Tailored chemotherapy for colorectal cancer peritoneal metastases based on a drug-screening platform in patient-derived organoids: a case report
• Drug testing in organoids established from peritoneal implants are useful to select chemotherapeutic agents and avoid unnecessary treatments and side effects for colorectal cancer patients with peritoneal metastases.
What is known and what is new?
• Few therapeutic options exist for colorectal cancer patients with peritoneal metastases and commonly used drugs show limited beneficial effects.
• This manuscript adds the utility of patient-derived organoid technology to improve the efficacy of chemotherapy for these patients.
What is the implication, and what should change now?
• Organoids constitute a convenient platform for selecting chemotherapeutic drugs. Generating organoids from peritoneal implants and, if possible, from primary tumors is recommendable for a personalized clinical management of colorectal cancer patients with peritoneal metastases.
Approximately 17% of patients with metastatic colorectal cancer (CRC) have peritoneal carcinomatosis. However, only 2% have the peritoneum as the only site of metastasis (1,2). Peritoneal carcinomatosis in patients with CRC is considered a fatal disease with a median overall survival (OS) of 6–9 months without treatment; however, it could reach 24 months in patients subjected to palliative surgery and systemic treatment. This scenario changed with the development of cytoreductive surgery (CRS), a combination of peritonectomy procedures and multivisceral resection to accomplish the complete removal of visible disease from the abdominal cavity, followed by perfusion of hyperthermic intraperitoneal chemotherapy (HIPEC). Mitomycin or oxaliplatin are the common drugs for HIPEC for colorectal peritoneal metastases and an OS between 42 and 62 months has been reported with this multimodal approach (3). However, these promising results have not been confirmed in the recent PRODIGE7 phase 3 trial (4) evaluating the role of HIPEC and CRS vs. CRS alone, and therefore the study of peritoneal carcinomatosis remains open to the assessment of new drugs and approaches (5). For patients with no possibility of CRS and HIPEC due to high peritoneal carcinomatosis index (PCI) or unresectable disease, a new technique that is increasingly being used in experienced centers is pressurized intraperitoneal aerosol chemotherapy (PIPAC). Increased penetration of chemotherapy in the tumors, reduced toxicity, an important rate of clinical responses, improves quality of life, and prolonged progression free-survival (PFS) have been reported as the most relevant benefits of this technique (6).
Organoids have recently emerged as a suitable platform to test the chemosensitivity of a variety of solid tumor types. They have the potential to avoid futile treatments and harmful side effects for the patients. Studies addressing the use of organoids to treat patients with CRC are emerging in the literature (7). Patients with CRC peritoneal metastases treated with HIPEC or PIPAC represent a unique subgroup of patients in which tumor specimen from the metastatic site can be sampled during the intervention in order to generate organoids and perform the drug-screening analysis.
We report a case of a patient with multiple colorectal peritoneal metastases that was not candidate for CRS and HIPEC due to non-resectable extent of the disease. Following standard FOLFOXIRI plus Bevacizumab treatment, the patient was subjected to palliative PIPAC. Additionally, we generated organoids from a peritoneal implant in order to determine the sensitivity to cytotoxic agents that could guide our therapeutic strategy. This case report is one of the very few studies performed in a patient with early-onset (<50 years) CRC with peritoneal carcinomatosis. We present the following article in accordance with the CARE reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-599/rc).
In January 2020, a 45-year-old man presented to the emergency room with rectal bleeding and hematuria. A colonoscopy performed during the diagnostic work-up revealed a histologically-confirmed upper rectal mucinous adenocarcinoma. Clinical staging evaluation with computed tomography (CT) scan and pelvic magnetic resonance imaging (MRI) classified the tumor as cT4b N2 M1 according to the American Joint Committee on Cancer (AJCC) tumor-node-metastasis (TNM) v.8, and showed involvement of hepatic hilum and gastrohepatic lymph nodes, and a solitary liver metastasis in segment V (Figure 1). The patient started on systemic chemotherapy with FOLFOXIRI plus Bevacizumab. After 3 cycles, the positron emission tomography (PET)-CT scan showed partial morphologic and metabolic response. In July 2020, after 8 cycles and a complete metabolic response on the subsequent PET-CT scan, an exploratory laparotomy was performed. Multiple peritoneal carcinomatosis were confirmed with a PCI of 27 involving the peritoneal surface of both diaphragms, mesentery, small intestine, gallbladder, hepatic hilum, stomach and omentum. We also found scarce ascites and massive dilatation of the descending colon that required segmental colonic resection and terminal stoma creation. Anatomopathological study of the surgical piece revealed a fragment of the large intestine extensively infiltrated by a mucinous adenocarcinoma. Cytological and molecular studies showed malignant cells of a mucinous phenotype with CDX2 and EpCAM (BerEp4 positivity) expression. The tumor was microsatellite stable (MSS) and biomarker analysis revealed no mutation in KRAS, NRAS and BRAF genes. During laparotomy, after signing an informed consent, a biopsy of a peritoneal metastasis was obtained to generate patient-derived organoids (PDO) (Figure 2). All procedures performed in the study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of the Hospital Universitario La Paz (No. HULP-PI-3196). Written informed consent was obtained from the closer relative of the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the editorial office of this journal.
Subsequently, the patient started on maintenance chemotherapy with 5-fluorouracil/leucovorin (5-FU/LV) plus Bevacizumab. In October 2020, the patient underwent a combination treatment protocol with systemic biweekly 5-FU/LV and PIPAC using the standard-of-care chemotherapy (mitomycin at 1.5 mg/m2) initially programmed every three months. In order to identify a personalized chemotherapy for administration during the second PIPAC procedure, and to study the putative acquisition of resistance and sensitivity to previously unused drugs, we performed low-throughput drug screening following a previously published protocol of our group that renders high reproducibility (8).
PDO from the peritoneal implant were established following the protocol previously described for primary colorectal tumors (9). The mucinous feature and low cellularity of the implant required the culture in the presence of Wnt-conditioned media and R-spondin for optimal growth (spherical) of the organoids (Figure 3A). For drug screening analysis, over 10 weeks after surgery, organoids were treated with 5-FU, SN-38 (7-ethyl-10-hydroxycamptothecin, the active metabolite of the DNA topoisomerase I inhibitor irinotecan) or oxaliplatin. After 4 days of treatment, PDO in cultures treated with 5-FU or SN-38 were smaller than those in control untreated cultures, a result that was less evident for cultures treated with oxaliplatin (Figure 3B). Cytotoxic activity of the drugs was addressed by measuring the cell viability, and the quality of the assay was checked by analyzing the Z-score, a control parameter to ensure an adequate drug-response validation (10). Under the microscope, 5-FU and SN-38 displayed relatively strong cytotoxicity (IC50 =9.6 µM and IC50 =0.015 µM, respectively). Contrarily, the response to oxaliplatin was much lower and no IC50 could be stated (Figure 3B). In good agreement with this, the Z-score for the assays using 5-FU and SN-38 were >0.5 (excellent) but only 0.03 in the case of oxaliplatin (Figure 3B). In addition, the PDO mutational analysis using the Oncomine Comprehensive Assay v3, which contains 161 of the most relevant cancer genes, confirmed the wild-type status of KRAS, NRAS and BRAF previously found in the original surgical biopsy. The assay showed two genes mutated, PIK3CA (c.3140A>G, rs121913279) and SMAD4 (c.1082G>T, rs377767347), which are associated to mucinous histology and aggressiveness of colorectal tumors (11,12) (Figure 2). In summary, these data indicate that at least the original implant had acquired resistance to oxaliplatin, while it remained sensitive to irinotecan and 5-FU, which supports therapeutic options based on single or combined treatments with these two drugs.
After the first PIPAC, the patient developed a colo-cutaneous fistulae that prevented from further PIPAC treatment. In line with the results of the drug-screening in the PDO and standard clinical practice, the patient resumed systemic 5-FU/LV until February 2021, when the central venous catheter was removed due to a gram-negative bacilli catheter-related infection. The treatment was then switched to capecitabine until September 2021, when the patient unfortunately died due to a malignant pseudo-obstruction (Figure 2).
Organoids were first established as a three-dimensional culture system of mouse intestinal stem cells (SC) and their cell progeny and later of human small intestine and colon normal and cancer stem cells (CSC) (13). The CSC model proposes the conversion of tissue-resident SC into CSC as the key event in tumor initiation, progression, recurrence and chemoresistance. On the basis of this model, many laboratories have established PDO from different tumor types to study the cancer biology and in several cases their predictive value in drug sensitivity testing aiming to their use for personalized treatment (14). In line with their original intestinal development, this has been particularly frequent in CRC (7,15). According to the 100% negative predictive value of drug assays in PDO models (16), the lack of effect of oxaliplatin suggested that this drug was not a valid therapeutic option for our patient.
Patients with colorectal peritoneal metastasis have a poor prognosis even when are subjected to HIPEC and/or PIPAC, usually in combination with oxaliplatin or mitomycin. Consequently, novel approaches are urgently needed. Our data show that drug sensitivity can be estimated in organoid cultures from peritoneal metastases of patients at a late stage of the tumorigenic process. This supports the establishment of peritoneal organoids as a useful platform to screen for drug activity and so for personalized treatment of patients with colorectal carcinomatosis. As the subject of this study had been treated with several cycles of chemotherapy, this is particularly promising for treatment-naïve patients or patients treated with fewer cycles of standard chemotherapy. The chance of obtaining beneficial results for the patient is clearly greater if organoids are established at early stages of the carcinomatosis process, when the number of peritoneal implants is low. During the course of our study, a report using PDO from colorectal peritoneal metastases has been published (17). This is a multicenter prospective study aimed at direct personalized therapy that describes the successful generation of organoids from 19 out of 28 patients (68%). In a timeframe comparable to our study (8 weeks), the authors performed genomic and drug profiling that resulted in a treatment change for two patients. Other studies have investigated the response to antitumoral drugs of peritoneal metastasis organoids upon xenotransplantation in mice, which however loses clinical relevance (18).
As for primary tumors of diverse origin, the generation of organoids from peritoneal metastases needs to be optimized to increase the percentage of success to nearly 100% of normal colon tissue (15). The dependence on Wnt and R-spondin of the organoids in this study agrees with that reported for mucinous primary colorectal tumors by Sato’s group (19). It suggests the vulnerability of these Wnt-dependent neoplasias to Wnt inhibitors such as those under clinical development that target porcupine, an acyltransferase enzyme required for the secretion of active Wnt factors. Interestingly, recent data from Clevers’ group indicate that 5-FU induces the enrichment of colon CSC via p53-mediated Wnt activation, which contributes to tumor recurrence after treatment (20). This finding supports the use of therapeutic strategies combining 5-FU with Wnt inhibitors, which can be tested in PDO.
In conclusion, our study is a proof-of-concept strategy that supports the feasibility of the use of PDO for the personalized treatment in colorectal carcinomatosis; however, the precise contribution of organoid technology needs to be addressed in further well-designed prospective clinical studies.
We want to particularly acknowledge the patient and the Biobank IdiPAZ (PT20/00004) integrated in the Spanish National Biobanks Network for their collaboration. We also thank Dr. Gema Moreno-Bueno (CIBERONC) for the mutational analysis of the PDO and Lucille Banham for her valuable assistance in the preparation of the English manuscript.
Funding: The work in the authors’ laboratory is funded by Instituto de Salud Carlos III (ISCIII) (ICI20/00057, CIBERONC/CB16/12/00273, CIBERONC/CB16/12/00398) and co-funded by the European Union-Fondo Europeo de Desarrollo Regional (FEDER), and the Agencia Estatal de Investigación (PID2019-104867RB-I00/AEI/10.13039/501100011033). DV is supported by a Sociedad Española de Oncología Médica (SEOM)-Río Hortega contract.
Reporting Checklist: The authors have completed the CARE reporting checklist. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-599/rc
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-599/coif). IP has received a grant from ISCIII-FEDER (Spain; No. ICI20/00057). AB, AM and AFB report that they have received grants from Agencia Estatal de Investigación (Spain; No. PID2019-104867RB100/AEI/10.13039/501100011033), Instituto de Salud Carlos III (ISCIII)-Fondo Europeo de Desarrollo Regional (FEDER) (Spain; No. ICI20/00057) and ISCIII-CIBERONC (Spain; No. CIBERONC/CB16/12/00273). NRS has received two grants from ISCIII-FEDER (Spain; No. ICI20/00057) and ISCIII-CIBERONC (Spain; No. CIBERONC/CB16/12/00398). DV has been awarded with a Río Hortega contract from Sociedad Española de Oncología Médica (SEOM) (Spain) and received a grant from ISCIII-FEDER (Spain; No. ICI20/00057). DCG participates on the Board of Directors of the European Society of Surgical Oncology (ESSO) and the Board of Directors of the Spanish Society of Surgical Oncology (SEOQ). The authors have no other conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in the study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of the Hospital Universitario La Paz (No. HULP-PI-3196). Written informed consent was obtained from the closer relative of the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the editorial office of this journal.
Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.
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