Immunocytochemical Phenotyping of Disseminated Tumor Cells in Bone Marrow by uPA Receptor and CK18: Investigation of Sensitivity and Specificity of an Immunogold/Alkaline Phosphatase Double Staining Protocol

Phenotyping of cytokeratin (CK) 18-positive cells in bone marrow is gaining increasing importance for future prognostic screening of carcinoma patients. Urokinase-type plasminogen activator receptor (uPA-R) is one example of a potential aggressive marker for those cells. However, a valid and reliable double staining method is needed. Using monoclonal antibodies against uPA-R and CK18, we modified an immunogold/alkaline phosphatase double staining protocol. UPA-R/CK18-positive tumor cell controls exhibited black uPA-R staining in 15–80 of cases and red CK18 staining in almost 100 of tumor cells. Isotype- and cross-matched controls were completely negative. Bone marrow from healthy donors was always CK18-negative. Reproducibility of CK18-positive cell detection was estimated in a series of specimens from 61 gastric cancer patients comparatively stained with the single alkaline phosphatase-anti-alkaline phosphatase (APAAP) and our double staining method (106 bone marrow cells/patient). In four cases, double staining could not reproduce CK18-positive cells. In 34 cases it revealed fewer or equal numbers, and in 23 cases more CK18-positive cells than the APAAP method. Overall quantitative analysis of detected cell numbers (838 in APAAP, range 1–280 in 106; double staining 808, range 0–253) demonstrated relative reproducibility of APAAP results by double staining of 97. Correlation of results between both methods was significant (p<0.001, linear regression). Sensitivity of double staining tested in logarithmic tumor cell dilutions was one CK18-positive cell in 300,000. Specific uPA-R staining was seen on CK18-positive cells in bone marrow from 29 of 61 patients, and also on single surrounding bone marrow cells. To test the specificity of this staining, bone marrow cytospins from 10 patients without tumor disease were stained for uPA-R with the APAAP method. uPA-R expression was confirmed in all 10 cases, with a mean of 6.5 uPA-R-positive cells in 1000 bone marrow cells (SEM 1.2). These results suggest that our double staining protocol is a sensitive, reproducible, and specific method for routine uPA-R phenotyping of disseminated CK18-positive cells in bone marrow of carcinoma patients

Prognostic relevance of MMP-2 (72-kD collagenase IV) in gastric cancer

The association of MMP-2 (matrix metalloproteinase 2, 72-kD collagenase IV) with invasive and metastatic capacity of tumor cells has implicated a potential role in the prognosis for cancer patients. However, no larger study has been done to prove this hypothesis. The present study was therefore designed to investigate the prognostic impact of MMP-2 in a prospective series of 203 gastric cancer patients. MMP-2 expression was measured immunohistochemically and scored semiquantitatively (score 0-3) in carcinoma cells, and results were correlated with clinicopathological tumor parameters and parameters of the urokinase-type plasminogen activator (uPA) system. Survival analyses were done using the Kaplan-Meier method (log-rank statistics) and multivariate Cox analysis. Significant correlations were found for MMP-2 and Lauren's classification, M stage and proteases/inhibitors of the uPA system in the primary tumor. Kaplan-Meier analysis revealed an association of increasing MMP-2 expression with worse prognosis. This was especially seen in patients with a parallel high expression of uPA receptor. However, differences in survival probabilities between low and high MMP-2 levels were not significant. In a separate analysis of diffuse-type cancers, MMP-2 was significantly associated with disease-free (p = 0.0056) and overall survival (p = 0.0426). Multivariately, MMP-2 was not an independent parameter. Our results demonstrate that there is an association of immunohistochemical detection of MMP-2 with prognosis of cancer patients. For diffuse gastric cancers, it is a significant prognostic parameter, however, not of independent impact. The study further suggests that consideration of interrelated tumor-associated proteases like uPA receptor in combination with MMP-2 may improve its prognostic power

Immunotherapy of Peritoneal Carcinomatosis with the Antibody Catumaxomab in Colon, Gastric, or Pancreatic Cancer: An Open-Label, Multicenter, Phase I/II Trial

Background: Peritoneal carcinomatosis (PC) is common in gastrointestinal (GI) cancer and there is no effective standard treatment. We investigated the tolerability and maximum tolerated dose (MTD) of the trifunctional antibody catumaxomab in patients with PC. Methods: In this open-label, phase I/II clinical trial, patients with epithelial cell adhesion molecule (EpCAM)-positive PC from GI cancer received 4 sequential intraperitoneal catumaxomab infusions: day 0: 10 mu g; day 3: 10 or 20 mu g; day 7: 30, 50, or 100 mu g; and day 10: 50, 100, or 200 mu g. Dose escalation was guided by dose-limiting toxicities. Results: The MTD was 10, 20, 50, and 200 mu g on days 0, 3, 7, and 10, respectively. Catumaxomab had an acceptable safety profile: Most common treatment-related adverse events (at the MTD) were fever, vomiting, and abdominal pain. At final examination, 11/17 evaluable patients (65%) were progression free: 1 patient had a complete and 3 a partial response. Median overall survival from the time of diagnosis of PC was 502 days. Conclusions: Intraperitoneal catumaxomab is a promising option for the treatment of PC from GI cancer

Synthesis and Structure Elucidation of Glutamyl-Queuosine

Queuosine is one of the most complex hypermodified RNA nucleosides found in the Wobble position of tRNAs. In addition to Queuosine itself, several further modified derivatives are known, where the cyclopentene ring structure is additionally modified by a galactosyl-, a mannosyl-, or a glutamyl-residue. While sugar-modified Queuosine derivatives are found in the tRNAs of vertebrates, glutamylated Queuosine (gluQ) is only known in bacteria. The exact structure of gluQ, particularly with respect to how and where the glutamyl side chain is connected to the Queuosine cyclopentene side chain, is unknown. Here we report the first synthesis of gluQ and, using UHPLC-MS-coinjection and NMR studies, we show that the isolated natural gluQ is the α-allyl-connected gluQ compound

Synthesis of Galactosyl‐Queuosine and Distribution of Hypermodified Q‐Nucleosides in Mouse Tissues

Queuosine (Q) is a hypermodified RNA nucleoside that is found in tRNAHis, tRNAAsn, tRNATyr, and tRNAAsp. It is located at the wobble position of the tRNA anticodon loop, where it can interact with U as well as C bases located at the respective position of the corresponding mRNA codons. In tRNATyr and tRNAAsp of higher eukaryotes, including humans, the Q base is for yet unknown reasons further modified by the addition of a galactose and a mannose sugar, respectively. The reason for this additional modification, and how the sugar modification is orchestrated with Q formation and insertion, is unknown. Here, we report a total synthesis of the hypermodified nucleoside galactosyl‐queuosine (galQ). The availability of the compound enabled us to study the absolute levels of the Q‐family nucleosides in six different organs of newborn and adult mice, and also in human cytosolic tRNA. Our synthesis now paves the way to a more detailed analysis of the biological function of the Q‐nucleoside family

Preemptive endoluminal vacuum therapy with the VACStent—A pilot study to reduce anastomotic leakage after Ivor Lewis hybrid esophagectomy

IntroductionEndoscopic treatment by vacuum therapy (EVT) or covered stents has emerged as an improved treatment option for upper gastrointestinal wall defects and is regarded as an improved treatment option for anastomotic leakage (AL) after esophagectomy. However, endoluminal EVT devices may lead to obstruction of the GI tract; and a high rate of migration and missing functional drainage has been shown for covered stents. The recently developed VACStent, a combination of a fully covered stent within a polyurethane sponge cylinder may overcome these issues allowing EVT while stent passage is still open. Initial clinical applications have demonstrated efficacy, practicability and safety in the treatment of esophageal leaks (AL).MethodsIn this pilot study, 9 patients with high-risk anastomosis after neoadjuvant therapy undergoing hybrid esophagectomy received the VACStent in a preemptive setting for the assessment of the reduction of the AL rate, postoperative morbidity and mortality.ResultsTechnical success of the application of the VACStent® was achieved in all interventions. One patient experienced anastomotic leakage 10 days after esophagectomy and was successfully treated with two consecutive VACStents and a VAC Sponge. In summary, mortality in-hospital was 0% and anastomotic healing was uneventful without septic episodes. No severe device-related adverse events (SADE) nor significant local bleeding or erosion could be observed. Oral intake of liquids or food was documented in all patients. The device handling was regarded uncomplicated.DiscussionThe preemptive application of the VACStent offers a promising new option for improved clinical treatment avoiding of critical situations in hybrid esophagectomy, which should be validated in a large clinical study

KI-basierte Erstellung individualisierter Mathematikaufgaben für MINT-Fächer

Die Tendenz zum selbstgesteuerten Lernen an Hochschulen verstärkt sich durch die COVID-19-Pandemie. Allerdings weisen Studierende insbesondere in der Studieneingangsphase teils erhebliche Defizite im Hinblick auf Selbstlernkompetenzen auf. Daher wird in der Fachdidaktik Mathematik versucht, Wissenslücken ab der ersten Studienwoche unter anderem durch Feedback in Kleingruppen oder durch Korrekturen von Übungsaufgaben zu verhindern. Die trotzdem hohe Zahl an Studienabbrecher:innen in MINT-Fächern steht dem wachsenden Bedarf der Wirtschaft an qualifizierten Absolvent:innen gegenüber. Wir schlagen vor, diese Abbruchquote vermittels Methodiken des maschinellen Lernens zu adressieren: KI-Algorithmen erstellen Mathematikaufgaben mit passgenauem, individuellem Schwierigkeitsgrad für Studierende. Dies wirkt – so unsere Hypothese – Überforderungen entgegen und steigert die individuelle Studienmotivation. Sowohl der Aufgabentyp als auch der Schwierigkeitsgrad der Übungen hängen von vielen Parametern ab. Diese werden iterativ, beruhend auf End- oder Zwischenergebnissen zuvor bearbeiteter Aufgaben und Learning-Analytics-Daten, durch Bayes’sche Optimierung angepasst. (DIPF/Orig.)The COVID-19 pandemic has reinforced the tendency toward self-directed learning in higher education. During the first semesters, however, many students still lack the self-learning skills they need to thrive in this environment. For this reason, attempts are made in mathematics teaching to prevent gaps in studentsʼ knowledge by providing feedback in small groups or by correcting their homework. Still, dropout rates in STEM subjects remain high, strongly contrasting with the economy’s growing demand for qualified graduates from these academic fields. We therefore propose to address this issue through an instructional design based on AI algorithms that create mathematical exercises with a degree of difficulty tailored individually to students’ knowledge and skills. We hypothesise that this intervention will counteract self-assessed feelings of stress and overload while working on these exercises, instead increasing learners’ motivation to study. As both the type of exercise and its difficulty depend on many parameters, they are adjusted iteratively by the AI, based on final or preliminary results of previously processed tasks and Learning Analytics data through Bayesian optimisation. (DIPF/Orig.

Dual-Energy Lung Perfusion in Portal Venous Phase CT—A Comparison with the Pulmonary Arterial Phase

Pulmonary arterial dual-energy (aDE) CT is an established technique for evaluating pulmonary perfusion blood volume (PBV). As DECT protocols are increasingly used for thoraco-abdominal CT, this study assessed image quality and clinical findings in portal–venous phase dual-energy (vDE) CT and compared it to aDE. In 95 patients, vDE-CT was performed using a dual-source scanner (70/Sn150 kV, 560/140 ref.mAs). Pulmonary triggered aDE-CT served as reference (n = 94). PBV was reconstructed using a dedicated algorithm. Mean relative attenuation was measured in the pulmonary trunk, aorta, and segmented lung parenchyma. A distribution ratio (DL) between vessels and parenchyma was calculated to assess the iodine uptake of the lung parenchyma. Subjective overall diagnostic image quality was assessed for PBV images on a five-point Likert scale. Image artifacts were classified into five groups based on scale rating and compared between vDE and aDE. Pathological findings were correlated with the anatomical image datasets. Mean relative attenuation of the lung parenchyma was comparable in both groups (vDE: 23 ± 6 HU and aDE: 22 ± 7 HU), but significantly lower in the vessels of vDE. Therefore, iodine uptake of the lung parenchyma was significantly higher in vDE (DL: 10% vs. 8%, p 86%, both p > 0.6). Dual-source artifacts were found in only a few patients in both groups (vDE 5%, aDE 7%, p = 0.5). Recess and subpleural artifacts were increased in vDE (vDE 53/27%, aDE 24/7%, both p < 0.001). Pathological findings were found in 19% of the vDE patients and 59% of the aDE patients. Comparable objective and subjective image quality of lung perfusion can be obtained in vDE and aDE. Iodine uptake of the lung parenchyma is increased in vDE compared to aDE, suggesting an interstitial pooling effect. Knowledge of the different appearances of artifacts will aid in the interpretation of the images. Additional clinical information about the lung parenchyma can be provided by PBV evaluation in vDE