Acquired NF2 mutation confers resistance to TRK inhibition in an ex vivo LMNA::NTRK1-rearranged soft-tissue sarcoma cell model.

Genomic rearrangements of the neurotrophic receptor tyrosine kinase genes (NTRK1, NTRK2, and NTRK3) are the most common mechanism of oncogenic activation for this family of receptors, resulting in sustained cancer cell proliferation. Several targeted therapies have been approved for tumours harbouring NTRK fusions and a new generation of TRK inhibitors has already been developed due to acquired resistance. We established a patient-derived LMNA::NTRK1-rearranged soft-tissue sarcoma cell model ex vivo with an acquired resistance to targeted TRK inhibition. Molecular profiling of the resistant clones revealed an acquired NF2 loss of function mutation that was absent in the parental cell model. Parental cells showed continuous sensitivity to TRK-targeted treatment, whereas the resistant clones were insensitive. Furthermore, resistant clones showed upregulation of the MAPK and mTOR/AKT pathways in the gene expression based on RNA sequencing data and increased sensitivity to MEK and mTOR inhibitor therapy. Drug synergy was seen using trametinib and rapamycin in combination with entrectinib. Medium-throughput drug screening further identified small compounds as potential drug candidates to overcome resistance as monotherapy or in combination with entrectinib. In summary, we developed a comprehensive model of drug resistance in an LMNA::NTRK1-rearranged soft-tissue sarcoma and have broadened the understanding of acquired drug resistance to targeted TRK therapy. Furthermore, we identified drug combinations and small compounds to overcome acquired drug resistance and potentially guide patient care in a functional precision oncology setting. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Unravelling homologous recombination repair deficiency and therapeutic opportunities in soft tissue and bone sarcoma.

Defects in homologous recombination repair (HRR) in tumors correlate with poor prognosis and metastases development. Determining HRR deficiency (HRD) is of major clinical relevance as it is associated with therapeutic vulnerabilities and remains poorly investigated in sarcoma. Here, we show that specific sarcoma entities exhibit high levels of genomic instability signatures and molecular alterations in HRR genes, while harboring a complex pattern of chromosomal instability. Furthermore, sarcomas carrying HRDness traits exhibit a distinct SARC-HRD transcriptional signature that predicts PARP inhibitor sensitivity in patient-derived sarcoma cells. Concomitantly, HRDhigh sarcoma cells lack RAD51 nuclear foci formation upon DNA damage, further evidencing defects in HRR. We further identify the WEE1 kinase as a therapeutic vulnerability for sarcomas with HRDness and demonstrate the clinical benefit of combining DNA damaging agents and inhibitors of DNA repair pathways ex vivo and in the clinic. In summary, we provide a personalized oncological approach to treat sarcoma patients successfully.

Normalization of lipid oxidation defects arising from hypoxia early posthepatectomy prevents liver failure in mouse.

Surgical liver failure (SLF) develops when a marginal amount of hepatic mass is left after surgery, such as following excessive resection. SLF is the commonest cause of death due to liver surgery; however, its etiology remains obscure. Using mouse models of standard hepatectomy (sHx) (68%, resulting in full regeneration) or extended hepatectomy (eHx) (86%/91%, causing SLF), we explored the causes of early SLF related to portal hyperafflux. Assessing the levels of HIF2A with or without oxygenating agent inositol trispyrophosphate (ITPP) indicated hypoxia early after eHx. Subsequently, lipid oxidation (PPARA/PGC1α) was downregulated and associated with persisting steatosis. Mild oxidation with low-dose ITPP reduced the levels of HIF2A, restored downstream PPARA/PGC1α expression along with lipid oxidation activities (LOAs), and normalized steatosis and other metabolic or regenerative SLF deficiencies. Promotion of LOA with L-carnitine likewise normalized the SLF phenotype, and both ITPP and L-carnitine markedly raised survival in lethal SLF. In patients who underwent hepatectomy, pronounced increases in serum carnitine levels (reflecting LOA) were associated with better recovery. Lipid oxidation thus provides a link between the hyperafflux of O2-poor portal blood, the metabolic/regenerative deficits, and the increased mortality typifying SLF. Stimulation of lipid oxidation-the prime regenerative energy source-particularly through L-carnitine may offer a safe and feasible way to reduce SLF risks in the clinic.

Establishment, characterization and functional testing of two novel ex vivo extraskeletal myxoid chondrosarcoma (EMC) cell models.

Extraskeletal myxoid chondrosarcoma (EMC) is a malignant mesenchymal neoplasm of uncertain differentiation as classified by the WHO Classification of Tumours 2020. Although often associated with pronlonged survival, EMC has high rates of distant recurrences and disease-associated death. EMCs are translocation sarcomas and harbor in > 90% of the cases an NR4A3 rearrangement. The molecular consequences of the NR4A3 gene fusions are not yet fully elucidated as well-characterized ex vivo cell models for EMC are lacking. Patient-derived ex vivo models are important and essential tools for investigating disease mechanisms associated with diseases that are rare, that exhibit poor prognosis and for the identification of potential novel treatment options. We established two novel EMC ex vivo models (USZ20-EMC1 and USZ22-EMC2) for functional testing and research purposes. USZ20-EMC1 and USZ22-EMC2 were established and maintained as sarco-sphere cell models for several months in culture. The cells were molecularly characterized using DNA sequencing and methylation profiling. Both cell models represent their native tumor tissue as confirmed by histomorphology and their molecular profiles, suggesting that native tumor cell function can be recapitulated in the ex vivo models. Using a functional screening approach, novel anti-cancer drug sensitivities including potential synergistic combinations were identified. In conclusion, two novel EMC ex vivo cell models (USZ20-EMC1 and USZ22-EMC2) were successfully established and characterized from native tumor tissues. Both cell models will be useful tools for further investigating disease mechanisms and for answering basic and translational research questions.

Observations and findings during the development of a subnormothermic/normothermic long-term ex vivo liver perfusion machine.

BACKGROUND: Ex situliver machine perfusion at subnormothermic/normothermic temperature isincreasingly applied in the field of transplantation to store and evaluateorgans on the machine prior transplantation. Currently, various perfusionconcepts are in clinical and preclinical applications. Over the last 6 years ina multidisciplinary team, a novel blood based perfusion technology wasdeveloped to keep a liver alive and metabolically active outside of the bodyfor at least one week. METHODS: Within thismanuscript, we present and compare three scenarios (Group 1, 2 and 3) we werefacing during our research and development (R&D) process, mainly linked tothe measurement of free hemoglobin and lactate in the blood based perfusate. Apartfrom their proven value in liver viability assessment (ex situ), these twoparameters are also helpful in R&D of a long-term liver perfusion machine and moreover supportive in the biomedical engineering process. RESULTS: Group 1 ("good" liver on the perfusion machine) represents the best liver clearance capacity for lactate and free hemoglobin wehave observed. In contrast to Group 2 ("poor" liver on the perfusion machine), that has shown the worst clearance capacity for free hemoglobin. Astonishingly,also for Group 2, lactate is cleared till the first day of perfusion andafterwards, rising lactate values are detected due to the poor quality of theliver. These two perfusate parametersclearly highlight the impact of the organ quality/viability on the perfusion process. Whereas Group 3 is a perfusion utilizing a blood loop only (without a liver). CONCLUSION: Knowing the feasible ranges (upper- and lower bound) and the courseover time of free hemoglobin and lactate is helpful to evaluate the quality ofthe organ perfusion itself and the maturity of the developed perfusion device. Freehemoglobin in the perfusate is linked to the rate of hemolysis that indicates how optimizing (gentle blood handling, minimizing hemolysis) the perfusion machine actually is. Generally, a reduced lactate clearancecapacity can be an indication for technical problems linked to the blood supplyof the liver and therefore helps to monitor the perfusion experiments.Moreover, the possibility is given to compare, evaluate and optimize developed liverperfusion systems based on the given ranges for these two parameters. Otherresearch groups can compare/quantify their perfusate (blood) parameters withthe ones in this manuscript. The presented data, findings and recommendations willfinally support other researchers in developing their own perfusion machine ormodifying commercially availableperfusion devices according to their needs.

De novo expression of gastrokines in pancreatic precursor lesions impede the development of pancreatic cancer.

Molecular events occurring in stepwise progression from pre-malignant lesions (pancreatic intraepithelial neoplasia; PanIN) to the development of pancreatic ductal adenocarcinoma (PDAC) are poorly understood. Thus, characterization of early PanIN lesions may reveal markers that can help in diagnosing PDAC at an early stage and allow understanding the pathology of the disease. We performed the molecular and histological assessment of patient-derived PanINs, tumor tissues and pancreas from mouse models with PDAC (KC mice that harbor K-RAS mutation in pancreatic tissue), where we noted marked upregulation of gastrokine (GKN) proteins. To further understand the role of gastrokine proteins in PDAC development, GKN-deficient KC mice were developed by intercrossing gastrokine-deficient mice with KC mice. Panc-02 (pancreatic cancer cells of mouse origin) were genetically modified to express GKN1 for further in vitro and in vivo analysis. Our results show that gastrokine proteins were absent in healthy pancreas and invasive cancer, while its expression was prominent in low-grade PanINs. We could detect these proteins in pancreatic juice and serum of KC mice. Furthermore, accelerated PanIN and tumor development were noted in gastrokine deficient KC mice. Loss of gastrokine 1 protein delayed apoptosis during carcinogenesis leading to the development of desmoplastic stroma while loss of gastrokine 2 increased the proliferation rate in precursor lesions. In summary, we identified gastrokine proteins in early pancreatic precursor lesions, where gastrokine proteins delay pancreatic carcinogenesis.

Long-term Normothermic Machine Preservation of Partial Livers: First Experience With 21 Human Hemi-livers.

OBJECTIVE: The aim of this study was to maintain long-term full function and viability of partial livers perfused ex situ for sufficient duration to enable ex situ treatment, repair, and regeneration. BACKGROUND: Organ shortage remains the single most important factor limiting the success of transplantation. Autotransplantation in patients with nonresectable liver tumors is rarely feasible due to insufficient tumor-free remnant tissue. This limitation could be solved by the availability of long-term preservation of partial livers that enables functional regeneration and subsequent transplantation. METHODS: Partial swine livers were perfused with autologous blood after being procured from healthy pigs following 70% in-vivo resection, leaving only the right lateral lobe. Partial human livers were recovered from patients undergoing anatomic right or left hepatectomies and perfused with a blood based perfusate together with various medical additives. Assessment of physiologic function during perfusion was based on markers of hepatocyte, cholangiocyte, vascular and immune compartments, as well as histology. RESULTS: Following the development phase with partial swine livers, 21 partial human livers (14 right and 7 left hemi-livers) were perfused, eventually reaching the targeted perfusion duration of 1 week with the final protocol. These partial livers disclosed a stable perfusion with normal hepatic function including bile production (5-10 mL/h), lactate clearance, and maintenance of energy exhibited by normal of adenosine triphosphate (ATP) and glycogen levels, and preserved liver architecture for up to 1 week. CONCLUSION: This pioneering research presents the inaugural evidence for long-term machine perfusion of partial livers and provides a pathway for innovative and relevant clinical applications to increase the availability of organs and provide novel approaches in hepatic oncology.

Sources and prevention of graft infection during long-term ex situ liver perfusion.

INTRODUCTION: The use of normothermic liver machine perfusion to repair injured grafts ex situ is an emerging topic of clinical importance. However, a major concern is the possibility of microbial contamination in the absence of a fully functional immune system. Here, we report a standardized approach to maintain sterility during normothermic liver machine perfusion of porcine livers for one week. METHODS: Porcine livers (n = 42) were procured and perfused with blood at 34°C following aseptic technique and standard operating procedures. The antimicrobial prophylaxis was adapted and improved in a step-wise manner taking into account the pathogens that were detected during the development phase. Piperacillin-Tazobactam was applied as a single dose initially and modified to continuous application in the final protocol. In addition, the perfusion machine was improved to recapitulate partially the host's defense system. The final protocol was tested for infection prevention during one week of perfusion. RESULTS: During the development phase, microbial contamination occurred in 27 out of 39 (69%) livers with a mean occurrence of growth on 4 ± 1.6 perfusion days. The recovered microorganisms suggested an exogenous source of microbial contamination. The antimicrobial agents (piperacillin/tazobactam) could be maintained above the targeted minimal inhibitory concentration (8-16 mg/L) only with continuous application. In addition to continuous application of piperacillin/tazobactam, partial recapitulation of the host immune system ex situ accompanied by strict preventive measures for contact and air contamination maintained sterility during one week of perfusion. CONCLUSION: The work demonstrates feasibility of sterility maintenance for one week during ex situ normothermic liver perfusion.

Bile formation in long-term ex situ perfused livers.

BACKGROUND: Long-term ex situ liver perfusion may rescue injured grafts. Little is known about bile flow during long-term perfusion. We report the development of a bile stimulation protocol and motivate bile flow as a viability marker during long-term ex situ liver perfusion. METHODS: Porcine and human livers were perfused with blood at close to physiologic conditions. Our perfusion protocol was established during phase 1 with porcine livers (n = 23). Taurocholic acid was applied to stimulate bile flow. The addition of piperacillin-tazobactam (tazobac) and methylprednisolone was modified from daily bolus to controlled continuous application. We adapted the protocol to human livers (n = 12) during phase 2. Taurocholic acid was replaced with medical grade ursodeoxycholic acid. RESULTS: Phase 2: Despite administering taurocholic acid, bile flow declined from 29.3 ± 6.5 to 9.3 ± 1.4 mL/h (P < .001). Shortly after bolus of tazobac/methylprednisolone, bile flow recovered to 39.0 ± 9.7 mL/h with a decrease of solid bile components. This implied bile salt independent bile flow stimulation by tazobac/methylprednisolone. Phase 2: Ursodeoxycholic acid was shown to stimulate bile flow ex situ in human livers. Eight livers were perfused successfully for 1 week with continuous bile flow. The other 4 livers demonstrated progressive cell death, of which only 1 exhibited bile flow. CONCLUSION: A lack of bile flow stimulation leads to a decline in bile flow and is not necessarily a sign of deterioration in liver function. Proper administration of stimulators can induce constant bile flow during ex situ liver perfusion for up to 1 week. Medical grade ursodeoxycholic acid is a suitable replacement for nonmedical grade taurocholic acid. The presence of bile flow alone is not sufficient to assess liver viability.

Automated Insulin Delivery - Continuous Blood Glucose Control During Ex Situ Liver Perfusion.

OBJECTIVE: With the growing demand for livers in the field of transplantation, interest in normothermic ex situ machine perfusion (NMP) has increased in recent years. This may open the door for novel therapeutic interventions such as repair of suboptimal grafts. For successful long-term NMP of livers, blood glucose (BG) levels need to be maintained in a close to physiological range. METHODS: We present an "automated insulin delivery" (AID) system integrated into an NMP system, which automatically adjusts insulin infusion rates based on continuous BG measurements in a closed loop manner during ex situ pig and human liver perfusion. An online glucose sensor for continuous glucose monitoring was integrated and evaluated in blood. A model based and a proportional controller were implemented and compared in their ability to maintain BG within the physiological range. RESULTS: The continuous glucose sensor is capable of measuring BG directly in human and pig blood for multiple days with an average error of 0.6 mmol/L. There was no significant difference in the performance of the two controllers in terms of their ability to keep BG in the physiological range. With the integrated AID, BG was controlled within the physiological range on average in 80% and 76% of the perfusion time for human and pig livers, respectively. CONCLUSION: The presented work offers a method and shows the feasibility to maintain BG in the physiological range for multiple (up to ten) days during ex situ liver perfusion with the help of an automated AID. SIGNIFICANCE: Maintaining BG within the physiological range is required to enable long-term ex situ liver perfusion.

Hyperoxia in portal vein causes enhanced vasoconstriction in arterial vascular bed.

Long-term perfusion of liver grafts outside of the body may enable repair of poor-quality livers that are currently declined for transplantation, mitigating the global shortage of donor livers. In current ex vivo liver perfusion protocols, hyperoxic blood (arterial blood) is commonly delivered in the portal vein (PV). We perfused porcine livers for one week and investigated the effect of and mechanisms behind hyperoxia in the PV on hepatic arterial resistance. Applying PV hyperoxia in porcine livers (n = 5, arterial PV group), we observed an increased need for vasodilator Nitroprussiat (285 ± 162 ml/week) to maintain the reference hepatic artery flow of 0.25 l/min during ex vivo perfusion. With physiologic oxygenation (venous blood) in the PV the need for vasodilator could be reduced to 41 ± 34 ml/week (p = 0.011; n = 5, venous PV group). This phenomenon has not been reported previously, owing to the fact that such experiments are not feasible practically in vivo. We investigated the mechanism of the variation in HA resistance in response to blood oxygen saturation with a focus on the release of vasoactive substances, such as Endothelin 1 (ET-1) and nitric oxide (NO), at the protein and mRNA levels. However, no difference was found between groups for ET-1 and NO release. We propose direct oxygen sensing of endothelial cells and/or increased NO break down rate with hyperoxia as possible explanations for enhanced HA resistance.

An integrated perfusion machine preserves injured human livers for 1 week.

The ability to preserve metabolically active livers ex vivo for 1 week or more could allow repair of poor-quality livers that would otherwise be declined for transplantation. Current approaches for normothermic perfusion can preserve human livers for only 24 h. Here we report a liver perfusion machine that integrates multiple core physiological functions, including automated management of glucose levels and oxygenation, waste-product removal and hematocrit control. We developed the machine in a stepwise fashion using pig livers. Study of multiple ex vivo parameters and early phase reperfusion in vivo demonstrated the viability of pig livers perfused for 1 week without the need for additional blood products or perfusate exchange. We tested the approach on ten injured human livers that had been declined for transplantation by all European centers. After a 7-d perfusion, six of the human livers showed preserved function as indicated by bile production, synthesis of coagulation factors, maintained cellular energy (ATP) and intact liver structure.

Model Assisted Analysis of the Hepatic Arterial Buffer Response During Ex Vivo Porcine Liver Perfusion.

OBJECTIVE: The hepatic arterial buffer response is a well-known phenomenon in hepatic circulation, describing the response of hepatic arterial resistance to changes in portal vein flow. Several vasoactive metabolites underlying its mechanism have been proposed, however, there is currently no clear consensus. The aim of this study is to investigate the hepatic arterial buffer response of porcine livers preserved in a controlled ex vivo perfusion machine. METHODS: Porcine livers are perfused on an ex vivo perfusion machine and hemodynamic experiments investigating the hepatic arterial resistance response to portal vein flow and vena cava pressure variations are conducted. A simple hemodynamic model is developed to support the interpretation of the received measurements. Further, a mechanism is proposed that explains hepatic arterial resistance changes in response to vena cava pressure as myogenic and in response to portal vein flow as a combined washout and myogenic effect. RESULTS: A clear correlation between hepatic sinusoidal pressure levels and hepatic arterial resistance is observed where an increase of approximately 4 mmHg of hepatic sinusoidal pressure level results in doubling of the hepatic arterial resistance. This relation is considered during the analysis of the portal vein flow variations resulting in a reduced isolated effect of adenosine washout on hepatic arterial resistance. With an average buffer capacity of 27% during our experiments, the hepatic arterial buffer response shows to be unimpaired in the ex vivo scenario. CONCLUSION: First, washout and myogenic effects both influence the hepatic arterial buffer response; and second, hepatic sinusoidal pressure levels strongly influence the hepatic arterial resistance. SIGNIFICANCE: These results present new findings in hemodynamics of the liver, which are fundamental for successful ex vivo liver perfusion.