Stratifying esophago-gastric cancer treatment using a patient-derived organoid-based threshold

Abstract

Background and aims: This study sought to determine the value of patient-derived organoids (PDOs) from esophago-gastric adenocarcinoma (EGC) for response prediction to neoadjuvant chemotherapy (neoCTx).

Methods: Endoscopic biopsies of patients with locally advanced EGC (n = 120) were taken into culture and PDOs expanded. PDOs' response towards the single substances of the FLOT regimen and the combination treatment were correlated to patients' pathological response using tumor regression grading. A classifier based on FLOT response of PDOs was established in an exploratory cohort (n = 13) and subsequently confirmed in an independent validation cohort (n = 13).

Results: EGC PDOs reflected patients' diverse responses to single chemotherapeutics and the combination regimen FLOT. In the exploratory cohort, PDOs response to single 5-FU and FLOT combination treatment correlated with the patients' pathological response (5-FU: Kendall's τ = 0.411, P = 0.001; FLOT: Kendall's τ = 0.694, P = 2.541e-08). For FLOT testing, a high diagnostic precision in receiver operating characteristic (ROC) analysis was reached with an AUC_ROC of 0.994 (CI 0.980 to 1.000). The discriminative ability of PDO-based FLOT testing allowed the definition of a threshold, which classified in an independent validation cohort FLOT responders from non-responders with high sensitivity (90%), specificity (100%) and accuracy (92%).

Conclusion: In vitro drug testing of EGC PDOs has a high predictive accuracy in classifying patients' histological response to neoadjuvant FLOT treatment. Taking into account the high rate of successful PDO expansion from biopsies, the definition of a threshold that allows treatment stratification paves the way for an interventional trial exploring PDO-guided treatment of EGC patients.

Keywords: Gastric cancer; Patient-derived organoids; Personalized medicine; Response prediction.

Fig. 1

Trial flow chart and phenotypic, molecular as well as functional characterization of PDO cohort. A Flow chart of the study population. DD/HD: University hospital of Dresden (DD) and Heidelberg (HD). B Brightfield images of a representative EGC organoids line (OO4) and a normal gastric PDO line as a comparison (scale bar: 50 µm). Representative images of the histopathological characterization of primary tumor and corresponding PDO (OO4). The treatment naïve tumor biopsy, thereof derived organoid culture and the post-treatment resection specimen were characterized by hematoxylin and eosin (HE) staining, carcinoembryonic antigen (CEA) staining, periodic acid-Schiff reaction (PAS) as well as cytokeratin7 and cadherin17 (CK7/CADH17) double-staining (scale bar: 50 μm). C Oncoplot depicting prevalent genetic alterations in primary tumors and derived PDO lines. D Bar graph of mutational frequencies of this study. E Dose response curves from cell viability assay of 5-FU treated PDOs 144 h post treatment (average of n = 3 replicates per PDO). F Combined dose response curves from PDOs according to the patients' pathological response of 5-FU with standard deviations (repeated measures analysis of variance (ANOVA) of grouped PDOs, P = 0.001 for 5-FU). *P < 0.05; **P < 0.01. G Comparison of the relative area under the curve (AUC_rel) of PDOs grouped by the patients' pathological response (R: responder; NR: non-responder) for 5-FU (unpaired two-tailed student's t-test, P = 0.010 for 5-FU, *P < 0.05). Blue lines represent pathological non-responders, black lines are pathological responders

Fig. 2

Establishment and validation of an in vitro threshold value to predict in vivo FLOT response. A Dose response curves (DRC) from cell viability assay of PDOs treated with the standard FLOT mixture (n) in varying dilutions analyzed 144 h post treatment (average of n = 3 replicates per PDO). B Combined FLOT-DRC from PDOs according to the patients' pathological response after FLOT neoCTx with standard deviations (repeated measures analysis of variance (ANOVA) of grouped PDOs, P = 4.55e-06). ***P < 0.001 C, Comparison of the relative area under the curve (AUC_rel) of PDOs grouped by the patients' pathological response (R: responder; NR: non-responder) (two-tailed student's t-test, P = 3.94e-05). ***P < 0.001 D Dot plot of AUC_rel(FLOT) ordered by the patients’ pathological response. Correlation analysis was performed using Kendall ordinal correlation. P < 0.005 was considered statistically significant. E Heatmap of the patients' pathological response and AUC_rel derived Z scores from single drug and FLOT combination treatment. Color scale indicates Z score (± 2.5; -2.5: black, 0: white, + 2.5: blue) and scored patients' pathological response according to Becker et al. F ROC curve of the AUC_rel(FLOT) generated from single FLOT treatments of PDOs (n = 39, 3 replicates of 13 PDOs). G Summary graph of the AUC_rel(ROC) and confidence interval (CI) 95% for FLOT, 5-FU, oxaliplatin and docetaxel. H Dose response curve with CI of FLOT combination treatment grouped according to the patients' pathological response with calculated threshold curve and calculated AUC_threshold(FLOT). Blue lines represent pathological non-responders, black lines are pathological responders. I FLOT combination test results with subsequent classification according to the determined threshold value (AUC_threshold = 0.559) into responding (R) and non-responding (NR) patients. Correctness of classification was later assessed by comparison to patients' pathological response. Red dotted line indicates the threshold established in the exploratory cohort. J Achieved sensitivity, specificity and accuracy depending on the number of performed FLOT testings considered for classification. K ROC curve of the AUC_rel(FLOT) generated from the mean of three independent replicates for each PDO of the whole cohort (n = 26)