Organoids as a biomarker for personalized treatment in metastatic colorectal cancer: drug screen optimization and correlation with patient response

Abstract

Background: The inability to predict treatment response of colorectal cancer patients results in unnecessary toxicity, decreased efficacy and survival. Response testing on patient-derived organoids (PDOs) is a promising biomarker for treatment efficacy. The aim of this study is to optimize PDO drug screening methods for correlation with patient response and explore the potential to predict responses to standard chemotherapies.

Methods: We optimized drug screen methods on 5-11 PDOs per condition of the complete set of 23 PDOs from patients treated for metastatic colorectal cancer (mCRC). PDOs were exposed to 5-fluorouracil (5-FU), irinotecan- and oxaliplatin-based chemotherapy. We compared medium with and without N-acetylcysteine (NAC), different readouts and different combination treatment set-ups to capture the strongest association with patient response. We expanded the screens using the optimized methods for all PDOs. Organoid sensitivity was correlated to the patient's response, determined by % change in the size of target lesions. We assessed organoid sensitivity in relation to prior exposure to chemotherapy, mutational status and sidedness.

Results: Drug screen optimization involved excluding N-acetylcysteine from the medium and biphasic curve fitting for 5-FU & oxaliplatin combination screens. CellTiter-Glo measurements were comparable with CyQUANT and did not affect the correlation with patient response. Furthermore, the correlation improved with application of growth rate metrics, when 5-FU & oxaliplatin was screened in a ratio, and 5-FU & SN-38 using a fixed dose of SN-38. Area under the curve was the most robust drug response curve metric. After optimization, organoid and patient response showed a correlation coefficient of 0.58 for 5-FU (n = 6, 95% CI -0.44,0.95), 0.61 for irinotecan- (n = 10, 95% CI -0.03,0.90) and 0.60 for oxaliplatin-based chemotherapy (n = 11, 95% CI -0.01,0.88). Median progression-free survival of patients with resistant PDOs to oxaliplatin-based chemotherapy was significantly shorter than sensitive PDOs (3.3 vs 10.9 months, p = 0.007). Increased resistance to 5-FU in patients with prior exposure to 5-FU/capecitabine was adequately reflected in PDOs (p = 0.003).

Conclusions: Our study emphasizes the critical impact of the screening methods for determining correlation between PDO drug screens and mCRC patient outcomes. Our 5-step optimization strategy provides a basis for future research on the clinical utility of PDO screens.

Keywords: Cancer; Chemotherapy; Colorectal cancer; Drug screening; Oncology; Organoids; Precision medicine.

Fig. 1

Schematic overview of the study. Tissue from metastatic and primary CRC tumours was obtained via resection or biopsy prior to starting a new line of systemic treatment. PDOs were cultured and screened for standard-of-care treatments while the patient received standard systemic treatment. PDO drug screens were optimized by comparing various methods using a limited set of 5–11 PDOs, either among one another or by correlation with patient response. For all patients, organoid and patient response were compared for treatment given after the organoid was established

Fig. 2

Association of tumour size change and PFS with organoid response to 5-FU & oxaliplatin. A DRCs of organoid sensitivity to 5-FU & oxaliplatin combination treatment screened in a 1.8:1 ratio. B Scatterplots show the correlation between patient response (% size change during treatment) and organoid response measured (GR_AUC) for 5-FU & oxaliplatin combination treatment screened in a 1.8:1 ratio. C Kaplan-Meier progression-free survival curves of patients stratified by organoid sensitivity to 5-FU & oxaliplatin, based on normalized GR_AUC (cut-off upper tertile = 0.63)_. Censoring events are indicated by vertical bars on the corresponding curve. The table underneath each plot denotes the numbers at risk. Log-rank test-based p value is shown. Abbreviations: 5-FU (5-fluorouracil), DRC (drug response curve), GR_AUC (area under the growth rate inhibition curve), PFS (progression-free survival)

Fig. 3

Association of tumour size change and organoid response to irinotecan-based treatment and 5-FU monotherapy. A DRCs of organoid sensitivity to 5-FU & SN-38 combination treatment. B Scatterplots show the correlation between patient response (% size change during treatment) and organoid response measured (GR_AUC) for (5-FU &) SN-38. C DRCs of organoid sensitivity to 5-FU monotherapy. D Scatterplots show the correlation between patient response (% size change during treatment) and organoid response measured (GR_AUC) for 5-FU monotherapy. Abbreviations: 5-FU (5-fluorouracil), DRC (drug response curve), CTG (CellTiter-Glo), GR_AUC (area under the growth rate inhibition curve), SN-38 (active metabolite of irinotecan)

Fig. 4

Organoid sensitivity based on primary tumour location and mutational status. A Clustered heatmap of the normalized GR_AUC, with characteristics of the patient indicated in the first four columns for chemotherapy exposure prior to PDO establishment (adjuvant or first line), the primary tumour location (left- versus right-sided), mutational status (BRAF-mutant, RAS-mutant and RAS/BRAF-wildtype) and PDO origin (metastatic and primary tumour). The normalized GR_AUC are illustrated as a heatmap with a column for each treatment type examined, with PDOs that were not screened for oxaliplatin in grey. B and C) The DRCs for panitumumab (B) and 5-FU (C) treatment and boxplots of the GR_AUC for PDOs categorized according to the tumour’s mutational status and sidedness (RAS/BRAF-wildtype and left-sided; RAS/BRAF-wildtype and rectal; RAS/BRAF-wildtype and right-sided; RAS-mutant and BRAF-mutant. Boxplots show the minimum, median, maximum, upper and lower quartiles and individual data points. Abbreviations: 5-FU (5-flouruoracil), DRC (drug response curve), CTG (CellTiter-Glo), CQ (CyQUANT), GR_AUC (area under the growth rate inhibition curve), PDO (patient-derived organoid), SN-38 (active metabolite of irinotecan), wt (wildtype)

Fig. 5

Increased resistance to 5-FU after prior exposure to chemotherapy. The association between prior chemotherapy exposure to patients before the biopsy for PDOs was established and organoid response (GR_AUC) is shown for 5-FU, oxaliplatin and SN-38. A, C and D. The DRC of organoid sensitivity with red curves indicating 5-FU or capecitabine containing chemotherapy exposure. B, D and F Boxplots of normalized GR_AUC for patients that were exposed to chemotherapy versus chemotherapy-naïve patients. Boxplots show the minimum, median, maximum, upper and lower quartiles and individual data points. Abbreviations: 5-FU (5-flouruoracil), GR_AUC (area under the growth rate inhibition curve), SN-38 (active metabolite of irinotecan)