Human Metastatic Cholangiocarcinoma Patient-Derived Xenografts and Tumoroids for Preclinical Drug Evaluation

Human metastatic cholangiocarcinoma; Xenografts; TumoroidsColangiocarcinoma metastàtic humà; Xenoempelts; TumoroidesColangiocarcinoma metastásico humano; Xenoinjertos; TumoroidesPurpose: Cholangiocarcinoma (CCA) is usually diagnosed at advanced stages, with limited therapeutic options. Preclinical models focused on unresectable metastatic CCA are necessary to develop rational treatments. Pathogenic mutations in IDH1/2, ARID1A/B, BAP1, and BRCA1/2 have been identified in 30%–50% of patients with CCA. Several types of tumor cells harboring these mutations exhibit homologous recombination deficiency (HRD) phenotype with enhanced sensitivity to PARP inhibitors (PARPi). However, PARPi treatment has not yet been tested for effectiveness in patient-derived models of advanced CCA. Experimental Design: We have established a collection of patient-derived xenografts from patients with unresectable metastatic CCA (CCA_PDX). The CCA_PDXs were characterized at both histopathologic and genomic levels. We optimized a protocol to generate CCA tumoroids from CCA_PDXs. We tested the effects of PARPis in both CCA tumoroids and CCA_PDXs. Finally, we used the RAD51 assay to evaluate the HRD status of CCA tissues. Results: This collection of CCA_PDXs recapitulates the histopathologic and molecular features of their original tumors. PARPi treatments inhibited the growth of CCA tumoroids and CCA_PDXs with pathogenic mutations of BRCA2, but not those with mutations of IDH1, ARID1A, or BAP1. In line with these findings, only CCA_PDX and CCA patient biopsy samples with mutations of BRCA2 showed RAD51 scores compatible with HRD. Conclusions: Our results suggest that patients with advanced CCA with pathogenic mutations of BRCA2, but not those with mutations of IDH1, ARID1A, or BAP1, are likely to benefit from PARPi therapy. This collection of CCA_PDXs provides new opportunities for evaluating drug response and prioritizing clinical trials.This work was supported by grants from the Fundació Marató TV3 awarded to T. Macarulla, M. Melé, and S. Peiró; BeiGene research grant awarded to T. Macarulla and S. Peiró; AECC (INVES20036TIAN), Ramón y Cajal investigator program (RYC2020-029098-I), Proyecto de I+D+i (PID2019-108008RJ-I00), and FERO Foundation grant awarded to T.V. Tian; Proyecto de Investigación en Salud from the Instituto de Salud Carlos III (ISCIII) (PI20/00898) awarded to T. Macarulla; FIS/FEDER from the Instituto de Salud Carlos III (ISCIII) (PI12/01250; CP08/00223; PI16/00253 and CB16/12/00449) awarded to S. Peiró; and Ramón y Cajal investigator program (RYC-2017-22249) awarded to M. Melé. Q. Serra-Camprubí is a recipient of the Ph.D. fellowship from La Caixa Foundation (LCF/PR/PR12/51070001). A. Llop-Guevara was supported by the AECC (INVES20095LLOP) and V. Serra by the ISCIII (CPII19/00033). E.J. Arenas was funded by the AECC (POSTD211413AREN). J. Arribas is funded by the Instituto de Salud Carlos III (AC15/00062, CB16/12/00449, and PI22/00001). This publication is based upon the work of COST Action CA18122, European Cholangiocarcinoma Network, supported by the COST (European Cooperation in Science and Technology, www.cost.eu), a funding agency for research and innovation networks. The authors would like to thank Dr. V.A. Raker for manuscript editing and Drs. N. Herranz and J. Mateo for scientific discussions. The authors acknowledge the infrastructure and support of the FERO Foundation, La Caixa Foundation, and the Cellex Foundation

Early-Stage Breast Cancer Detection in Breast Milk

Breast cancer; Breast milkCáncer de mama; Leche maternaCàncer de mama; Llet maternaBreast cancer occurring during pregnancy (PrBC) and postpartum (PPBC) is usually diagnosed at more advanced stages compared with other breast cancer, worsening its prognosis. PPBC is particularly aggressive, with increased metastatic risk and mortality. Thus, effective screening methods to detect early PrBC and PPBC are needed. We report for the first time that cell-free tumor DNA (ctDNA) is present in breast milk (BM) collected from patients with breast cancer. Analysis of ctDNA from BM detects tumor variants in 87% of the cases by droplet digital PCR, while variants remain undetected in 92% of matched plasma samples. Retrospective next-generation sequencing analysis in BM ctDNA recapitulates tumor variants, with an overall clinical sensitivity of 71.4% and specificity of 100%. In two cases, ctDNA was detectable in BM collected 18 and 6 months prior to standard diagnosis. Our results open up the potential use of BM as a new source for liquid biopsy for PPBC detection. Significance: For the first time, we show that BM obtained from patients with breast cancer carries ctDNA, surpassing plasma-based liquid biopsy for detection and molecular profiling of early-stage breast cancer, even prior to diagnosis by image.We thank the patients who participated in the study and donated samples for analysis for their generous contribution, with particular thanks to the first patient, Maite, and her daughter Àneu, who inspired us to initiate this study (oral consent to name the patient and her daughter was provided by the patient, and her legal partner provided written consent after patient's exitus). We are grateful to Javier Carmona for his valuable contributions and support in the manuscript's conceptualization, preparation, and revision. VHIO would like to acknowledge the Cellex Foundation for providing research facilities and equipment and the CERCA Programme from the Generalitat de Catalunya for their support of this research. The authors from VHIO acknowledge the State Agency for Research (Agencia Estatal de Investigación) for the financial support as a Center of Excellence Severo Ochoa (CEX2020-001024-S/AEI/10.13039/501100011033). This research is financially supported by the “El paseíco de la mama” Foundation. C. Saura was the recipient of a II FERO-GHD grant from the FERO Foundation (FERO/5086), a Junior Clinical award from the Spanish Association Against Cancer Foundation (FAECC; CLJUN212026ORTI), and a SEOM-Daiichi Sankyo grant for its support on the Breast Cancer Research Projects 2021 (SEOM/FECMA2022) and received funding from the Department of Health (Generalitat de Catalunya SLT008/18/00198) and from the Instituto de Salud Carlos III (ISCIII) and Fondo Europeo de Desarrollo Regional (FEDER), cofunded by the European Union (PI21/01020). C. Ortiz was the recipient of a Junior Clinician award from the FAECC (CLJUN212026ORTI) and a SEOM-Daiichi Sankyo grant for its support on the Breast Cancer Research Projects 2021 (SEOM/FECMA2022), and received funding from the Department of Health (Generalitat de Catalunya SLT008/18/00198). N. Bayó-Puxan received funding from the Department of Health (Generalitat de Catalunya SLT008/18/00205), MCIN/AEI/10.13039/501100011033 (GPE2022-001029) and MCIN/AEI/10.130.39/501100011033, and the European Union “Next GenerationEU/PRTR” (ECT2020-000827). J.M. Miquel received funding from the Department of Health (Generalitat de Catalunya SLT008/18/00205), MCIN/AEI/10.130.39/501100011033, and the European Union “Next GenerationEU/PRTR” (ECT2020-000827). J. Arribas is funded by the Breast Cancer Research Foundation (BCRF-23-008), Instituto de Salud Carlos III (project reference numbers AC15/00062, CB16/12/00449, and PI22/00001), and the European Commission under the framework of the ERA-NET TRANSCAN-2 initiative cofinanced by FEDER and Asociación Española Contra el Cáncer. A. Vivancos was the recipient of a project award from the FAECC (AVP/18/AECC/3219) and received funding from the Advanced Molecular Diagnostic (DIAMAV) program from the FERO Foundation (8361) and from ISDIN for supporting the development of liquid biopsy applications at the Cancer Genomics Lab (1848). M. Sansó was the recipient of a II FERO-GHD grant from the FERO Foundation (FERO/5086) and an investigator award from the FAECC (INVES19056SANS), and received funding from the Health Research Institute of the Balearic Islands (IdISBa), the RADIX-Janssen program (RADIX/JANSSEN21/01), and the Miguel Servet Program funded by the ISCIII (CP22/00131)