Development and Expansion of Patient-Derived Xenografts for Endometrial Cancer.

Patient-Derived Xenografts (PDXs) are established by implanting a fragment of a patient tumor into rodents either subcutaneously or orthotopically. PDX models faithfully recapitulate the histologic and molecular profile of the donor patient's cancer and are regarded as authentic preclinical models for drug testing, understanding of tumor biology and biomarker discovery. This Chapter describes the detailed method for establishing robust PDXs for endometrial cancer and provide important notes for users of the protocol to consider during PDXs development.

Endometrial cancer PDX-derived organoids (PDXOs) and PDXs with FGFR2c isoform expression are sensitive to FGFR inhibition.

Endometrial cancer (EC) patients with metastatic/recurrent disease have limited treatment options and poor survival outcomes. Recently, we discovered the FGFR2c splice isoform is associated with poor prognosis in EC patients. Here we report the establishment of 16 EC patient-derived xenografts (PDX)-derived organoids (PDXOs) with or without FGFR2c expression. In vitro treatment of 5 EC PDXOs with BGJ398 showed significant cell death in 3 models with FGFR2c expression. PDXs with high/moderate FGFR2c expression showed significant tumour growth inhibition (TGI) following 21-day treatment with FGFR inhibitors (BGJ398 or pemigatinib) and significantly prolonged survival in 4/5 models. Pemigatinib + cisplatin combination therapy (n = 5) resulted in significant TGI and prolonged survival in one of two p53abn PDXs. All five models treated with cisplatin alone showed de novo resistance and no survival benefit. Seven-day treatment with BGJ398 revealed a significant reduction in angiogenesis and CD206 + M2 macrophages. These data collectively support the evaluation of FGFR inhibitors in a clinical trial.

Spatial expression of the FGFR2b splice isoform and its prognostic significance in endometrioid endometrial carcinoma.

Endometrial carcinoma (EC) is the most common gynecological malignancy and fibroblast growth factor receptor 2 (FGFR2) is a frequently dysregulated receptor tyrosine kinase. FGFR2b and FGFR2c are the two main splice isoforms of FGFR2 and are normally localized in epithelial and mesenchymal cells, respectively. Previously, we demonstrated that FGFR2c mRNA expression was associated with aggressive tumor characteristics, shorter progression-free survival (PFS), and disease-specific survival (DSS) in endometrioid ECs (EECs). The objectives of this study were to investigate the spatial expression of FGFR2b in normal and hyperplasia with and without atypia of human endometrium and to assess the prognostic significance of FGFR2b expression in EC. FGFR2b and FGFR2c mRNA expression was evaluated in normal (proliferative [n = 10], secretory [n = 15], and atrophic [n = 10] endometrium), hyperplasia with and without atypia (n = 19) as well as two patient cohorts of EC samples (discovery [n = 78] and Vancouver [n = 460]) using isoform-specific BaseScope RNA in situ hybridization assays. Tumors were categorized based on FGFR2 isoform expression (one, both, or neither) and categories were correlated with clinicopathologic markers, molecular subtypes, and clinical outcomes. The FGFR2b splice isoform was exclusively expressed in the epithelial compartment of normal endometrium and hyperplasia without atypia. We observed FGFR2c expression at the basalis layer of glands in 33% (3/9) of hyperplasia with atypia. In patients with EEC, FGFR2b+/FGFR2c- expression was found in 48% of the discovery cohort and 35% of the validation Vancouver cohort. In univariate analyses, tumors with FGFR2b+/FGFR2c- expression had longer PFS (hazard ratio [HR] 0.265; 95% CI 0.145-0.423; log-rank p < 0.019) and DSS (HR 0.31; 95% CI 0.149-0.622; log-rank p < 0.001) compared to tumors with FGFR2b-/FGFR2c+ expression in the large EEC Vancouver cohort. In multivariable Cox regression analyses, tumors with FGFR2b+/FGFR2c- expression were significantly associated with longer DSS (HR 0.37; 95% CI 0.153-0.872; log-rank p < 0.023) compared to FGFR2b-/FGFR2c+ tumors. In conclusion, FGFR2b+/FGFR2c- expression is associated with favorable clinicopathologic markers and clinical outcomes suggesting that FGFR2b could play a role in tailoring the management of EEC patients in the clinic if these findings are confirmed in an independent cohort.

Patient-derived xenograft models capture genomic heterogeneity in endometrial cancer.

BACKGROUND: Endometrial cancer (EC) is a major gynecological cancer with increasing incidence. It comprises four molecular subtypes with differing etiology, prognoses, and responses to chemotherapy. In the future, clinical trials testing new single agents or combination therapies will be targeted to the molecular subtype most likely to respond. As pre-clinical models that faithfully represent the molecular subtypes of EC are urgently needed, we sought to develop and characterize a panel of novel EC patient-derived xenograft (PDX) models. METHODS: Here, we report whole exome or whole genome sequencing of 11 PDX models and their matched primary tumor. Analysis of multiple PDX lineages and passages was performed to study tumor heterogeneity across lineages and/or passages. Based on recent reports of frequent defects in the homologous recombination (HR) pathway in EC, we assessed mutational signatures and HR deficiency scores and correlated these with in vivo responses to the PARP inhibitor (PARPi) talazoparib in six PDXs representing the copy number high/p53-mutant and mismatch-repair deficient molecular subtypes of EC. RESULTS: PDX models were successfully generated from grade 2/3 tumors, including three uterine carcinosarcomas. The models showed similar histomorphology to the primary tumors and represented all four molecular subtypes of EC, including five mismatch-repair deficient models. The different PDX lineages showed a wide range of inter-tumor and intra-tumor heterogeneity. However, for most PDX models, one arm recapitulated the molecular landscape of the primary tumor without major genomic drift. An in vivo response to talazoparib was detected in four copy number high models. Two models (carcinosarcomas) showed a response consistent with stable disease and two models (one copy number high serous EC and another carcinosarcoma) showed significant tumor growth inhibition, albeit one consistent with progressive disease; however, all lacked the HR deficiency genomic signature. CONCLUSIONS: EC PDX models represent the four molecular subtypes of disease and can capture intra-tumor heterogeneity of the original primary tumor. PDXs of the copy number high molecular subtype showed sensitivity to PARPi; however, deeper and more durable responses will likely require combination of PARPi with other agents.

Fibroblast Growth Factor Receptor 2 Isoforms Detected via Novel RNA ISH as Predictive Biomarkers for Progestin Therapy in Atypical Hyperplasia and Low-Grade Endometrial Cancer.

Women with atypical hyperplasia (AH) or well-differentiated early-stage endometrioid endometrial carcinoma (EEC) who wish to retain fertility and/or with comorbidities precluding surgery, are treated with progestin. Clinically approved predictive biomarkers for progestin therapy remain an unmet need. The objectives of this study were to document the overall response rate (ORR) of levonorgestrel intrauterine device (LNG-IUD) treatment, and determine the association of FGFR2b and FGFR2c expression with treatment outcome. BaseScope RNA ISH assay was utilized to detect expression of FGFR2b and FGFR2c mRNA in the diagnostic biopsies of 89 women (40 AH and 49 EEC) treated with LNG-IUD. Detailed clinical follow-up was available for 69 women which revealed an overall response rate (ORR) of 44% (30/69) with a higher ORR seen in AH (64%) compared to EEC (23%). The recurrence rate in women who initially responded to LNG-IUD was 10/30 (33.3%). RNA ISH was successful in 72 patients and showed FGFR2c expression in 12/72 (16.7%) samples. In the 59 women with detailed clinical follow-up and RNA-ISH data, women with tumours expressing FGFR2c were 5-times more likely to have treatment failure in both univariable (HR 5.08, p < 0.0001) and multivariable (HR 4.5, p < 0.002) Cox regression analyses. In conclusion, FGFR2c expression appears to be strongly associated with progestin treatment failure, albeit the ORR is lower in this cohort than previously reported. Future work to validate these findings in an independent multi-institutional cohort is needed.

FGFR2c Mesenchymal Isoform Expression Is Associated with Poor Prognosis and Further Refines Risk Stratification within Endometrial Cancer Molecular Subtypes.

PURPOSE: The two most common molecular subtypes of endometrial cancers, mismatch repair deficient (MMRd) and p53 wild-type (p53wt) comprise the majority of endometrial cancers and have intermediate prognoses where additional risk stratification biomarkers are needed. Isoform switching of FGFR2 from FGFR2b to FGFR2c (normally expressed in mesenchymal cells), has been reported in other solid carcinomas. The objective of this study was to investigate the role of FGFR2c in risk stratification of endometrial cancer. EXPERIMENTAL DESIGN: We have developed and optimized a BaseScope RNA ISH assay to detect FGFR2c. FGFR2c expression was determined in a preliminary screening cohort of 78 endometrial cancers and a clinically and molecularly annotated Vancouver cohort (n = 465). Cox regression model analyses were performed to assess the prognostic value of FGFR2c. RESULTS: Univariate and multivariate analyses revealed FGFR2c expression was significantly associated with shorter disease-specific survival (DSS) and progression-free survival (PFS) in endometrioid endometrial cancer (EEC, n = 302). Notably, FGFR2c expression was significantly associated with shorter PFS and DSS in patients with grade 3 EECs (P < 0.003 and P < 0.002) and the European Society Medical Oncology (ESMO) high-risk group (P < 0.0001 and P < 0.002), respectively. Moreover, within the MMRd subtype, FGFR2c expression was significantly associated with shorter PFS (P < 0.048) and DSS (P < 0.001). CONCLUSIONS: FGFR2c expression appears an independent prognostic biomarker in patients with EEC and further discerns the outcomes within grade 3 tumors, ESMO high-risk groups, as well as within the MMRd and p53wt subtypes. FGFR2c inclusion into future molecular subtyping can further refine risk stratification of EEC.