Zebrafish Patient-Derived Xenografts Identify Chemo-Response in Pancreatic Ductal Adenocarcinoma Patients

Alice Usai, Gregorio Di Franco, Margherita Piccardi, Perla Cateni, Luca Emanuele Pollina, Caterina Vivaldi, Enrico Vasile, Niccola Funel, Matteo Palmeri, Luciana Dente, Alfredo Falcone, Dimitri Giunchi, Alessandro Massolo, Vittoria Raffa, Luca Morelli, Alice Usai, Gregorio Di Franco, Margherita Piccardi, Perla Cateni, Luca Emanuele Pollina, Caterina Vivaldi, Enrico Vasile, Niccola Funel, Matteo Palmeri, Luciana Dente, Alfredo Falcone, Dimitri Giunchi, Alessandro Massolo, Vittoria Raffa, Luca Morelli

Abstract

It is increasingly evident the necessity of new predictive tools for the treatment of pancreatic ductal adenocarcinoma in a personalized manner. We present a co-clinical trial testing the predictiveness of zPDX (zebrafish patient-derived xenograft) for assessing if patients could benefit from a therapeutic strategy (ClinicalTrials.gov: XenoZ, NCT03668418). zPDX are generated xenografting tumor tissues in zebrafish embryos. zPDX were exposed to chemotherapy regimens commonly used. We considered a zPDX a responder (R) when a decrease ≥50% in the relative tumor area was reported; otherwise, we considered them a non-responder (NR). Patients were classified as Responder if their own zPDX was classified as an R for the chemotherapy scheme she/he received an adjuvant treatment; otherwise, we considered them a Non-Responder. We compared the cancer recurrence rate at 1 year after surgery and the disease-free survival (DFS) of patients of both groups. We reported a statistically significant higher recurrence rate in the Non-Responder group: 66.7% vs. 14.3% (p = 0.036), anticipating relapse/no relapse within 1 year after surgery in 12/16 patients. The mean DFS was longer in the R-group than the NR-group, even if not statistically significant: 19.2 months vs. 12.7 months, (p = 0.123). The proposed strategy could potentially improve preclinical evaluation of treatment modalities and may enable prospective therapeutic selection in everyday clinical practice.

Keywords: chemosensitivity; pancreatic cancer; personalized medicine; preclinical model; zebrafish avatar.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Zebrafish patient-derived xenograft model and type of response to chemotherapy drugs. (A) zPDXs were established from the transplantation of fresh PDAC human tissue (DiI-labeled) into the perivitelline space of 2 dpf zebrafish embryos. zPDXs were then treated with GEMOX, GEM/nab-P, GEM and FOLFOXIRI for two days to detect their chemosensitivity profile. Representative image on the bottom (the right image is the magnification of the white-delimited area). Scale bars = 50 µm. (B) Percentage of progressive disease (PD), stable disease (SD), minor response (MR), partial response (PR) and complete response (CR). No statistically significant differences were detected by a Chi-square test. (n = 26, 24, 26, 27 zPDXs, respectively, for GEMOX, GEM/nab-P, GEM and FOLFOXIRI).
Figure 2
Figure 2
Effects of chemotherapy drugs in the linear mixed effect model. (A) Effect displays. The treatments are displayed on the x-axis. Dots identify the fixed effect values of %ΔV estimated by LMM. The bars are the 95% CI of fixed values. (B) Post-hoc test results. Pairwise comparisons are on the y-axis, and the differences of marginal means between treatments are on the x-axis. Blue bars represent the 95% CI of means differences. The dashed line is a difference equal to zero between means.
Figure 3
Figure 3
Analysis of LMM predicted %ΔV and 95% CI for each chemotherapy scheme. Predicted values of %ΔV and 95% CI in (A) FOLFOXIRI, (B) GEM/nab-P, (C) GEMOX and (D) GEM. These values were obtained by adding the LMM fitted values of fixed effects to random values and to their simulated 95% CI. The dashed line is 0 on the log scale. The 95% CI above and intersecting the line identify zPDXs with a non-significant reduction of tumor volume. The 95% CI below and not intersecting the line are zPDXs with a significant reduction of tumor volume. Green bars are zPDXs in which tumor mass is significantly reduced compared to control. To determine the significant reduction of tumor volume with respect to control, we overlapped the 95% CI of treatments and control groups. Patient enrollment codes are reported (P = pancreas). (E) Intersection sets of zPDX classified as “non-significant” and (F) “significant” in Figure 3 (table data with the list of patient codes is provided as Table S1).
Figure 4
Figure 4
Analysis of the follow-up data in comparison to the prediction of the zPDX. (A) Relapse/non-relapse (r/nr) information for 16 PDAC patients enrolled in Table 2 versus the respective responder/non-responder (R/NR) zPDXs. We considered relapse when the patient has the clinical evidence of recurrence within one year after surgery. The zPDX treatment response may predict an early relapse (r) or a better response to therapy (non-relapse, nr). Sixteen PDAC zPDX, corresponding to patients subjected to curative surgery and postoperative adjuvant treatment, were treated with GEM, GEM/nab-P, GEMOX and FOLFOXIRI for 2 days. The zPDX response to treatment was analyzed and quantified adapting the WHO criteria for tumor response. We considered responder (R) zPDX with a decrease ≥50% in the relative tumor area. (B) Confusion matrix highlights the number of no cancer relapse (nr) in patients with the own responder (R) zPDX and the number of cancer relapse (r) in patients with the own non-responder (NR) zPDX. (C) Disease-free survival difference in R group (green) and NR group (blue), p = 0.123 by log-rank test.

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