Rapid Capture of Cancer Extracellular Vesicles by Lipid Patch Microarrays

Extracellular vesicles (EVs) contain various bioactive molecules such as DNA, RNA, and proteins, and play a key role in the regulation of cancer progression. Furthermore, cancer-associated EVs carry specific biomarkers and can be used in liquid biopsy for cancer detection. However, it is still technically challenging and time consuming to detect or isolate cancer-associated EVs from complex biofluids (e.g., blood). Here, a novel EV-capture strategy based on dip-pen nanolithography generated microarrays of supported lipid membranes is presented. These arrays carry specific antibodies recognizing EV- and cancer-specific surface biomarkers, enabling highly selective and efficient capture. Importantly, it is shown that the nucleic acid cargo of captured EVs is retained on the lipid array, providing the potential for downstream analysis. Finally, the feasibility of EV capture from patient sera is demonstrated. The demonstrated platform offers rapid capture, high specificity, and sensitivity, with only a small need in analyte volume and without additional purification steps. The platform is applied in context of cancer-associated EVs, but it can easily be adapted to other diagnostic EV targets by use of corresponding antibodies

Biomarkers in Liquid Biopsies for Prediction of Early Liver Metastases in Pancreatic Cancer

Individualized diagnostics approaches in modern cancer therapy require predictive and prognostic biomarkers that are easily accessible and stratify patients for optimal and individualized treatment. Pancreatic ductal adenocarcinoma (PDAC) is still a life-threatening disease mainly because of its late diagnosis in advanced stages or rapid progress even in patients with curative resection of the primary tumor. Moreover, patients with liver metastases exhibit an even worse prognosis. Hence, this retrospective multi-center study aims to identify biomarkers in perioperative serum of PDAC patients predicting early liver metastasis. A highly sensitive biomarker analysis was performed using two different methodological approaches. Olink® analysis, which was also used to validate LEGENDplexTM results, identified significant differences in proteins involved in chemotaxis and migration of immune cells as well as cell growth in serum of patients with early versus late onset of liver metastasis. Further studies with larger cohorts are required to validate these findings for clinical translation

BRAFΔβ3−αC^{Δβ3-αC} in-frame deletion mutants differ in their dimerization propensity, HSP90 dependence, and druggability

In-frame BRAF exon 12 deletions are increasingly identified in various tumor types. The resultant BRAF$^{Δβ3-αC}$ oncoproteins usually lack five amino acids in the β3-αC helix linker and sometimes contain de novo insertions. The dimerization status of BRAF$^{Δβ3-αC}$ oncoproteins, their precise pathomechanism, and their direct druggability by RAF inhibitors (RAFi) has been under debate. Here, we functionally characterize BRAF$^{ΔLNVTAP>F}$ and two novel mutants, BRAF$^{delinsFS}$ and BRAF$^{ΔLNVT>F}$, and compare them with other BRAF$^{Δβ3-αC}$ oncoproteins. We show that BRAF$^{Δβ3-αC}$ oncoproteins not only form stable homodimers and large multiprotein complexes but also require dimerization. Nevertheless, details matter as aromatic amino acids at the deletion junction of some BRAF$^{Δβ3-αC}$ oncoproteins, e.g., BRAF$^{ΔLNVTAP>F}$, increase their stability and dimerization propensity while conferring resistance to monomer-favoring RAFi such as dabrafenib or HSP 90/CDC37 inhibition. In contrast, dimer-favoring inhibitors such as naporafenib inhibit all BRAF$^{Δβ3-αC}$ mutants in cell lines and patient-derived organoids, suggesting that tumors driven by such oncoproteins are vulnerable to these compounds

Potentials of Plasma NGAL and MIC-1 as Biomarker(s) in the Diagnosis of Lethal Pancreatic Cancer

Pancreatic cancer (PC) is lethal malignancy with very high mortality rate. Absence of sensitive and specific marker(s) is one of the major factors for poor prognosis of PC patients. In pilot studies using small set of patients, secreted acute phase proteins neutrophil gelatinase associated lipocalin (NGAL) and TGF-β family member macrophage inhibitory cytokine-1 (MIC-1) are proposed as most potential biomarkers specifically elevated in the blood of PC patients. However, their performance as diagnostic markers for PC, particularly in pre-treatment patients, remains unknown. In order to evaluate the diagnostic efficacy of NGAL and MIC-1, their levels were measured in plasma samples from patients with pre-treatment PC patients (n = 91) and compared it with those in healthy control (HC) individuals (n = 24) and patients with chronic pancreatitis (CP, n = 23). The diagnostic performance of these two proteins was further compared with that of CA19-9, a tumor marker commonly used to follow PC progression. The levels of all three biomarkers were significantly higher in PC compared to HCs. The mean (± standard deviation, SD) plasma NGAL, CA19-9 and MIC-1 levels in PC patients was 111.1 ng/mL (2.2), 219.2 U/mL (7.8) and 4.5 ng/mL (4.1), respectively. In comparing resectable PC to healthy patients, all three biomarkers were found to have comparable sensitivities (between 64%-81%) but CA19-9 and NGAL had a higher specificity (92% and 88%, respectively). For distinguishing resectable PC from CP patients, CA19-9 and MIC-1 were most specific (74% and 78% respectively). CA19-9 at an optimal cut-off of 54.1 U/ml is highly specific in differentiating resectable (stage 1/2) pancreatic cancer patients from controls in comparison to its clinical cut-off (37.1 U/ml). Notably, the addition of MIC-1 to CA19-9 significantly improved the ability to distinguish resectable PC cases from CP (p = 0.029). Overall, MIC-1 in combination with CA19-9 improved the diagnostic accuracy of differentiating PC from CP and HCs

Outcomes After Minimally-invasive Versus Open Pancreatoduodenectomy: A Pan-European Propensity Score Matched Study

OBJECTIVE: To assess short-term outcomes after minimally invasive (laparoscopic, robot-assisted, and hybrid) pancreatoduodenectomy (MIPD) versus open pancreatoduodenectomy (OPD) among European centers. BACKGROUND: Current evidence on MIPD is based on national registries or single expert centers. International, matched studies comparing outcomes for MIPD and OPD are lacking. METHODS: Retrospective propensity score matched study comparing MIPD in 14 centers (7 countries) performing ≥10 MIPDs annually (2012-2017) versus OPD in 53 German/Dutch surgical registry centers performing ≥10 OPDs annually (2014-2017). Primary outcome was 30-day major morbidity (Clavien-Dindo ≥3). RESULTS: Of 4220 patients, 729/730 MIPDs (412 laparoscopic, 184 robot-assisted, and 130 hybrid) were matched to 729 OPDs. Median annual case-volume was 19 MIPDs (interquartile range, IQR 13-22), including the first MIPDs performed in 10/14 centers, and 31 OPDs (IQR 21-38). Major morbidity (28% vs 30%, P = 0.526), mortality (4.0% vs 3.3%, P = 0.576), percutaneous drainage (12% vs 12%, P = 0.809), reoperation (11% vs 13%, P = 0.329), and hospital stay (mean 17 vs 17 days, P > 0.99) were comparable between MIPD and OPD. Grade-B/C postoperative pancreatic fistula (POPF) (23% vs 13%, P < 0.001) occurred more frequently after MIPD. Single-row pancreatojejunostomy was associated with POPF in MIPD (odds ratio, OR 2.95, P < 0.001), but not in OPD. Laparoscopic, robot-assisted, and hybrid MIPD had comparable major morbidity (27% vs 27% vs 35%), POPF (24% vs 19% vs 25%), and mortality (2.9% vs 5.2% vs 5.4%), with a fewer conversions in robot-assisted- versus laparoscopic MIPD (5% vs 26%, P < 0.001). CONCLUSIONS: In the early experience of 14 European centers performing ≥10 MIPDs annually, no differences were found in major morbidity, mortality, and hospital stay between MIPD and OPD. The high rates of POPF and conversion, and the lack of superior outcomes (ie, hospital stay, morbidity) could indicate that more experience and higher annual MIPD volumes are needed

Robot-assisted versus laparoscopic pancreatoduodenectomy:a pan-European multicenter propensity-matched study

Background: The use of robot-assisted and laparoscopic pancreatoduodenectomy is increasing, yet large adjusted analyses that can be generalized internationally are lacking. This study aimed to compare outcomes after robot-assisted pancreatoduodenectomy and laparoscopic pancreatoduodenectomy in a pan-European cohort. Methods: An international multicenter retrospective study including patients after robot-assisted pancreatoduodenectomy and laparoscopic pancreatoduodenectomy from 50 centers in 12 European countries (2009–2020). Propensity score matching was performed in a 1:1 ratio. The primary outcome was major morbidity (Clavien–Dindo ≥III). Results: Among 2,082 patients undergoing minimally invasive pancreatoduodenectomy, 1,006 underwent robot-assisted pancreatoduodenectomy and 1,076 laparoscopic pancreatoduodenectomy. After matching 812 versus 812 patients, the rates of major morbidity (31.9% vs 29.6%; P = .347) and 30-day/in-hospital mortality (4.3% vs 4.6%; P = .904) did not differ significantly between robot-assisted pancreatoduodenectomy and laparoscopic pancreatoduodenectomy, respectively. Robot-assisted pancreatoduodenectomy was associated with a lower conversion rate (6.7% vs 18.0%; P &lt; .001) and higher lymph node retrieval (16 vs 14; P = .003). Laparoscopic pancreatoduodenectomy was associated with shorter operation time (446 minutes versus 400 minutes; P &lt; .001), and lower rates of postoperative pancreatic fistula grade B/C (19.0% vs 11.7%; P &lt; .001), delayed gastric emptying grade B/C (21.4% vs 7.4%; P &lt; .001), and a higher R0-resection rate (73.2% vs 84.4%; P &lt; .001). Conclusion: This European multicenter study found no differences in overall major morbidity and 30-day/in-hospital mortality after robot-assisted pancreatoduodenectomy compared with laparoscopic pancreatoduodenectomy. Further, laparoscopic pancreatoduodenectomy was associated with a lower rate of postoperative pancreatic fistula, delayed gastric emptying, wound infection, shorter length of stay, and a higher R0 resection rate than robot-assisted pancreatoduodenectomy. In contrast, robot-assisted pancreatoduodenectomy was associated with a lower conversion rate and a higher number of retrieved lymph nodes as compared with laparoscopic pancreatoduodenectomy.</p

Pathobiological Implications of MUC16 Expression in Pancreatic Cancer

MUC16 (CA125) belongs to a family of high-molecular weight O-glycosylated proteins known as mucins. While MUC16 is well known as a biomarker in ovarian cancer, its expression pattern in pancreatic cancer (PC), the fourth leading cause of cancer related deaths in the United States, remains unknown. The aim of our study was to analyze the expression of MUC16 during the initiation, progression and metastasis of PC for possible implication in PC diagnosis, prognosis and therapy. In this study, a microarray containing tissues from healthy and PC patients was used to investigate the differential protein expression of MUC16 in PC. MUC16 mRNA levels were also measured by RT-PCR in the normal human pancreatic, pancreatitis, and PC tissues. To investigate its expression pattern during PC metastasis, tissue samples from the primary pancreatic tumor and metastases (from the same patient) in the lymph nodes, liver, lung and omentum from Stage IV PC patients were analyzed. To determine its association in the initiation of PC, tissues from PC patients containing pre-neoplastic lesions of varying grades were stained for MUC16. Finally, MUC16 expression was analyzed in 18 human PC cell lines. MUC16 is not expressed in the normal pancreatic ducts and is strongly upregulated in PC and detected in pancreatitis tissue. It is first detected in the high-grade pre-neoplastic lesions preceding invasive adenocarcinoma, suggesting that its upregulation is a late event during the initiation of this disease. MUC16 expression appears to be stronger in metastatic lesions when compared to the primary tumor, suggesting a role in PC metastasis. We have also identified PC cell lines that express MUC16, which can be used in future studies to elucidate its functional role in PC. Altogether, our results reveal that MUC16 expression is significantly increased in PC and could play a potential role in the progression of this disease

Robot-assisted versus laparoscopic pancreatoduodenectomy:a pan-European multicenter propensity-matched study

Background: The use of robot-assisted and laparoscopic pancreatoduodenectomy is increasing, yet large adjusted analyses that can be generalized internationally are lacking. This study aimed to compare outcomes after robot-assisted pancreatoduodenectomy and laparoscopic pancreatoduodenectomy in a pan-European cohort. Methods: An international multicenter retrospective study including patients after robot-assisted pancreatoduodenectomy and laparoscopic pancreatoduodenectomy from 50 centers in 12 European countries (2009–2020). Propensity score matching was performed in a 1:1 ratio. The primary outcome was major morbidity (Clavien–Dindo ≥III). Results: Among 2,082 patients undergoing minimally invasive pancreatoduodenectomy, 1,006 underwent robot-assisted pancreatoduodenectomy and 1,076 laparoscopic pancreatoduodenectomy. After matching 812 versus 812 patients, the rates of major morbidity (31.9% vs 29.6%; P = .347) and 30-day/in-hospital mortality (4.3% vs 4.6%; P = .904) did not differ significantly between robot-assisted pancreatoduodenectomy and laparoscopic pancreatoduodenectomy, respectively. Robot-assisted pancreatoduodenectomy was associated with a lower conversion rate (6.7% vs 18.0%; P &lt; .001) and higher lymph node retrieval (16 vs 14; P = .003). Laparoscopic pancreatoduodenectomy was associated with shorter operation time (446 minutes versus 400 minutes; P &lt; .001), and lower rates of postoperative pancreatic fistula grade B/C (19.0% vs 11.7%; P &lt; .001), delayed gastric emptying grade B/C (21.4% vs 7.4%; P &lt; .001), and a higher R0-resection rate (73.2% vs 84.4%; P &lt; .001). Conclusion: This European multicenter study found no differences in overall major morbidity and 30-day/in-hospital mortality after robot-assisted pancreatoduodenectomy compared with laparoscopic pancreatoduodenectomy. Further, laparoscopic pancreatoduodenectomy was associated with a lower rate of postoperative pancreatic fistula, delayed gastric emptying, wound infection, shorter length of stay, and a higher R0 resection rate than robot-assisted pancreatoduodenectomy. In contrast, robot-assisted pancreatoduodenectomy was associated with a lower conversion rate and a higher number of retrieved lymph nodes as compared with laparoscopic pancreatoduodenectomy.</p

Hemoadsorption in a Case of Severe Septic Shock and Necrotizing Fasciitis Caused by Nontraumatic Renal Rupture due to Pyelonephritis with Obstructive Uropathy

Background. Nontraumatic renal rupture due to pyelonephritis with obstructive uropathy is an uncommon but life-threatening situation. Case Presentation. A 25-year-old female presented to the emergency department with acute worsening of abdominal pain that began four weeks earlier. She was found to have peritonitis, leukocytosis, severe lactic acidosis, and a pronounced anemia and imaging was consistent with nontraumatic renal rupture with retroperitoneal abscess, perforation of the colon, and severe necrotizing fasciitis of the right lower limb. She underwent a right nephrectomy, a right hemicolectomy, surgical debridement of the retroperitoneum, and an upper thigh amputation. Due to severe septic shock and rhabdomyolysis with acute renal failure we performed a combined treatment of hemoadsorption using a Cytosorb hemoadsorber and continuous venovenous hemodialysis (CVVHD). Subsequently the patient recovered and was discharged home with no signs of infections and with normal renal function. Conclusion. We present a case of pyelonephritis with nontraumatic renal rupture leading to necrotizing fasciitis with osteomyelitis of the lower limb. The early treatment of the patient with a Cytosorb hemoadsorber led to a rapid hemodynamic and metabolic stabilization and preservation of the renal function, suggesting that hemoadsorption might be a rescue therapy in patients with severe septic shock and traumatic rhabdomyolysis