Patient-derived Preclinical Models Research Articles

Myxofibrosarcoma primary cultures: molecular and pharmacological profile.

Background:Myxofibrosarcoma (MFS), formerly considered as a myxoid variant of malignant fibrous histiocytoma, is the most common sarcoma of the extremities in adults and is characterized by a high frequency of local recurrence. The clinical behavior of MFS is unpredictable and the efficacy of chemotherapy is still not well documented. Furthermore, given the relatively recent recognition of MFS as a distinct pathologic entity its cellular and molecular biology has still not been extensively studied in patient-derived preclinical models. We examined the molecular biology and treatment outcomes of high-grade, patient-derived MFS primary cultures.Methods:A total of three patient-derived MFS primary cultures were analyzed. We evaluated the role of CD109 expression and also looked for a correlation between transforming growth factor-beta (TGF-β) expression and sensitivity of the primary cultures to different drugs.Results:CD109 was a promising marker for the identification of more aggressive high-grade MFS and a potential therapeutic target. The results also highlighted the potential role of TGF-β in chemoresistance. Pharmacological analysis confirmed the sensitivity of the cultures to chemotherapy. The most active treatments were epirubicin alone and epirubicin in combination with ifosfamide, the latter representing the current standard of care for soft tissue sarcomas (STSs), including MFS.Conclusions:Our results provide a starting point for further research aimed at improving the management of MFS patients undergoing chemotherapy.

Abstract LB-130: Tumor regression in novel triple therapy BRAF mutant metastatic melanoma patient-derived preclinical models

Abstract Metastatic melanoma (MM) is the most aggressive type of skin cancer contributing to approximately 80% of all skin cancer related deaths. In 2016, it was estimated that more 76,000 cases would be diagnosed in the United States. The high metastatic potential and aggressive clinical behavior of this disease makes it a major health problem. As a consequence there has been notable development in for novel targeted (i.e. BRAF inhibitors) and immune therapies (i.e. anti-PD1 and CTLA4 inhibitors). However, despite improvements in patient outcomes, most patients develop resistance within 6-11 months on dual BRAF/MEK inhibition, and 5 months on immune therapy, highlighting the need to identify new therapies that improve disease management and patient survival. Increased expression of CyclinD1 has been reported to occur as a mechanism of resistance to BRAF inhibition, resulting in reactivation of the canonical activating pathway in melanoma, MAPK. Thus, we hypothesize that combined inhibition of CDK4/6 and MAPK signaling will provide a novel anti-tumor synergy drug combination superior to current standard of care in BRAF mutant patient-derived preclinical models. Using MM human tissue from patients we developed 3 BRAF mutant preclinical models investigating a novel triple therapy drug combination BRAFi/MEKi/CDK4/6i. Protein analysis and drug proliferation assays were used to design the novel combination. In vivo, we found statistically significant differences in tumor inhibition between the novel combination and the current standard of care. The triple therapy also showed promising results, in vivo, when assessed as a salvage therapy following tumor escape from BRAFi/MEKi combination treatment. Citation Format: Antoneicka L. Harris, Michael Thompson, Svetomir Markovic, William Durham, Louis Dawson, Daniel Small, John A. Copland. Tumor regression in novel triple therapy BRAF mutant metastatic melanoma patient-derived preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-130. doi:10.1158/1538-7445.AM2017-LB-130

Establishment of patient-derived renal cell carcinoma (RCC) models based on orthotopic xenografts (PDX) and cancer stem cell (CSC) isolation to provide prognostic and predictive information.

e16055 Background: The introduction of VEGF/VEGFR- and mTOR, and immune checkpoint-targeted agents has radically changed metastatic RCC treatment; however, predictive biomarkers are still lacking and the potential for curative impact of such treatments in earlier disease stages is hotly debated. To identify potential diagnostic, prognostic, and predictive biomarkers, we developed patient-derived preclinical RCC models based on PDX obtained from freshly dissociated cancer tissues and combining isolation of cell populations endowed with self renewal and multi-lineage differentiation abilities (CSC). Methods: 182 surgical RCC specimens (clear cell, n = 110; papillary type I/II, n = 23, chromophobe/oncocytoma, n = 26, other, n = 23; stage I-III, n = 124, stage IV, n = 28, n/a n = 30) were collected; tumor spheroids were obtained and characterized in 57 cases, using a stem-cell isolating medium supplemented with EGF/b-FGF; 30 cancer samples were orthotopically injected in immunocompromised mice and were able to engraft in 67% of cases (G3-G4 cases more frequently than G1-G2 cases, p = 0.04). Results: PDX tumors recapitulated histological appearance, sarcomatoid features when they were present, and tumor heterogeneity of their human counterpart; interestingly, PDX engraftment was significantly correlated with shorter DFS in the corresponding patient population (p = 0.12). We conducted proteomic analysis by Reverse Phase Protein Array (RPPA) in 21 patient-derived CSC models in vitro: a panel of established (HIF, VEGFR, mTOR) and novel (EGFR(Y1148)), AKT, PI3K) oncogenic signals were deregulated in our isolated cell populations; molecular endpoints were correlated with grading and with angiogenesis and mTOR pathways (p < 0.05). Data analysis is ongoing to ascertain whether specific RPPA-signatures can potentially complement clinical prognostic information and suggest candidate biomarkers and potential targets for therapy. Conclusions: In the era of personalized therapy, the combined use of PDX and RPPA from tumor specimens may be very useful for drug testing and patient stratification in RCC.

Development of Novel Patient-Derived Preclinical Models from Malignant Effusions in Patients with Tyrosine Kinase Inhibitor–Resistant Clear Cell Renal Cell Carcinoma

PURPOSE: Although targeting angiogenesis with tyrosine kinase inhibitors (TKIs) has become standard of care in the treatment of clear cell renal cell carcinoma (RCC), resistance mechanism are not fully understood, and there is a need to develop new therapeutic options overcoming them. METHODS AND MATERIALS: To develop a preclinical model that predicts clinical activity of novel agents in 19 RCC patients, we established patient-derived cell (PDC) and xenograft (PDX) models derived from malignant effusions or surgical specimen. RESULTS: Successful PDCs, defined as cells that maintained growth following two passages, were established in 5 of 15 malignant effusions and 1 of 4 surgical specimens. One PDC, clinically refractory to TKIs, was implanted and engrafted in mice, resulting in a comparable histology to the primary tumor. The PDC-PDX model also showed similar genomic features when tested using targeted sequencing of cancer-related genes. When we examined the drug effects of the PDX model, the tumor cells showed resistance to TKIs and everolimus in vitro. CONCLUSION: The results suggest that the PDC-PDX preclinical model we developed using malignant effusions can be a useful preclinical model to interrogate sensitivity to targeted agents based on genomic alterations.

Abstract P6-12-06: Nonsteroidal, tissue selective androgen receptor modulator (SARM), enobosarm, reduces growth of androgen receptor-positive breast cancer in patient-derived preclinical models

Abstract Introduction: In breast cancer the androgen receptor (AR) is the most abundantly expressed steroid receptor with 75-95% of estrogen receptor (ER)-positive and 40-70% of ER-negative breast cancers expressing the AR. Historically, advanced breast cancer has been treated with androgens, resulting in significant clinical response. However, the use of steroidal androgens fell from favor as a result of their virilizing side effects. Nonsteroidal, tissue selective androgen receptor modulators (SARMs) will provide a novel targeted approach to exploit the therapeutic benefits of androgens in breast cancer. Aims: To test the effects of enobosarm (a first-in-class SARM) and enzalutamide (AR antagonist) on the growth of patient-derived breast cancer xenografts (PDX) and to discern the mechanism of action of AR-targeted therapies in AR-positive breast cancer. Materials and Methods: AR-positive PDXs with varying receptor expression (ER, progesterone receptor (PR), and HER2) were implanted in immunecompromised mice. Mice carrying PDXs were treated with vehicle, 10 mg/kg/day (mpk) enobosarm (GTx, Inc., Memphis, TN), or 20 mpk enzalutamide (Medivation Inc.), orally. Tumor volume was measured twice or thrice weekly. Tumors that received enobosarm were further analyzed to determine the mechanism of action. Results: Enobosarm significantly (p<0.01) inhibited the growth of ER-, PR-, and HER2- positive HCI-7 and ER- and PR- negative and HER2-positive HCI-12 PDX. While enobosarm inhibited the growth of HCI-12 by ~80% and HCI-7 by ~60%, enzalutamide failed to inhibit the growth of the HCI-7 PDX. In contrast, neither enobosarm nor enzalutamide inhibited the growth of ER- and PR-negative and HER2-positive HCI-9 PDX, consistent with the heterogeneity of AR-positive breast cancers. Growth of two triple-negative breast cancer (TNBC) PDXs were inhibited by 30-40% by enobosarm, but not by enzalutamide. These results were reproduced in xenografts developed with breast cancer cell lines, MCF-7 and MDA-MB-231 expressing the AR. Gene expression studies conducted with the HCI-12 tumors indicated that enobosarm inhibited the expression of various proliferative genes (MUC2, IL10RA, IGSF1, SLC6A4, and others) and increased the expression of growth inhibitory genes (CYP4F8, MYBPC1, and others). Ingenuity pathway analysis demonstrated that enobosarm inhibited genes that are downstream of HER2 signaling. Interestingly, miR-21-3p, which has been implicated in chemo-resistance, was consistently expressed at approximately 10-50-fold higher than miR-21-5p in PDXs. This imbalance was partially reversed by enobosarm. Conclusion: These results indicate that AR-positive breast cancers are highly heterogeneous and that enobosarm has promise as novel targeted therapy to treat AR-positive breast cancer. Enobosarm is currently in phase II clinical trial in both ER-positive breast cancer and in TNBC patients. Citation Format: Narayanan R, Ponnusamy S, Fan M, Yang CH, Grimes BL, Fleming MD, Pritchard EF, Berry MP, Oswaks RM, Fine RE, Loiseau J-C, Schwartzberg LS, Pfeffer LM. Nonsteroidal, tissue selective androgen receptor modulator (SARM), enobosarm, reduces growth of androgen receptor-positive breast cancer in patient-derived preclinical models [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-12-06.

Establishment of a large panel of patient-derived preclinical models of human renal cell carcinoma.

The objective of the present work was to establish a large panel of preclinical models of human renal cell carcinoma (RCC) directly from patients, faithfully reproducing the biological features of the original tumor. RCC tissues (all stages/subtypes) were collected for 8 years from 336 patients undergoing surgery, xenografted subcutaneously in nude mice, and serially passaged into new mice up to 13 passages. Tissue samples from the primary tumor and tumors grown in mice through passages were analyzed for biological tissue stability by histopathology, mRNA profiling, von Hippel-Lindau gene sequencing, STR fingerprinting, growth characteristics and response to current therapies. Metastatic models were also established by orthotopic implantation and analyzed by imagery. We established a large panel of 30 RCC models (passage > 3, 8.9% success rate). High tumor take rate was associated with high stage and grade. Histopathologic, molecular and genetic characteristics were preserved between original tumors and case-matched xenografts. The models reproduced the sensitivity to targeted therapies observed in the clinic. Overall, these models constitute an invaluable tool for the clinical design of efficient therapies, the identification of predictive biomarkers and translational research.

Abstract 19: Antitumor activity of selective inhibitors of XPO1/CRM1-mediated nuclear export in diffuse malignant peritoneal mesothelioma: the role of survivin

Abstract Survivin, which is highly expressed and promotes cell survival in diffuse malignant peritoneal mesothelioma (DMPM), exclusively relies on the nuclear exportin 1 (XPO1/CRM1) to be released in the cytoplasm and perform its anti-apoptotic function. Here, we explored the efficacy of selective inhibitors of nuclear export (SINEs) in patient-derived DMPM preclinical models. Exposure to individual SINE (KPT-251, KPT-276, KPT-330) was able to induce a time- and dose-dependent inhibition of the growth of two DMPM cell lines without affecting normal cell proliferation. Such a cell growth inhibition was preceded by a decline in the nuclear XPO1/CRM1 levels and an increase in the nuclear accumulation of its cargo proteins p53 and p21, which led to a cell cycle arrest at G1-phase. Our results also indicated that survivin is an essential component of the downstream signaling pathway of XPO1/CRM1 inhibition in DMPM cells. In fact, in both cell lines, exposure to SINEs led to a time-dependent reduction of cytoplasmic survivin levels and, after an initial survivin nuclear accumulation, also to a progressive decrease in the nuclear protein abundance, through the ubiquitin-proteasomal degradation pathway, leading to the complete depletion of total survivin levels. In both DMPM cell models, according to survivin anti-apoptotic activity, drug-induced reduction of cytoplasmic survivin levels correlated with the onset of caspase-dependent apoptosis. We further observed that SINEs can be combined with other survivin inhibitors, such as the survivin suppressant YM155 to achieve enhanced growth inhibition in DMPM cells. Initial in vivo experiments with orally administered KPT-251, KPT-276 and the orally available, clinical stage KPT-330 (selinexor) indicated that each compound was able to significantly reduce the growth of early-stage subcutaneous DMPM xenografts. Interestingly, additional experiments carry out with selinexor demonstrated that the compound was also able to inhibit the growth of late-stage subcutaneous DMPM xenografts in nude mice. Most importantly, oral administration of selinexor to SCID mice reduced the growth of orthotopic DMPM xenografts, which properly recapitulate the dissemination pattern in the peritoneal cavity of human DMPM and, for this reason, represent a valuable model for investigating novel therapeutic approaches for the disease. Consistent with an important role of survivin as a determinant of anti-cancer activity of SINE compounds, a reduction of the protein expression was observed in tumor specimens obtained from selinexor treated mice. Overall, our results (i) demonstrate a marked efficacy of SINEs in DMPM preclinical models, which is, at least in part, dependent on the interference with survivin intracellular distribution and function, and (ii) suggest SINE-mediated XPO1/CRM1 inhibition as a novel therapeutic option for the disease. Citation Format: Nadia Zaffaroni, Michelandrea De Cesare, Denis Cominetti, Valentina Doldi, Alessia Lopergolo, Marcello Deraco, Paolo Gandellini, Yosef Landesman, Sharon Friedlander, Michael G. Kauffman, Sharon Shacham, Marzia Pennati. Antitumor activity of selective inhibitors of XPO1/CRM1-mediated nuclear export in diffuse malignant peritoneal mesothelioma: the role of survivin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 19. doi:10.1158/1538-7445.AM2015-19

High-Resolution Mutational Profiling Suggests the Genetic Validity of Glioblastoma Patient-Derived Pre-Clinical Models

Recent advances in the ability to efficiently characterize tumor genomes is enabling targeted drug development, which requires rigorous biomarker-based patient selection to increase effectiveness. Consequently, representative DNA biomarkers become equally important in pre-clinical studies. However, it is still unclear how well these markers are maintained between the primary tumor and the patient-derived tumor models. Here, we report the comprehensive identification of somatic coding mutations and copy number aberrations in four glioblastoma (GBM) primary tumors and their matched pre-clinical models: serum-free neurospheres, adherent cell cultures, and mouse xenografts. We developed innovative methods to improve the data quality and allow a strict comparison of matched tumor samples. Our analysis identifies known GBM mutations altering PTEN and TP53 genes, and new actionable mutations such as the loss of PIK3R1, and reveals clear patient-to-patient differences. In contrast, for each patient, we do not observe any significant remodeling of the mutational profile between primary to model tumors and the few discrepancies can be attributed to stochastic errors or differences in sample purity. Similarly, we observe ∼96% primary-to-model concordance in copy number calls in the high-cellularity samples. In contrast to previous reports based on gene expression profiles, we do not observe significant differences at the DNA level between in vitro compared to in vivo models. This study suggests, at a remarkable resolution, the genome-wide conservation of a patient’s tumor genetics in various pre-clinical models, and therefore supports their use for the development and testing of personalized targeted therapies.

Abstract 1754: Identification of BAY 94-9343, a mesothelin antibody-drug conjugate (ADC): Characterization and anti-tumor activity in mesothelin-positive preclinical tumor models

Abstract Monoclonal antibodies have proven to be very effective in the treatment of various cancers, including solid tumors. For example, HERCEPTIN® and Erbitux® are successfully used to treat HER2-positive breast cancer and EGFR-positive colorectal cancer, respectively. Conjugation of cytotoxic drugs to antibodies represents a promising approach to improve cancer therapy. Antibody-drug conjugates (ADCs) are able to deliver highly potent toxophores to tumors while at the same time reducing systemic toxicity. Promising efficacy and tolerability profiles of ADCs have been observed in clinical trials including Hodgkin lymphoma (brentuximab vedotin) and breast cancer (trastuzumab-DM1), thus, development of new ADCs targeting tumor- associated antigens has potential to identifiy novel cancer therapeutics. Mesothelin, a glycoprotein expressed in mesothelial cells found in the membrane lining of the peritoneal and pleural cavities, is overexpressed in all mesotheliomas as well as many ovarian and pancreatic cancers. Due to its limited expression on normal tissues and higher expression in a number of tumor types, mesothelin represents an attractive ADC target. BAY 94-9343 consists of a fully human anti-mesothelin IgG1 antibody conjugated to the potent tubulin-binding drug DM4 with an average of 3.2 drug molecules per antibody. The resulting ADC bound to human recombinant mesothelin with high affinity (Kd = 15nM) leading to antigen-dependent internalization and potent cytotoxicity (nanomolar range in vitro IC50) in tumor cells that express mesothelin either endogenously or exogenously, but not in mesothelin-negative cells. In vivo, BAY 94-9343 demonstrated dose-dependent, mesothelin-specific anti-tumor efficacy in subcutaneous and orthotopic xenograft models at doses between 2.5 and 10 mg/kg using a Q3Dx3 schedule. Endogenously expressing mesothelin tumor models included sc and orthotopic OVCAR3 (ovarian), sc BxPC-3 (pancreatic) and sc NCI-H226 (mesothelioma). Furthermore, in mesothelin-positive patient-derived preclinical tumor models of both platinum-resistant ovarian cancer and gemcitabine-resistant pancreatic cancer, BAY 94-9343 exhibited high anti-tumor efficacy leading to partial and complete tumor regressions with a 10mg/kg Q3Dx3 dosing schedule. This ADC was well tolerated in mice at 10mg/kg (Q3Dx3) without any evidence of body weight loss, compared to either cisplatin or gemcitabine treatments. In summary, BAY 94-9343 is a mesothelin-targeted ADC with promising preclinical anti-tumor activity for mesothelin-positive tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1754. doi:10.1158/1538-7445.AM2011-1754