 mg/kg) and docetaxel (100 mg/m2) every 3 weeks. Daily amlodipine was given to prevent hypertension from L-NMMA. Primary objective was to assess clinical benefit rate (CBR), as assessed by the Response Evaluation Criteria in solid tumors (RECIST). Secondary objectives were to study overall response rate (ORR), progression free survival (PFS), overall survival (OS), dose limiting toxicities (DLT), response correlation with type of MpBC, body mass index (BMI) and ethnicity. Exploratory analysis included immune correlates for clinical response; staining for iNOS, M2 macrophages (CD 68, CD 163), N2 neutrophils (CD 15, arginase), immune infiltration (PD-L1, CD 8) and fibrosis marker (α- SMA). Fisher’s exact test was used to find the association between different patient’s characteristics and the main outcome. A p-value of 0.05 was considered statistically significant and all analyses were conducted using Stata V16.1 (StataCorp, College Station, Texas 77845 USA) RESULTS Of the total 35 TNBC patients recruited, 15 patients had MpBC (Phase 1B, n= 4; Phase 2, n=11); 86.6% (13/15) patients had metastatic breast cancer (MBC), with a median of 2 prior lines of therapy (range 0-5) and 13.3% (2/15) had anthracycline-refractory locally advanced breast cancer (LABC). The CBR was 40% (6/15); the ORR was 20% (3/15) with 1 PR in MBC , 1 pathological CR and 1 PR in LABC. Grade 3 or more toxicity was seen in 13.3% (2/15) patients; however, none was attributed to L-NMMA. The mPFS and mOS for MBC patients were 4.5 months (range 3-7m) and 12.8 months, respectively. The response was more likely to be in women of Caucasian ethnicity, BMI&amp;gt; 25 and non-spindle pathological features such as squamous differentiation, keratinized and myxo-chondroid tumors; albeit these were not statistically significant (Table 1). CONCLUSIONS Inhibition of iNOS pathway in MpBC is a promising and novel therapeutic option in this very challenging breast cancer subtype. The small study size is an impediment in identifying clinical factors which can predict a response. This warrants further evaluation of treatment with L-NMMA in chemo-refractory MpBC patients in a larger clinical trial. Table 1.Association between ethnicity, BMI and pathological characteristics with responseTotalNo responderResponderp-valueN=13N=7N=6Ethnicity0.19Caucasian10 (76.92%)4 (57.14%)6 (100.00%)Other3 (23.08%)3 (42.86%)0 (0.00%)Obesity1.00&amp;lt;25 BMI3 (23.08%)2 (28.57%)1 (16.67%)&amp;gt;=25 BMI10 (76.92%)5 (71.43%)5 (83.33%)Pathological features0.27Spindle5 (38.46%)4 (57.14%)1 (16.67%)Other8 (61.54%)3 (42.86%)5 (83.33%)Data are presented as n (%). *P-value from Fisher’s exact test. The 2 patients with adverse events were excluded from the analysis. Citation Format: Akshjot Puri, Adriana Ordonez, Ann C. Anselme, Liliana Guzman, Polly Niravath, Jenny C. Chang. Phase 1B/2 clinical trial targeting nitric oxide in the treatment of chemo-refractory metaplastic triple-negative breast cancer patients [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 P5-17-07.

The receptor tyrosine kinase RET is implicated in the progression of luminal breast cancers (BC) but its role in estrogen receptor (ER) negative tumors is unknown. Here we investigated the expression of RET in breast cancer patients tumors and patient-derived xenografts (PDX) and evaluated the therapeutic potential of Vandetanib, a tyrosin kinase inhibitor with strong activity against RET, EGFR and VEGFR2, in ER negative breast cancer PDX. The RT-PCR analysis of RET expression in breast tumors of 446 patients and 57 PDX, showed elevated levels of RET in ER+ and HER2+ subtypes and in a small subgroup of triple-negative breast cancers (TNBC). The activity of Vandetanib was tested in vivo in three PDX models of TNBC and one model of HER2+ BC with different expression levels of RET and EGFR. Vandetanib induced tumor regression in PDX models with high expression of RET or EGFR. The effect was associated with inhibition of RET/EGFR phosphorylation and MAP kinase pathway and increased necrosis. In a PDX model with no expression of RET nor EGFR, Vandetanib slowed tumor growth without inducing tumor regression. In addition, treatment by Vandetanib decreased expression of murine Vegf receptors and the endothelial marker Cd31 in the four PDX models tested, suggesting inhibition of tumor vascularization. In summary, these preclinical results suggest that Vandetanib treatment could be useful for patients with ER negative breast cancers overexpressing Vandetanib's main targets.

Background: Patient derived xenografts (PDX) are regarded as the gold-standard for translational preclinical cancer research due to their preservation of tumor heterogeneity and propagation in vivo. Similarly, grafting human tumor cells of a particular tissue origin into the corresponding orthotopic context enhances translational accuracy; with recapitulation of tumor::microenvironment biology, as well as more accurate tumor progression kinetics and metastasis, a s well as for systemic delivery of therapies as compared to traditional subcutaneous xenografts. Bioluminescent luciferase-reporters into traditional cancer cell lines has allowed for in-life imaging of tumor growth kinetics in real-time, within obfuscated internal organs, improving experimental precision. We have reported on the development and validation of bioluminescent pancreatic cancer and breast cancer PDX in clinically recapitulative orthotopic models. Here, we characterize these bioluminescent PDX by gene-expression profiling; comparing orthotopic and ectopically propagated bioluminescent PDX to their non-transduced parental PDX counterparts. Methods: Pancreatic (PANx-005-Luc) or metastatic breast (HBCx-14-Luc) PDXs stably transduced with lentiviral luciferase were dissociated and implanted orthotopically into pancreas or mammary fat pad respectively or subcutaneously into the flank of NOD scid gamma (NSG) mice. Likewise, the parental reporter-free PANx-005 or HBCx-14 PDX were implanted subcutaneously into NSG mice. For luciferase-expressing PDX implanted orthotopically, tumor growth was monitored in-life (Xenogen IVIS® Lumina Series III instrument (IVIS)). For the PANx-005-Luc, orthotopic tumors were harvested at a mean tumor radiance (TR) of 2.53 × 106 + 1.63 × 106 ph/s, while ectopic luciferase-transduced and parental tumors were harvested at a mean tumor volume of 530 + 337mm3. For the HBCx-14-Luc, orthotopic tumors were harvested individually when TR reached 6.5 × 109 ph/s while ectopic luciferase-transduced or parental tumors were harvested when tumor volumes reached 1500 + 362.5mm3. Tumor samples were fixed in 10% neutral buffered formalin and embedded in paraffin-blocks for histological analysis. RNA was extracted from all tumors and full-coverage human genome gene-expression profiling was performed. Major differences observed in microarray data was validated via quantitative biochemical assays. Results: Stable expression of lentiviral luciferase did not result in significant gene-expression changes to PDX lines, while site of implantation altered some gene-expression characteristics of the PDX. Conclusions: The introduction of an integrating stable bioluminescent reporter does not significantly alter the gene-expression characteristics of PANx-005 or HBCx-14 PDX. Site of implantation plays a role in gene-expression of PDX tumor models. Citation Format: Benjamin G. Cuiffo, Olivier Deas, Ingrid Rankin, Gregory D. Lyng, Stephano Cairo, Katie Pedrick, Alexandra Kury, Enora Le Ven, Jean-Gabriel Judde, Stephen T. Sonis. Gene-expression characterization of bioluminescent PDX in orthotopic and ectopic models of pancreatic cancer and metastatic triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 633.

Triple negative breast cancer (TNBC) is an aggressive and difficult-to-treat subtype of breast cancer that typically exhibits rapid growth rates, high rates of metastasis, and resistance to commonly used oncological drugs. Historically, cell lines have been utilized in order to study TNBC; recently, however, patient derived xenografts (PDX) models have evolved as the new standard that offers a translational approach to the research and subsequent treatment of breast cancer. Here, we characterize two novel PDX models for TNBC: TU-BcX-4QA and TU-BcX-4QAN. The former derived from a biopsy specimen prior to any therapies, and the latter derived from a mastectomy of the same patient after three rounds of AC-T therapy (doxorubicin and cyclophosphamide followed by paclitaxel). In establishing a treatment naïve and post-neoadjuvant therapy PDX model pair, we created a prime model that examines the effects of chemotherapy on tumor heterogeneity, clonal selection, and the overall characteristics of a tumor. Furthermore, we examined the evolution of the characteristics of the post-neoadjuvant therapy PDX model after continual passaging within the SCID/Beige murine models. Through serial implantation in SCID/Beige murine models for tissue propagation, we observed that TU-BcX-4QAN consistently had a higher tumor growth rate and a smaller number of metastatic lesions that developed on the lungs and liver in comparison to the TU-BcX-4QA model. In treating the tumor derived cell lines with NCI-approved oncological drugs, we distinguished the variations in their responses to various, commonly used therapies, and determined that TU-BcX-4QAN had a more resistant profile. Using qRT-PCR, we further discovered the differences between the two models in their contrasting gene expression; preliminary data indicates an increase in certain mesenchymal genes (CDH2, VIM, and ZEB2), a decrease in cell cycle genes (p21, p53), and an increase in proliferation genes (MK167) in TU-BcX-4QAN compared to TU-BcX-4QA. Additionally, serial passages are correlated with a decrease trend in human gene expression within TU-BcX-4QAN. This suggest that treatment can select for certain cancer cells within the primary tumor that allows for the growth of a different tumor altogether and illustrates both the advantages and limitations of the TU-BcX-4QA andTU-BcX-4QAN model pair in translational research. Citation Format: Gabrielle Olivia Windsor, Margarite Matossian, Maryl Wright, Steven Elliott, Khoa Nguyen, Bridgette Collins-Burow, Matthew Burow. Treatment naïve patient-derived xenograft model compared to the post-neoadjuvant model from the same patient diagnosed with triple negative breast cancer [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 PS17-47.