Generation of Metastatic Cholangiocarcinoma Patient-Derived Xenograft Models.

Cholangiocarcinoma (CCA) poses a substantial clinical hurdle as it is often detected at advanced metastatic stages with limited therapeutic options. To enhance our understanding of advanced CCA, it is imperative to establish preclinical models that faithfully recapitulate the disease's characteristics. Patient-derived xenograft (PDX) models have emerged as a valuable approach in cancer research, offering an avenue to reproduce and study the genomic, histologic, and molecular features of the original human tumors. By faithfully preserving the heterogeneity, microenvironmental interactions, and drug responses observed in human tumors, PDX models serve as highly relevant and predictive preclinical tools. Here, we present a comprehensive protocol that outlines the step-by-step process of generating and maintaining PDX models using biopsy samples from patients with advanced metastatic CCA. The protocol encompasses crucial aspects such as tissue processing, xenograft transplantation, and subsequent monitoring of the PDX models. By employing this protocol, we aim to establish a robust collection of PDX models that accurately reflect the genomic landscape, histologic diversity, and therapeutic responses observed in advanced CCA, thereby enabling improved translational research, drug development, and personalized treatment strategies for patients facing this challenging disease.

Clinical and genomic characterisation of early-onset pancreatic cancer.

BACKGROUND: The incidence of early-onset pancreatic cancer (EOPC) has risen dramatically in recent years. We aimed to characterise the clinical and genomic features of EOPC and evaluate their therapeutic implications. METHODS: We performed a comparative, single-centre, retrospective analysis of clinical, germline, and genomic features in EOPC (≤50 years) patients and compared them with a control group of average-onset pancreatic cancer patients (AOPC, ≥70 years). Key molecular findings were compared with an external, publicly available cohort. RESULTS: We reviewed 336 patients who met all inclusion criteria (EOPC N = 139, AOPC N = 197). EOPC was associated with smoking status, lower prevalence of diabetes, better performance status, higher CA19.9 levels, and higher albumin levels at diagnosis. After adjustment for baseline covariates, we observed no differences in overall survival (OS). Age was associated with an increase in the incidence of KRASMUT both in our cohort and the validation cohort. EOPC were enriched in potentially actionable alterations according to ESCAT tiers I-IIIA when compared with AOPC in discovery and validation cohorts (19% versus 14% and 14% versus 8%, respectively). In the first-line metastatic setting, EOPC had a longer progression-free survival (hazard ratio [HR] 0.61, 95% confidence interval (CI) 0.43-0.87) and OS (HR 0.65, 95% CI 0.45-0.95), although there were no differences in response rate. After adjusting for the number of treatment lines, EOPC patients who did receive targeted therapies exhibited longer OS compared with EOPC who did not (HR 0.34, 95% CI 0.12-0.93). CONCLUSIONS: EOPC patients have improved outcomes in the metastatic setting when compared to AOPC and are enriched for targetable alterations that open opportunities for precision oncology-based approaches.

Liquid biopsy after resection of pancreatic adenocarcinoma and its relation to oncological outcomes. Systematic review and meta-analysis.

BACKGROUND: It has been hypothesised that manipulation during surgery releases tumoral components into circulation. We investigate the effect of surgery on plasma-borne DNA biomarkers and the oncological outcomes in resectable pancreatic ductal adenocarcinoma (PDAC). We also compare non-touch isolation techniques (NTIT) with standard techniques. MATERIALS AND METHODS: We performed a systematic review and a meta-analysis of studies analysing liquid biopsy as circulating tumour DNA (ctDNA), circulating tumour cells (CTCs), and messenger RNA (mRNA) in resectable PDAC patients who underwent surgery and its association with overall survival (OS) and disease-free survival (DFS). Research in EMBASE, Web of Science and PubMed was performed. The ctDNA shift negative-to-positive (ctDNA -/+) or ctDNA shift positive-to-negative (ctDNA +/-) before and after surgery was evaluated. RESULTS: Twelve studies comprising 413 patients were included. Shorter OS and DFS were identified in patients with positive ctDNA status before (HR = 2.28, p = 0.005 and HR = 2.16, p = 0.006) or after surgery (HR = 3.88, p < 0.0001 and HR = 3.81, p = 0.03), respectively. Surgical resection increased the rate of ctDNA +/-. There were no differences in OS or DFS in the ctDNA +/- group compared with ctDNA +/+ or ctDNA -/+. However, there was a trend to shorter OS in the ctDNA -/+ group (HR = 5.00, p = 0.09). No differences between NTIT and standard techniques on liquid biopsy status were found. CONCLUSION: Positive ctDNA in the perioperative period is associated with a worse prognosis. Surgical resection has a role in the negativisation of liquid biopsy status. More studies are needed to assess the potential of minimally invasive techniques on ctDNA dynamics.

Clinical Outcomes After Progression on First-Line Therapies in IDH1 Mutated Versus Wild-Type Intrahepatic Cholangiocarcinoma Patients.

BACKGROUND: Isocitrate dehydrogenase-1 (IDH1) mutations occur in a significant proportion of intrahepatic cholangiocarcinomas (iCCAs). No data are available regarding the prognostic impact of IDH1 mutations in advanced iCCA patients after progression on first-line therapies. OBJECTIVE: We investigated the role of IDH1 mutation in advanced iCCA after progression on first-line therapies. PATIENTS AND METHODS: After progression on first-line therapies for advanced iCCA, consecutive patients were retrospectively collected. The IDH1 status was tested at baseline. This analysis aimed to examine the association between the presence of IDH1 missense mutations and survival outcomes in patients with advanced iCCA treated with a second-line therapy. RESULTS: The analysis included 119 patients; 56/119 (47%) were IDH1 mutated (IDH1m) and 63/119 (53%) were IDH1 wild type (IDH1 WT). At univariate analysis for overall survival (OS), the presence of IDH1 mutation was associated with a worse median OS (mOS; 8.2 vs. 14.1 months; hazard ratio [HR] 1.9, 95% confidence interval [CI] 1.2-3.0, p = 0.0047). Patients harboring IDH1 mutations showed a worse objective response rate (ORR) compared with patients without IDH1 mutation, whereas no significant differences in disease control rate (DCR) were found. Multivariate analysis confirmed IDH1 mutations as an independent negative prognostic factor for OS (HR 1.7, 95% CI 1.1-2.7, p = 0.0256). By evaluating only patients receiving FOLFOX as second-line therapy, no statistically significant differences were found in terms of both OS and PFS between IDH1m and IDH1 WT patients. In this subset of patients, those harboring an IDH1 mutation showed a worse ORR and DCR compared with those without. Finally, at univariate analysis for OS from third-line treatment, the presence of an IDH1 mutation was associated with a trend toward a worse mOS (6.0 vs. 11.9 months; HR 1.6, 95% CI 0.8-3.2, p = 0.25). CONCLUSION: The present analysis constitutes the first evidence of a negative prognostic impact of IDH1 mutations in a cohort of patients treated after progression on first-line therapies in contrast to IDH1 inhibitors.

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

PURPOSE: 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.

Molecular profile and its clinical impact of IDH1 mutated versus IDH1 wild type intrahepatic cholangiocarcinoma.

IDH1-mutated cholangiocarcinomas (CCAs) are an interesting group of neoplasia with particular behavior and therapeutic implications. The aim of the present work is to highlight the differences characterizing IDH1m and IDH1wt CCAs in terms of genomic landscape. 284 patients with iCCA treated for resectable, locally advanced or metastatic disease were selected and studied with the FOUNDATION Cdx technology. A comparative genomic analysis and survival analyses for the most relevant altered genes were performed between IDH1m and IDH1wt patients. Overall, 125 patients were IDH1m and 122 IDH1wt. IDH1m patients showed higher mutation rates compared to IDH1wt in CDKN2B and lower mutation rates in several genes including TP53, FGFR2, BRCA2, ATM, MAP3K1, NOTCH2, ZNF703, CCND1, NBN, NF1, MAP3KI3, and RAD21. At the survival analysis, IDH1m and IDH1wt patients showed no statistically differences in terms of survival outcomes, but a trend in favor of IDH1wt patients was observed. Differences in prognostic values of the most common altered genes were reported. In surgical setting, in IDH1m group the presence of CDKN2A and CDKN2B mutations negatively impact DFS, whereas the presence of CDKN2A, CDKN2B, and PBRM1 mutations negatively impact OS. In advanced setting, in the IDH1m group, the presence of KRAS/NRAS and TP53 mutations negatively impact PFS, whereas the presence of TP53 and PIK3CA mutations negatively impact OS; in the IDH1wt group, only the presence of MTAP mutation negatively impact PFS, whereas the presence of TP53 mutation negatively impact OS. We highlighted several molecular differences with distinct prognostic implications between IDH1m and IDH1wt patients.

Next-generation sequencing analysis of cholangiocarcinoma identifies distinct IDH1-mutated clusters.

BACKGROUND: IDH1-mutated intrahepatic cholangiocarcinomas (IDH1m iCCAs) could be treated with anti-IDH1 drugs, although the high heterogeneity in this class of tumours could limit treatment efficacy. METHODS: We selected 125 IDH1m iCCAs that were treated as resectable, locally advanced, or metastatic and were screened by the NGS-based FoundationOne gene panel. We conducted a mutation-based clustering of tumours and survival analysis. RESULTS: Three main clusters were identified. The most altered pathways in cluster 1 were cell cycle and apoptosis, RTK/RAS, PI3K, and chromatin modification. Of note, CDKN2A/2B were mutated in 41/44 patients of this cluster. In cluster 2, the most affected pathways were as follows: Chromatin modification, DNA damage control, PI3K, and RTK/RAS. In this cluster, the most frequently mutated genes were ARID1A and PBRM1. The most altered pathways in cluster 3 were as follows: Cell cycle and apoptosis, DNA damage control, TP53, and chromatin modification. Importantly, TP53 was mutated only in cluster 3 patients. In the cohort of patients treated with surgery, cluster 2 showed statistically significant better disease-free survival (DFS) and overall survival (OS) compared with patients in cluster 3 and cluster 1 (p = 0.0014 and p = 0.0003, respectively). In the advanced setting, cluster 2 experienced a statistically significant better PFS (p = 0.0012), a tendency toward a better OS from first-line treatment, and a better OS from first-line progression compared with patients in cluster 1 and cluster 3 (p = 0.0017). We proposed an easy-to-use algorithm able to stratify patients in the three clusters on the basis of the genomic profile. CONCLUSION: We highlighted three different mutation-based clusters with prognostic significance in a cohort of IDH1m iCCAs.