PURPOSE:To assess associations between six diagnostic, staging, and coordination of care indicators in pancreatic cancer, and: (1) surgery being abandoned intraoperatively; or (2) a positive macroscopic margin (R2 resection) or a positive microscopic pathological margin (R1 resection). METHODS:Data was provided by the Upper Gastrointestinal Cancer Registry operating across two Australian states. Associations were tested using multivariable logistic regression. RESULTS:704 patients underwent an attempted surgical resection (54 % male; median age 69 years). Of the completed resections (n = 585) with a known margin status (n = 513), 54 % (n = 278) were reported as having a negative pathological (R0) margin, 41 % (n = 211) had an R1 margin, and 5 % (n = 24) had an R2 margin. Patients who underwent surgery or neoadjuvant therapy within 60 days from referral had double the odds of a complete resection (OR=2.12, 95 % CI, 1.19 - 3.76). Imaging undertaken beyond 30 days prior to surgery had a 40 % reduction in the odds of a completed resection (OR=0.58, 95 % CI, 0.37 - 0.92). Patients with their ECOG and/or ASA documented at presentation had 90 % increased odds of a R0 margin resection (OR=1.90, 95 % CI, 1.32 - 2.73). CONCLUSIONS:Timely progression to primary treatment had the most significant association with achieving complete resection status.

392 Background: Prostate cancer (PCa) remains the second leading cause of cancer death in men. Neoadjuvant pharmacodynamic studies facilitate rational decisions about which therapies should progress to phase 2/3 trials. CDK4/6 inhibitors are effective in breast cancer, but have not demonstrated the same efficacy in metastatic PCa. LEEP assessed the pharmacodynamic effects of ribociclib, a highly selective oral CDK4/6 inhibitor, in hormone-naïve, high-risk, localised PCa. Methods: This open-label, window of opportunity trial randomised participants at 3 Australian sites in a 4:1 ratio to an experimental or control group. Participants in the experimental group were to be treated with ribociclib 400mg daily for 21 days prior to radical prostatectomy (RP). The primary endpoint was a 50% reduction in Ki-67 expression from the pre-treatment biopsy compared to the RP. Immune cell changes in peripheral blood and tissue were also examined. Results: Between November 2018 and October 2022,33 patients were randomised to either ribociclib or control. 17 participants in the ribociclib group and 7 in the control group were evaluable for response (9 not evaluable due to surgical delays (including COVID shutdowns) or insufficient cancer in the biopsy). When examining a random selection of cancer areas, 10/17 (58%) participants had a greater than 50% reduction in Ki-67, compared to only 2/7 (29%) in the control group. When examining hot spot areas of cancer (i.e. areas with the highest proportion of positive cells on low power estimation), only 5/17 (29%) participants had a greater than 50% reduction in Ki-67, compared to 0/7 (0%) in the control group. In peripheral blood, there were reductions in CD1c and CD141 dendritic cells and myeloid derived suppressor cells over 4 times points in the ribociclib (p>0.05, 0.05 and 0.05 respectively) vs. control groups (p=0.46, 0.14 and 0.14). In patients treated with ribociclib, there was no difference in circulating regulatory T cells (p=0.67). Within the PCa tissue, there was a reduction in regulatory T cells in the ribociclib group compared to controls (p=0.07), but no change in adjacent non-cancerous tissue (p=0.701). There was no difference in M1 or M2 macrophages between the ribociclib and control groups (M1: tumour p=0.307, adjacent non-cancer p=0.334; M2: tumour p=0.168, adjacent non-cancer p=0.130). Conclusions: Following neoadjuvant treatment with ribociclib, >50% reductions in Ki-67 were more frequent among those treated with ribociclib than controls. Reductions in Ki-67 were uncommon in more proliferative areas. Changes in the immune environment were consistent with breast cancer studies. We hypothesise that the more proliferative areas of cancer are less responsive to CDK4/6 inhibitors and this may in part explain the lower observed efficacy in mCRPC than metastatic breast cancer. Clinical trial information: ACTRN12618000354280 .

Localised prostate cancers (PCa) are heterogeneous and multifocal, with diverse outcomes. Current prognostic methods are epithelium-centric, overlooking the complex cellular landscape within the tumour microenvironment (TME), which remains incompletely characterised. We performed a comprehensive analysis of cancerous and adjacent-benign cores from 24 patients with hormone therapy-naive localised PCa using single-cell RNA-sequencing. By integrating copy number variation and transcriptional signatures, we classified epithelial cells across a malignant spectrum, revealing widespread molecular perturbation. We found an expansion of Club cell phenotypes, suggestive of Luminal dedifferentiation. We also performed a detailed annotation of stromal phenotypes, focusing on fibroblasts, and identified a novel peri-neural fibroblast population. Spatial transcriptomics elucidated the precise anatomical distribution of CAFs within the PCa TME. This study provides a valuable foundation for advancing our understanding of PCa pathobiology and developing a comprehensive cellular model of the disease. ### Competing Interest Statement M.H. is a scientific consultant to 10x Genomics, Inc. J.L. is an author on patents owned by Spatial Transcriptomics AB covering technology presented in this paper. A.S. has received research funding from 10x Genomics, Inc. The remaining authors declare no potential conflicts of interest.

Abstract Introduction: Appendiceal tumors (AT) are rare and often develop peritoneal disease (PD), with 5 year survival of 15-63% for high grade adenocarcinomas (HG) and 46-96% for low grade mucinous neoplasms (LG). While the mainstay of treatment is cytoreductive surgery and heated intraperitoneal chemotherapy (CRS/HIPEC), the role of systemic chemotherapy is less clear with limited biological characterization of AT and its tumor microenvironment (TME). The purpose of this study was to evaluate the TME of AT PD. Here we present an atlas of AT PD cells alongside a spatially resolved transcriptomic profile comparing HG and LG. Methods: We collected tissue from 29 patients with AT PD during CRS prior to HIPEC (18 LG, nine HG and two no tumor control). We performed comprehensive single-cell RNA sequencing (38224 cells) on fresh tissue specimens from nine patients (four HG, five LG), and whole transcriptome spatial analysis (>18000 genes) using the GeoMx Digital Spatial Profiler on formalin-fixed paraffin-embedded tissue from 22 patients (five HG, 13 LG). In the spatial profiling, we used anti-CK20, anti-CD45 and SYTO 83 as morphology markers to select areas of interest (AOIs): tumor (CK20), immune (CD45) and stroma (non-CK20/CD45). Analyses were performed with R and a false discovery rate threshold of 0.05. Results: Single-cell RNA sequencing showed that both LG and HG have a preponderance of T-lineage cells including CD8, CD4 and NK T-cells. Both LG and HG tumors display association of mesothelial cells with cancer-associated fibroblasts (CAF), and CAF subsets expressing genes of epithelial-mesenchymal transition (EMT). LG and HG tumors display different patterns of mucin gene expression - epithelial cells of HG express the secreted mucin MUC20 in addition to MUC2 and MUC5B also expressed in LG; a subset of mesothelial cells in LG but not HG express the transmembrane mucin genes MUC1, MUC3A, MUC12 and MUC16. Spatial analysis of tumor AOIs revealed HG compared to LG had increased expression of genes associated with MYC targets, oxidative phosphorylation, anti-tumor immune pathways (IFNα/γ response, intestinal immune network for IGA production), cell cycle pathways (E2F targets, G2M checkpoint) and EMT. LG compared to HG had increased expression of genes associated with inflammation-mediated disease (TNFα signaling via NFKB, inflammatory response), regulation of tumor growth (P53), hypoxia and early cell cycle (estrogen response) and glycosylation pathways. Immune and stromal AOIs did not have significant transcriptomic differences between LG and HG. Conclusion: This study provides novel insights into the biological profile of AT PD by describing TME cell types, immune function, tumor growth and cell cycle pathways. These findings may be used to identify clinically relevant biomarkers and new therapeutic targets. Citation Format: Madeleine C. Strach, Nicole Yeung, Eva Apostolov, Tony Wang, Hui-Ming Lin, Nabila Ansari, Cherry Koh, Joo-Shik Shin, James Kench, Alexander Swarbrick, Lisa Horvath, Kate L. Mahon. Single cell and spatial transcriptomic profile of appendiceal tumour peritoneal disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5480.

Acute allograft rejection is a well-known complication of liver transplantation (LT). The incidence, epidemiology, and outcomes of acute rejection have not been well described in Australia. We retrospectively studied consecutive adults who underwent deceased donor LT at a single center between 2010 and 2020. Donor and recipient data at the time of LT and recipient outcomes were collected from a prospective LT database. Liver biopsy reports were reviewed, and only a graft’s first instance of biopsy-proven acute rejection was analyzed. During the study period, 796 liver transplants were performed in 770 patients. Biopsy-proven rejection occurred in 34.9% of transplants. There were no significant changes in the incidence of rejection over time (linear trend p=0.11). The median time to the first episode of rejection was 71 days after LT: 2.2% hyperacute, 50.4% early (≤90 d), and 47.5% late rejection (>90 d). Independent risk factors for rejection were younger recipient age at transplant (aHR 0.98 per year increase, 95% CI: 0.97–1.00, p=0.01), and ABO-incompatible grafts (aHR 2.55 vs. ABO-compatible, 95% CI: 1.27–5.09, p<0.01) while simultaneous multiorgan transplants were protective (aHR 0.21 vs. LT only, 95% CI: 0.08–0.58, p<0.01). Development of acute rejection (both early and late) was independently associated with significantly reduced graft (aHR 3.13, 95% CI: 2.21–4.42, p<0.001) and patient survival (aHR 3.42, 95% CI: 2.35–4.98, p<0.001). In this 11-year Australian study, acute LT rejection occurred in 35%, with independent risk factors of younger recipient age and ABO-incompatible transplant, while having a simultaneous multiorgan transplant was protective. Acute rejection was independently associated with reduced graft and patient survival after adjustment for other factors.

AbstractObjectivesWe aim to determine the prognostic significance of glutathione S‐transferase Pi 1 DNA methylation (mGSTP1) in two independent prostate cancer cohorts with long‐term clinical follow‐up data.Subjects/Patients and MethodsWe first re‐examined a published, in‐house whole genome bisulphite sequencing (WGBS) prostate cancer dataset, derived from radical prostatectomy (RP) tissue (n = 15) with median follow‐up 19.5 years, to confirm and visualise the association between mGSTP1 and patient mortality. To validate prognostic significance, we used a quantitative methylation‐specific head‐loop (MS‐HL) assay to measure mGSTP1 levels in a larger, independent cohort (n = 186), and performed univariable and multivariable Cox survival analysis.ResultsRe‐analysis of WGBS data showed a significant increase in mGSTP1 in RP samples from patients with lethal versus non‐lethal disease. Subsequent analysis in the larger cohort using the MS‐HL assay confirmed that mGSTP1 was detectable in 97% of RP samples, validating the diagnostic potential of mGSTP1. Univariable Cox survival analysis revealed a significant association between mGSTP1 levels and biochemical recurrence and metastatic relapse free survival, with a near‐significant association with prostate cancer specific mortality. Notably, multivariable Cox models demonstrated that mGSTP1 did not add independent prognostic value beyond standard clinicopathological features.ConclusionOur study supports the importance of DNA methylation as a tissue‐based prostate tumour biomarker. GSTP1 methylation is well established as a diagnostic marker, and in this study, we find that GSTP1 methylation levels are also associated with disease prognosis. Further research is required into the clinical utility of prognostic methylation markers and their functional role in disease progression.

Supplementary Tables 1-3 from Epigenetic Deregulation Across Chromosome 2q14.2 Differentiates Normal from Prostate Cancer and Provides a Regional Panel of Novel DNA Methylation Cancer Biomarkers

AIM The 5-year survival rate of pancreatic ductal adenocarcinoma (PDAC) is approximately 11% and has only improved marginally over the last three decades. For operable PDAC, resection and adjuvant FOLFIRINOX chemotherapy is standard of care. There is growing interest in perioperative regimens to improve outcomes. The non-randomized Phase II study "Gemcitabine and Abraxane for resectable Pancreatic cancer" (GAP) demonstrated the feasibility of perioperative gemcitabine/abraxane. Long-term survival in PDAC requires an effective immune response; hence, we undertook this translational study of the GAP trial cohort to identify immune-oncology biomarkers for clinical use. METHODS We combined Nanostring nCounter technology with immunohistochemistry to investigate the correlation between gene expression and overall patient survival. Findings were investigated in samples from the International Cancer Genome Consortium (ICGC, n = 88) and the Australian Pancreatic Genome Initiative (APGI, n = 227). RESULTS We confirmed that human equilibrative nucleoside transporter 1 (hENT1) expression was not a prognostic marker in PDAC but patients with high levels of hENT1 were more likely to live longer than 24 months post-surgery. Additionally, CD274 (PD-L1) and two novel biomarkers of survival, cathepsin W (CTSW) and C-reactive protein (CRP), were identified in the GAP cohort (n = 19). CRP expression was confirmed in data from the ICGC. Although PD-L1 and CTSW proteins were not significant across all three cohorts, results show that low CRP mRNA and protein expression are associated with longer overall survival in all three patient groups. CONCLUSION PDAC patients with long survival have higher hENT1 expression levels. Furthermore, CRP expression is a biomarker of poor prognosis following perioperative chemotherapy and resection in PDAC patients and thus may be useful for identifying patients who could benefit from more aggressive adjuvant strategies.

Current machine perfusion technology permits livers to be preserved ex situ for short periods to assess viability prior to transplant. Long-term normothermic perfusion of livers is an emerging field with tremendous potential for the assessment, recovery, and modification of organs. In this study, we aimed to develop a long-term model of ex situ perfusion including a surgical split and simultaneous perfusion of both partial organs. Human livers declined for transplantation were perfused using a red blood cell-based perfusate under normothermic conditions (36 °C) and then split and simultaneously perfused on separate machines. Ten human livers were split, resulting in 20 partial livers. The median ex situ viability was 125 h, and the median ex situ survival was 165 h. Long-term survival was demonstrated by lactate clearance, bile production, Factor-V production, and storage of adenosine triphosphate. Here, we report the long-term ex situ perfusion of human livers and demonstrate the ability to split and perfuse these organs using a standardised protocol.

Introduction. Biliary complications are a common cause of morbidity after liver transplantation and associated with bile duct injury. To reduce injury, a bile duct flush is performed with high-viscosity preservation solution. It has been suggested that an earlier additional bile duct flush with low-viscosity preservation solution may reduce bile duct injury and biliary complications. This study aimed to investigate whether an earlier additional bile duct flush would reduce bile duct injury or biliary complications. Methods. A randomized trial was conducted using 64 liver grafts from brain dead donors. The control group received a bile duct flush with University of Wisconsin (UW) solution after donor hepatectomy. The intervention group received a bile duct flush using low-viscosity Marshall solution immediately after the onset of cold ischemia and a bile duct flush with University of Wisconsin solution after donor hepatectomy. The primary outcomes were the degree of histological bile duct injury, assessed using the bile duct injury score, and biliary complications within 24 mo of transplant. Results. Bile duct injury scores were not different between the 2 groups. Similar rates of biliary complications occurred in the intervention group (31% [n = 9]) and controls (23% [n = 8]) (P = 0.573). No difference between groups was observed for anastomotic strictures (24% versus 20%, P = 0.766) or nonanastomotic strictures (7% versus 6%, P = 1.00). Conclusions. This is the first randomized trial to investigate an additional bile duct flush using low-viscosity preservation solution during organ procurement. The findings from this study suggest that performing an earlier additional bile duct flush with Marshall solution does not prevent biliary complications and bile duct injury.

Background: Acinar cystic transformation (ACT) of the pancreas is a non-neoplastic cystic lesion lined by acinar and ductal epithelium. Only 84 cases including this report have been described in the literature. Case report: The clinical history, imaging, macroscopic, histologic and immunohistochemical features were consistent with acinar cystic transformation of the pancreas in a 24-year-old female. Discussion: Although rare, ACT of the pancreas should be considered as a differential diagnosis when considering cystic lesions of the pancreas. Cytological examination of aspirated fluid is often inconclusive or may not correlate with the final histological diagnosis. Micro biopsies via EUS examined histologically with immunohistochemical support may help distinguish ACT from relevant premalignant differentials including an intraductal papillary mucinous neoplasm and mucinous cystic neoplasm. References 1. WHO Classification of Tumours Editorial Board. Digestive system tumours. Lyon (France): International Agency for Research on Cancer 2019. WHO classification of tumours series, 5th ed, Vol 1). 2. Rift CV, Hasselby JP, Hansen CP, Federspiel B. Acinar cystic transformation of the pancreas: report of a case and a review of the literature. Pathol Res Pract 2020; 216: 152928. 3. Lee JH, Jung SJ, Park YH, Park SJ, Choi JS. Pancreatic Acinar Cell Cystadenoma Mimicking Pancreatic Serous Cystadenoma. Korean J Gastroenterol 2021; 78: 138–143.

Introduction: Histological injury to the biliary tree during organ preservation leads to biliary strictures after liver transplantation. The Bile Duct Injury (BDI) score was developed to assess histological injury and identify the grafts most likely to develop biliary strictures. The BDI score evaluates the bile duct mural stroma, peribiliary vascular plexus (PVP) and deep peribiliary glands (DPGs), which were correlated with post-transplant biliary strictures. However, the BDI score has not been externally validated. The aim of this study was to verify whether the BDI score could predict biliary strictures at our transplant centre. Methods: Brain-dead donor liver grafts transplanted at a single institution from March 2015 to June 2016 were included in this analysis. Bile duct biopsies were collected immediately before transplantation and assessed for bile duct injury by two blinded pathologists. The primary outcome was the development of clinically significant biliary strictures within 24 months post-transplant. Results: Fifty-seven grafts were included in the study which included 16 biliary strictures (28%). Using the BDI score, mural stromal, PVP and DPG injury did not correlate with biliary strictures including Non-Anastomotic Strictures. Severe inflammation (>50 leucocytes per HPF) was the only histological feature inversely correlated with the primary outcome (absent in the biliary stricture group vs. 41% in the no-stricture group, p = 0.001). Conclusions: The current study highlights limitations of the histological assessment of bile duct injury. Although all grafts had bile duct injury, only inflammation was associated with biliary strictures. The BDI score was unable to predict post-transplant biliary strictures in our patient population.

Background: Split liver transplantation permits the transplant of two recipients using a single donor liver. Liver splitting can be performed using the ex-vivo technique (more convenient), or the in-situ technique (shorter cold ischaemic time). We aimed to develop a technique for liver splitting during normothermic machine perfusion which combines the advantages of both techniques and permits graft assessment prior to transplant. Methods: Human livers declined for transplantation were perfused at 36 degrees C using a modified -commercial perfusion machine. We developed a six-step method to split whole livers into left lateral segment grafts and extended right grafts. Both partial livers were then perfused on separate machines for individual assessment. Results: Using our technique, 10 whole livers were successfully split during normothermic perfusion resulting in 20 partial grafts. Apart from a single graft which failed due to a technical error, all grafts survived for 24-h after splitting. Survival was demonstrated by lactate clearance, bile production and synthesis of coagulation factors. Conclusions: Liver splitting during normothermic machine perfusion has the potential to revolutionise split liver transplantation. We describe a novel technique that reliably achieves two grafts from a single donor liver. This raises the possibility of semi-elective transplantation, and sophisticated graft assess-ment prior to implant.

Drug reaction with eosinophilia and systemic symptoms (DRESS) is a rare and potentially life-threatening T-cell mediated hypersensitivity reaction involving the skin and visceral organs.1 The syndrome is heterogenous, unpredictable, and difficult to diagnose. Three cases of DRESS due to rivaroxaban have been reported, including two with a mild eosinophilia <1.1×109/L.2–4 One case of DRESS due to apixaban, another factor Xa inhibitor, has been reported.5 Cases of drug-induced liver injury (DILI) due to rivaroxaban without DRESS have also been reported.

Abstract Current perfusion technology only allows livers to be preserved ex-vivo for short periods. Long-term normothermic perfusion of livers is an emerging field with tremendous potential for the assessment, recovery, and modification of organs. In this study, we aimed develop a long-term model of ex-vivo perfusion including a surgical split and simultaneous perfusion of both partial grafts. Our long-term perfusion system included long-term oxygenators, a gas-mixer and a dialysis filter. Human livers declined for transplantation were perfused using a red-cell based perfusate under normothermic conditions (36°C) and then split and simultaneously perfused on separate machines. Ten human livers were split resulting in 20 partial grafts. The median ex-vivo survival was 165 hours (7 days). Long-term graft survival was demonstrated by lactate clearance, bile production, Factor-V production, and storage of adenosine triphosphate. The grafts that survived > 7 days demonstrated significantly higher bile production, Factor-V production, and hepatic arterial flow and significantly lower microvesicular steatosis. We report reliable long-term ex-vivo perfusion of human livers and demonstrate the ability to split and perfuse these organs using a reproducible protocol. This provides the opportunity for improved assessment of organs and could act as a model for the testing of therapeutics with a matched control.

Introduction . Histopathological assessment of liver biopsies is the current “gold standard” for diagnosing graft dysfunction after liver transplantation (LT), as graft dysfunction can have nonspecific clinical presentations and inconsistent patterns of liver biochemical dysfunction. Most commonly, post-LT, graft dysfunction within the first year, is due to acute T-cell mediated rejection (TCMR) which is characterised histologically by the degree of portal inflammation (PI), bile duct damage (BDD), and venous endothelial inflammation (VEI). This study aimed to establish the relationship between global assessment, which is the global grading of rejection using a “gestalt” approach, and the rejection activity index (RAI) of each component of TCMR as described in revised Banff 2016 guidelines. Methods . Liver biopsies ( n  = 90) taken from patients who underwent LT in 2015 and 2016 at the Australian National Liver Transplant Unit were identified from the electronic medical records. All biopsy slides were microscopically graded by at least two assessors independently using the revised 2016 Banff criteria. Data were analysed using IBM SPSS v21. A Fisher–Freeman–Halton test was performed to assess the correlation between the global assessment and the RAI scores for each TCMR biopsy. Results . Within the cohort, 60 (37%, n  = 164) patients underwent at least 1 biopsy within 12 months after LT. The most common biopsy outcome (total n  = 90) was acute TCMR (64, 71.1%). Global assessment of TCMR slides strongly positively correlated with PI ( p value <0.001), BDD ( p value <0.001), VEI ( p value <0.001), and total RAI ( p value <0.001). Liver biochemistry of patients with TCMR significantly improved within 4 to 6 weeks post-biopsy compared to the day of the biopsy. Conclusion . In acute TCMR, global assessment and total RAI are strongly correlated and can be used interchangeably to describe the severity of TCMR.

IntroductionLiver cancers exhibit abnormal (leaky) vasculature, hypoxia and an immunosuppressive microenvironment. Normalization of tumor vasculature is an emerging approach to treat many cancers. Blockmir CD5-2 is a novel oligonucleotide-based inhibitor of the miR-27a interaction with VE-Cadherin, the endothelial-specific cadherin. The combination of a vasoactive medication with inhibition of immune checkpoints such as programmed cell death protein 1 (PD1) has been shown to be effective in treating liver cancer in humans. We aimed to study the effect of CD5-2 combined with checkpoint inhibition (using an antibody against PD1) on liver tumor growth, vasculature and immune infiltrate in the diethylnitrosamine (DEN)-induced liver tumor mouse model.MethodsWe first analyzed human miR-27a and VE-Cadherin expression data from The Cancer Genome Atlas for hepatocellular carcinoma. CD5-2 and/or anti-PD1 antibody were given to the DEN-treated mice from age 7-months until harvest at age 9-months. Tumor and non-tumor liver tissues were analyzed using histology, immunohistochemistry, immunofluorescence and scanning electron microscopy.ResultsHuman data showed high miR-27a and low VE-Cadherin were both significantly associated with poorer prognosis. Mice treated with CD5-2 plus anti-PD1 antibody had significantly smaller liver tumors (50% reduction) compared to mice treated with either agent alone, controls, or untreated mice. There was no difference in tumor number. Histologically, tumors in CD5-2-treated mice had less leaky vessels with higher VE-Cadherin expression and less tumor hypoxia compared to non-CD5-2-treated mice. Only tumors in the combination CD5-2 plus anti-PD1 antibody group exhibited a more favorable immune infiltrate (significantly higher CD3+ and CD8+ T cells and lower Ly6G+ neutrophils) compared to tumors from other groups.DiscussionCD5-2 normalized tumor vasculature and reduced hypoxia in DEN-induced liver tumors. CD5-2 plus anti-PD1 antibody reduced liver tumor size possibly by altering the immune infiltrate to a more immunosupportive one.