The majority of colorectal cancers (CRC) arise from somatic mutations occurring throughout a patient’s life. And yet, rare germline variants also contribute to more rapid progression of cancer and drug resistance. Based on computational analysis of 3806 patients from three independent cohorts (DFCI profile, Hartwig Medical Foundation, TCGA), we have identified potential germline modifiers that influence the incidence of activating KRAS mutations. KRAS somatic driver mutations are present in nearly 50% of CRC patients, with the KRAS-G12D variant being one of the more frequently observed. In this study, we are validating this in silico modeling through experimental studies. This combined computational and experimental approach will have implications at the basic translational and clinical levels.

We researched the effect of Kras^G12D on RNA and protein expression of our gene of interest in our existing mouse data sets and mouse organoids, derived from tumors resulting from Fabp1-Cre;Apc(lox/+);Kras(lsl-G12D/+) mice. We next generated a series of lentivirus constructs of our modifier of interest carrying single nucleotide polymorphisms (SNPs) observed in human patients and infected isogenic SW48 colon cancer cells that express either wild-type KRAS or KRAS^G12D. Immunoblots were then performed to determine the effects of these SNPs on the expression of both the modifier and KRAS, as well as downstream pathways. Lastly, we tested the sensitivity of the isogenic SW48 cells to inhibitors of the target receptor using CellTiterGlo, to determine if different KRAS alleles have differential dependency on the function of the wild-type receptor. These experiments were followed up by immunoblot analysis of the downstream pathways following inhibition.

In accordance with data from B. Shui et al., Kras^G12D expressing tumors upregulate the Fgfr4. However, Fgfr4 SNP variants had no effect on protein expression levels, regardless of the presence or absence of Kras in HEK293T cells. Furthermore, Kras^WT and Kras^G12D SW48 cells exhibited similar sensitivity to the Fgfr4 inhibitors. The investigation into the effects of inhibitors on downstream pathways is ongoing, with additional experiments in colon cancer cells currently underway. To further explore these germline modifiers, we anticipate forthcoming results from a murine model.

BACKGROUND: During anoxia, flavin mononucleotide (FMN) is released from complex I. This reflects directly on mitochondrial preservation damage and the cells metabolic capability. Measurement of FMN is currently the only possibility for graft assessment during hypothermic oxygenated machine perfusion (HOPE).
METHODS: During 50 HOPE, FMN was measured and correlated with standard laboratory and outcome parameters including early allograft dysfunction (EAD), biliary complications, 1-year graft and patient survival. FMN was measured via fluorescence spectroscopy, levels were displayed in artificial units and concentrations were determined.
RESULTS: The area under the curve (AUC) for prediction of EAD at 5 minutes of perfusion was 0.744, the thereby determined cut-off of 10.65 ng/mL (sensitivity 87%, specificity 63%), identified 52% of recipients that developed an EAD. FMN levels at 5 minutes were higher in grafts whose recipients did not survive the first year after liver transplantation (p<0.001. Patient one-year survival was predicted at 5 minutes with an AUC of 0.890. The determined cut-off of 23.5ng/mL (sensitivity 83.3 % and specificity 92.7 %) was used to identify patients with a high risk for early mortality. Below the cut-off, 98% of recipients survived whereas only 37.5% of recipients above the cut-off survived. When combined, both cut-offs allowed for risk stratification in high, intermediate and low risk. EAD (p=0.004), 1-year mortality (p<0.001), need for dialysis (0.019) and re-transplantation (p=0.001) were highest in the high-risk group followed by intermediate and low risk.
CONCLUSIONS: FMN allows for excellent risk stratification during HOPE. Categorization into risk groups helps to identify grafts with an especially high risk for graft loss and early mortality. The threshold identified in this study enables reliable prediction of one-year patient survival at 5 minutes of perfusion. FMN levels correlate to increasing complication rates and higher morbidity, underlining its relevance.

BACKGROUND:

Sub-normothermic machine perfusion (SNMP) is used to preserve and evaluate liver grafts at temperatures above hypothermic and below normothermic conditions, especially evaluating its temperature stage in controlled oxygenated rewarming in combined perfusion protocols such as DHOPE-COR-NMP. However, the experience in preserving and assessing steatotic livers during SNMP is limited. This study focused on comparing the performance of steatotic versus healthy liver grafts during SNMP to provide a more comprehensive understanding during this specific temperature setting.

METHODS:

Steatotic livers were obtained from pigs fed with a specifically designed high-fat diet over a median of 32 weeks to induce steatosis. Healthy liver grafts were obtained from pigs receiving a standard diet. Both steatotic and healthy livers underwent SNMP for 12 hours. During perfusion, key parameters such as perfusion dynamics, biliary tree secretion and lactate clearance were monitored. Histopathological analysis and immunofluorescence assays were performed post-hoc to assess steatosis levels and liver condition.

RESULTS:

Steatotic livers exhibited impaired perfusion dynamics compared to healthy livers, with higher vascular resistance and lower flow rates. Biliary tree secretion and overall metabolic activity, measured by lactate clearance and glucose metabolism, was also lower in steatotic livers compared to healthy controls. Histopathological analysis confirmed the presence of macrovesicular steatosis, with associated cellular damage.

CONCLUSIONS:

SNMP showed differences in the performance of steatotic versus healthy liver grafts. Steatotic livers showed a tendency of impaired perfusion highlighting the challenges associated with steatotic livers for transplantation. This study underscores the importance of individualized assessment and optimization of liver grafts during machine perfusion. Further research is needed to refine SNMP techniques and improve the viability of steatotic liver grafts.

BACKGROUND:

The increasing need for extended criteria donor (ECD) organs stems from the disparity between transplant waiting lists and organ availability. While donor livers with microsteatosis or moderate steatosis generally show favorable outcomes, severely macrosteatotic livers carry a higher risk of primary non-function or early allograft dysfunction, complicating clinical transferability due to subjective differentiation between steatosis types. Hyperspectral imaging (HSI) offers a non-invasive approach for assessing steatosis, potentially aiding in pre-transplant evaluation. This study investigated the feasibility and accuracy of using HSI in steatosis assessment.

METHODS:

Livers from a previously established non-alcoholic steatosis pig model were utilized for this study. As part of this model, blood was retrieved prior to the start of perfusion. In this context, HSI data was collected using the TIVITA® 2.0 device to observe the consequent reduction in blood flow and precisely determine the onset of warm ischemia. The collected data was then analyzed to quantify steatosis levels, and the results were compared with histopathological findings to validate the correlation.

RESULTS:

HSI successfully differentiated between steatotic livers and healthy control livers, providing quantitative data on steatosis with high correlation to histopathological results. During blood retrieval, HSI detected changes in tissue composition and accurately identified the onset of warm ischemia by assessing the decline in blood flow and oxygen supply, with the real-time data limited to the time needed for taking one picture.

CONCLUSIONS:

Additionally, HSI demonstrated its capability to assess microperfusion, oxygenation, and organ morphology, including steatosis. This highlights its potential to enhance clinical decision-making by enabling non-invasive and nearly continuous monitoring regarding organ suitability during various stages of the transplantation process. The implementation of HSI in the clinical settings could improve the assessment of liver grafts prior to transplantation, potentially increasing the pool of acceptable organs and improving transplant outcomes.

BACKGROUND:

Steatosis, characterized by the accumulation of lipids in hepatocytes, markedly affects liver transplantation outcomes. The development of a reliable and reproducible animal model of steatosis facilitates research using not only healthy but also steatotic pig livers, thereby advancing the understanding of extended criteria donor organs. This study aimed to establish an effective pig model of steatosis to further evaluate the performance of these organs during different machine perfusion techniques, in comparison to healthy livers.

METHODS:

A cohort of pigs was subjected to a specifically designed high-fat diet over a median of 32 weeks to induce steatosis, with dietary intake and health parameters continuously monitored. Liver biopsies were performed at defined time points to assess steatosis progression. Post-induction, livers were evaluated using conventional laboratory values to confirm steatosis. Following these assessments, the livers were explanted according to standard clinical protocols and subjected to sub-normothermic machine perfusion (SNMP) after cold ischemia. Key perfusion metrics were monitored throughout the process.

RESULTS:

The high-fat diet successfully induced several degrees of steatosis in the pig model, with histopathological analysis revealing macrovesicular steatosis similar to human non-alcoholic fatty liver disease, along with signs of inflammation and commencing fibrosis. Additionally, immunofluorescence staining was performed. During SNMP, steatotic livers exhibited impaired perfusion dynamics compared to healthy controls.

CONCLUSIONS:

The study successfully established a pig steatosis model that aimed to mimic steatosis in humans, providing a valuable tool for transplantation research. The model demonstrated consistent and reproducible induction of steatosis. Comparative analysis under SNMP revealed differences in the performance of steatotic versus healthy livers. This model holds potential to serve as a platform for investigating steatosis pathophysiology and improving machine perfusion strategies of such livers.