Alva‐Ruiz, Roberto

Background and Aims: Cholangiocarcinoma (CCA) is one of the most lethal cancers, characterized by molecular heterogeneity and treatment resistance. To uncover new biological signals and therapeutic opportunities, we employed multiomic characterization combined with machine learning. Approach and Results: We profiled all anatomical CCA subtypes using whole exome sequencing, mRNA sequencing, and proteome/phosphoproteome analysis. Integrative dimensional reduction revealed RNA, protein, and phosphoprotein features driving tumor heterogeneity, enabling clustering. Machine learning algorithms identified molecular features for each cluster and mapped external datasets and patient-derived xenograft (PDX) models onto these clusters. Kinase enrichment analysis highlighted targetable kinases active in each cluster. In vivo validation was performed in cluster-specific PDX models using the selective TNK1 inhibitor, TP-5801. We identified 3 molecular clusters with distinct pathway characterization: immunomodulatory (cluster 1), metabolic (cluster 2), and gene regulation/cellular fate (cluster 3). Cluster assignment was independent of anatomic subtype but correlated with overall survival following curative-intent resection. We also identified multiomic features and pathways linked to overall survival and lymph node metastases, crucial for patient treatment selection. Kinase enrichment analysis pinpointed TNK1 as a highly active kinase in the metabolic cluster. Treatment with TP-5801 significantly reduced tumor growth in a metabolic PDX model, but not in models representing the other clusters. Combining internal data with publicly available datasets, we identified the immunomodulatory cluster as most responsive to gemcitabine/cisplatin therapy, confirmed in vivo using cluster-specific PDX models. Conclusions: Integrated multiomic characterization provides translational insights by defining unique molecular subtypes associated both with therapeutic response and overall clinical outcomes. This approach identified TNK1 as a previously unrecognized therapeutic target in a defined subset of CCA tumors.

Abstract Acute appendicitis is a common surgical emergency, most often caused by luminal obstruction from fecaliths. Rarely, foreign body ingestion can lead to appendicitis, particularly when small objects like lead shot become lodged in the appendix. We present the case of a 30-year-old man with chronic intermittent right lower quadrant pain who presented with an acute exacerbation. Imaging revealed two metallic densities in the appendix without overt signs of inflammation. He underwent laparoscopic appendectomy, which confirmed the presence of two retained lead pellets and histologic evidence of acute appendicitis. The patient had a history of frequent game meat consumption. This case underscores a rare but important etiology of appendicitis and the potential for systemic lead exposure from retained foreign bodies. In patients with relevant social histories and suggestive imaging, clinicians should consider this diagnosis and proceed with timely surgical management to prevent complications.

Cholangiocarcinoma (CCA) is a highly aggressive form of liver cancer and is an increasing cause of cancer‐related death worldwide. Despite its increasing incidence globally and alarming mortality, treatment options for CCA have largely remained unchanged, stressing the importance of developing new effective therapies. YAP activation is common in CCA, and its major transcriptional signaling partners are the TEAD proteins. CA3 is a small‐molecule YAP‐TEAD disrupter discovered utilizing a TEAD reporter assay. Utilizing CCA, gastric cancer cell lines, and patient‐derived xenograft models (PDX), we demonstrate that CA3 is effective in inducing cell death and delaying tumor growth in both FGFR2 fusion and wild‐type models. CA3 was associated with on‐target decreases in YAP‐TEAD target gene expression, TEAD reporter activity, and overall TEAD levels. Hippo pathway signaling was not altered as there was no change in YAP phosphorylation status in the cells exposed to CA3. RNA sequencing of gastric cancer and CCA models demonstrated upregulation of an androgen receptor–mediated transcriptional program following exposure to CA3 in five unique models tested. Consistent with this upstream regulator analysis, CA3 exposure in CCA cells was associated with increased AR protein levels, and combinatorial therapy with CA3 and androgen receptor blockade was associated with increased cancer cell death. CA3 behaves functionally as a YAP‐TEAD disrupter in the models tested and demonstrated therapeutic efficacy. Exposure to CA3 was associated with compensatory androgen receptor signaling and dual inhibition improved the therapeutic effect.

Objective: The aim of this study is to determine perioperative outcomes and the patency of interposition conduits for visceral arterial reconstruction in this setting. Background: Visceral arterial encasement in locally advanced pancreatic cancer was historically a contraindication for surgery. With modern, effective neoadjuvant strategies, our recent experience has made advanced vascular resection and reconstruction feasible in selected patients. Methods: A retrospective review was performed of patients undergoing pancreatic tumor resection with en bloc arterial resection and interposition revascularization between June 2002 and October 2022. Endpoints included graft patency, vascular-related complications, reinterventions, morbidity, and mortality. Results: Visceral arterial reconstruction with interposition grafting was performed in 111 patients undergoing en bloc arterial resections for pancreatic cancer. Graft types included autologous arterial conduits (n=66, 58 superficial femoral arteries (SFA) and 8 splenic arteries), cryopreserved arterial allografts (n=24), autologous saphenous veins (n=12), synthetic conduits (n=8), and composite autologous artery and synthetic (n=1). Perioperative 90-day mortality decreased significantly over time to 5% in the last 6 years. Vascular complications related to arterial reconstruction occurred in 11% (n=12) and included pseudoaneurysm (n=6), graft thrombus (n=2), stenosis requiring reintervention (n=2), hepatic failure (n=1), and hepatic and intestinal ischemia (n=1). Nine (8%) patients underwent vascular-related reinterventions. After a median follow-up of 17 months, primary patency was 81% for the entire cohort and was highest in the SFA group (95%). The donor limb/harvest site complication rate was 8% with 100% primary patency. Conclusion: Visceral arterial resection with interposition reconstruction for locally advanced pancreatic cancer can be performed with acceptable vascular morbidity and durable patency. Autologous SFA was the most suitable conduit for reconstructions in our experience, with the highest primary patency.

BACKGROUND: Pancreaticobiliary (PB) cancers are a diverse group of cancers with poor prognoses and high rates of recurrence after resection. Patient-derived xenografts (PDX), created from surgical specimens, provide a reliable preclinical research platform and high-fidelity cancer model from which to study these malignancies with consistent recapitulation of their original patient tumors in vivo. However, the relationship between PDX engraftment success (growth or no growth) and patient oncologic outcomes has not been well studied. We sought to evaluate the correlation between successful PDX engraftment and survival in several PB exocrine carcinomas, including the pancreatic and biliary tract. STUDY DESIGN: In accordance with IRB and Institutional Animal Care and Use Committee protocols and with appropriate consent and approval, excess tumor tissue obtained from surgical patients was implanted into immunocompromised mice. Mice were monitored for tumor growth to determine engraftment success. PDX tumors were verified to recapitulate their tumors of origin by a hepatobiliary pathologist. Xenograft growth was correlated with clinical recurrence and overall survival data. RESULTS: A total of 384 PB xenografts were implanted. The successful engraftment rate was 41% (158/384). We found that successful PDX engraftment was highly associated with both recurrence-free survival (p < 0.001) and overall survival (p < 0.001) outcomes. Successful PDX tumor generation occurs significantly in advance of clinical recurrences in their corresponding patients (p < 0.001). CONCLUSIONS: Successful PB cancer PDX models predict recurrence and survival across tumor types and may provide critical lead time to alter patients’ surveillance or treatment plans before cancer recurrence.

Abstract A major hurdle to the application of precision oncology in pancreatic cancer is the lack of molecular stratification approaches and targeted therapy for defined molecular subtypes. In this work, we sought to gain further insight and identify molecular and epigenetic signatures of the Basal-like A pancreatic ductal adenocarcinoma (PDAC) subgroup that can be applied to clinical samples for patient stratification and/or therapy monitoring. We generated and integrated global gene expression and epigenome mapping data from patient-derived xenograft models to identify subtype-specific enhancer regions that were validated in patient-derived samples. In addition, complementary nascent transcription and chromatin topology (HiChIP) analyses revealed a Basal-like A subtype-specific transcribed enhancer program in PDAC characterized by enhancer RNA (eRNA) production that is associated with more frequent chromatin interactions and subtype-specific gene activation. Importantly, we successfully confirmed the validity of eRNA detection as a possible histologic approach for PDAC patient stratification by performing RNA-ISH analyses for subtype-specific eRNAs on pathologic tissue samples. Thus, this study provides proof-of-concept that subtype-specific epigenetic changes relevant for PDAC progression can be detected at a single-cell level in complex, heterogeneous, primary tumor material. Implications: Subtype-specific enhancer activity analysis via detection of eRNAs on a single-cell level in patient material can be used as a potential tool for treatment stratification.

BACKGROUND: Portal or superior mesenteric vein (PV-SMV) resection and reconstruction is sometimes required during pancreatic tumor resection. In patients requiring segmental venous resection with interposition grafting, the left renal vein (LRV) is an accessible autologous solution. However, long-term patency outcomes of the LRV as an interposition conduit in this setting have not been analyzed. STUDY DESIGN: We conducted a retrospective analysis of patients undergoing pancreatic resection with PV-SMV reconstruction using LRV between 2002 and 2022. The primary outcome was PV-SMV patency at last follow-up, assessed with postoperative CT scans and analyzed using Kaplan-Meier survival methods that account for variation in follow-up duration. Development of any postoperative acute kidney injury within 7 days of surgery and morbidity were secondary outcomes. RESULTS: The study cohort includes 65 patients who underwent LRV harvest; 60 (92%) ultimately underwent successful reconstruction with harvested LRV graft. Kaplan-Meier 2-year estimated patency rate of the LRV graft was 88%, with no cases of complete occlusion. Six (10%) patients experienced graft stenosis. Nine of 61 (15%) patients experienced grade II or III acute kidney injury, 6 of 9 returning to normal renal function before discharge. No difference in median serum creatinine was observed at baseline, 6 and 12 months from surgery. LRV remnant thrombosis was seen in 7 of 65 (11%) patients. Only 3 of 61 (5%) patients had persistent acute kidney injury caused by complications unrelated to LRV harvesting. CONCLUSIONS: Autologous LRV graft was a reliable conduit for segmental PV-SMV reconstruction, resulting in a high patency rate and marginal impact on renal function. LRV harvest is a safe and potentially ideal surgical option for PV-SMV reconstruction in pancreatic surgery.

BACKGROUND: Accurate staging prior to resection of pancreatic ductal adenocarcinoma (PDAC) is imperative to avoid unnecessary operative morbidity and oncologic futility in patients with occult intra-abdominal distant metastases. We aimed to determine the diagnostic yield of staging laparoscopy (SL) and to identify factors associated with increased risk of positive laparoscopy (PL) in the modern era. STUDY DESIGN: Patients with radiographically localized PDAC who underwent SL from 2017 to 2021 were retrospectively reviewed. The yield of SL was defined as the proportion of patients with PL, including gross metastases and/or positive peritoneal cytology. Factors associated with PL were assessed using univariate analysis and multivariable logistic regression. RESULTS: Of 1,004 patients who underwent SL, 180 (18%) had PL due to gross metastases (n = 140) and/or positive cytology (n = 96). Patients who had neoadjuvant chemotherapy prior to laparoscopy had lower rates of PL (14% vs 22%, p = 0.002). When the analysis was restricted to chemo-naive patients who had concurrent peritoneal lavage performed, 95 of 419 patients (23%) had PL. In multivariable analysis, PL was associated with younger (<60) age, indeterminate extrapancreatic lesions on preoperative imaging, body/tail tumor location, larger tumor size, and elevated serum CA 19-9 (all p < 0.05). Among patients with no indeterminate extrapancreatic lesions on preoperative imaging, the rate of PL ranged from 1.6% in patients with no risk factors to 42% in young patients with large body/tail tumors and elevated serum CA 19-9. CONCLUSIONS: The rate of PL in patients with PDAC remains high in the modern era. SL with peritoneal lavage should be considered for the majority of patients prior to resection, specifically those with high-risk features, and ideally prior to neoadjuvant chemotherapy.

Abstract Background: Cholangiocarcinoma (CCA) is a heterogeneous malignancy arising from the biliary epithelium. Its diverse molecular landscape and aggressive biology render many anti-cancer therapies ineffective. Nanovesicle technology provides an opportunity for therapeutic inhibition of oncogenic targets that have been previously classified as undruggable. EpCAM is an epithelial-specific, transmembrane glycoprotein with increased expression in human and murine CCA which can be used for nanovesicle targeting. As a proof of concept study, we designed and validated a novel strategy to direct therapeutic milk-derived nanovesicles (tMNVs) to CCA tumors. Methods: tMNVs were decorated with RNA nanoparticles containing a validated aptamer (EpDT3) against EpCAM conjugated to a cholesterol-triethylene-glycol (TEG) scaffold containing an Alexa647 fluorophore. Human and murine CCA cell lines were treated with aptamer directed tMNVs and assessed for nanovesicle uptake by fluorescent microscopy. CCA tumor tissue, derived from orthotopic implantation of a syngeneic CCA cell line, SB1, into a C57BL/6 mouse, was collected and treated with either aptamer-directed or bare tMNVs ex vivo, and compared with adjacent normal liver tissue. Flow cytometry was utilized to characterize tMNVs absorption profile. C57BL/6 mice who had previously undergone SB1 orthotopic and flank implantation were treated with aptamer-directed tMNVs by tail-vein injection and subsequently euthanized. Tissue was collected for biodistribution analyses by fluorescent microscopy. The experiment was repeated in NOD-scid mice following orthotopic implantation of patient derived xenograft (PDX) CCA tumor. Results: Both human and murine CCA cells treated with aptamer-directed tMNVs demonstrated high fluorescent signal consistent with tMNV absorption within 12 hours of application. Flow cytometry analysis showed aptamer-directed tMNVs were absorbed at a higher proportion by CCA tumors than bare tMNVs ex vivo. Aptamer-directed tMNVs also had better absorption by CCA tumors compared to adjacent normal liver tissue. Following treatment with aptamer-directed or bare tMNVs in vivo, fluorescent microscopy demonstrated that aptamer-directed tMNVs were significantly better absorbed in the orthotopic SB1 tumors, followed by the subcutaneous tumors. Minimal fluorescent signal was noted in the normal adjacent liver. Orthotopically implanted PDX tumors also demonstrated high fluorescent signals following intravenous treatment with aptamer-directed tMNVs. Conclusions: Utilizing a novel targeting strategy, we were able to design tMNVs capable of reliably and specifically targeting CCA in preclinical models. This work is foundational to the future application of nanovesicle technology in the CCA treatment paradigm. Citation Format: Mincheng Yu, Jennifer L. Tomlinson, Emilien J. Loeuillard, Ryan D. Watkins, Caitlin B. Conboy, Shohei Takaichi, Nathan W. Werneburg, Roberto Alva-Ruiz, Amro Abdelrahman, Danielle M. Carlson, Jingchun Yang, Sumera I. Ilyas, Gregory J. Gores, Tushar Patel, Rory L. Smoot. Novel strategy for aptamer-directed nanovesicle targeting in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 814.

Objective Pancreatic ductal adenocarcinoma (PDAC) displays a remarkable propensity towards therapy resistance. However, molecular epigenetic and transcriptional mechanisms enabling this are poorly understood. In this study, we aimed to identify novel mechanistic approaches to overcome or prevent resistance in PDAC. Design We used in vitro and in vivo models of resistant PDAC and integrated epigenomic, transcriptomic, nascent RNA and chromatin topology data. We identified a JunD-driven subgroup of enhancers, called interactive hubs (iHUBs), which mediate transcriptional reprogramming and chemoresistance in PDAC. Results iHUBs display characteristics typical for active enhancers (H3K27ac enrichment) in both therapy sensitive and resistant states but exhibit increased interactions and production of enhancer RNA (eRNA) in the resistant state. Notably, deletion of individual iHUBs was sufficient to decrease transcription of target genes and sensitise resistant cells to chemotherapy. Overlapping motif analysis and transcriptional profiling identified the activator protein 1 (AP1) transcription factor JunD as a master transcription factor of these enhancers. JunD depletion decreased iHUB interaction frequency and transcription of target genes. Moreover, targeting either eRNA production or signaling pathways upstream of iHUB activation using clinically tested small molecule inhibitors decreased eRNA production and interaction frequency, and restored chemotherapy responsiveness in vitro and in vivo. Representative iHUB target genes were found to be more expressed in patients with poor response to chemotherapy compared with responsive patients. Conclusion Our findings identify an important role for a subgroup of highly connected enhancers (iHUBs) in regulating chemotherapy response and demonstrate targetability in sensitisation to chemotherapy.