[Trans-Sectoral Patient Care in Pediatric Oncology: Quality Assurance by Novel Reimbursement Models for Outpatient Services]. / Sektorenübergreifende Versorgung in der Pädiatrischen Onkologie: Qualitätssicherung durch neue Vergütungsmodelle für nicht-vollstationäre Leistungen.

BACKGROUND: The medical care of pediatric cancer patients in the German health care system relies on special structures. All children and adolescents with a diagnosis of cancer receive uniform treatment within clinical studies or registers and exclusively at centers which can ensure interdisciplinary care by a multiprofessional team. Reimbursement of outpatient services is highly heterogeneous among the centers, and the expenses are often not adequately compensated. METHOD: A nation-wide survey was performed among all centers of the German Society of Pediatric Hematology and Oncology, with a standardized questionnaire inquiring which reimbursement models are used to finance outpatient treatment and whether full coverage of the expenses is achieved. RESULTS: Of 58 Pediatric Oncology Centers in Germany 18 (33%) participated in the survey, including 8 (44%) University Hospitals. The use of available reimbursement tools was highly heterogeneous. Reimbursement for outpatient service was based on a mean of 3,33±1,49 individual components. Of the 18 responding centers, 17 indicated that the revenues do not fully cover the expenses. DISCUSSION AND CONCLUSION: Pediatric oncology centers in Germany can not achieve full coverage of expenses in the outpatient setting. Nationally uniform cost-covering remuneration strategies are needed. This article proposes three individual models for an adequate nationwide financial framework for the outpatient care of pediatric cancer patients.

Correction: Complex Epigenetic Regulation of Chemotherapy Resistance and Biology in Esophageal Squamous Cell Carcinoma via MicroRNAs. Int. J. Mol. Sci. 2018, 19, 499.

We would like to submit a correction to the published paper [...].

Complex Epigenetic Regulation of Chemotherapy Resistance and Biohlogy in Esophageal Squamous Cell Carcinoma via MicroRNAs.

BACKGROUND: Resistance towards chemotherapy is a major obstacle in the treatment of esophageal squamous cell carcinoma (ESCC). We investigated the role of specific microRNAs in chemotherapy resistance and tumor biology. METHODS: We selected three microRNAs from characteristic microRNA signatures of resistant ESCC (hsa-miR-125a-5p, hsa-miR-130a-3p, hsa-miR-1226-3p), and hsa-miR-148a-3p. Effects on chemotherapy, adhesion, migration, apoptosis and cell cycle were assessed in six ESCC cell lines. Target analyses were performed using Western blotting and luciferase techniques. RESULTS: MiR-130a-3p sensitized cells towards cisplatin in 100% of cell lines, miR-148a-3p in 83%, miR-125a-5p in 67%, miR-1226-3p in 50% (p ≤ 0.04). MiR-130a-3p sensitized 83% of cell lines towards 5-FU, miR-148a-3p/miR-125a-5p/miR-1226-3p only 33% (p ≤ 0.015). Several resistance-relevant pathways seem to be targeted on various levels. Bcl-2 was confirmed as a direct target of miR-130a-3p and miR-148a-3p, and p53 as a target of miR-125a-5p. All microRNAs decreased migration and adhesion, except miR-130a-3p, and increased apoptosis. Simultaneous manipulation of two microRNAs exhibited additive sensitizing effects towards cisplatin in 50% (miR-125a-5p/miR-148a-3p), and 75% (miR-148a-3p/miR-130a-3p) of cell lines (p ≤ 0.016) [corrected] CONCLUSION: Our data present strong evidence that specific microRNA signatures are responsible for drug resistance and aggressiveness of ESCC. Final functional readout of these complex processes appears to be more important than single microRNA-target interactions.

miR clusters target cellular functional complexes by defining their degree of regulatory freedom.

Using the two paralog miR-23∼27∼24 clusters as an example and combining experimental and clinical data in a systematical approach to microRNA (miR) function and dysregulation, a complex picture of their roles in cancer is drawn. Various findings appear to be contradictory to a larger extent and cannot be fully explained by the classical regulatory network models and feedback loops that are mainly considered by one-to-one regulatory interactions of the involved molecules. Here, we propose an extended model of the regulatory role of miRs that, at least, supplements the usually considered single/oligo-target regulation of certain miRs. The cellular availability of the participating miR members in this model reflects an upper hierarchy level of intracellular and extracellular environmental influences, such as neighboring cells, soluble factors, hypoxia, chemotherapeutic drugs, and irradiation, among others. The novel model is based on the understanding of cellular functional complexes, such as for apoptosis, migration, and proliferation. These complexes consist of many regulatory components that can be targeted by miR cluster members to a different extent but may affect the functional complex in different ways. We propose that the final miR-related effect is a result of the possible degree of regulatory freedom provided by the miR effects on the whole functional complex structure. This degree of regulatory freedom defines to which extent the cellular functional complex can react in response to regulatory triggers, also understood as sensitization (more regulatory response options) or de-sensitization (less regulatory response options) of the system rather than single molecules.

Esophageal Cancer Specific Risk Score Is Associated with Postoperative Complications Following Open Ivor-Lewis Esophagectomy for Adenocarcinoma.

BACKGROUND/AIMS: Surgery for esophageal cancer is associated with a high morbidity and mortality. With this study, we investigated if a validated preoperative risk score correlates with overall morbidity, mortality, anastomotic insufficiency, respiratory complications and with the severity of complications after open Ivor-Lewis esophagectomy. METHODS: A total of 94 patients undergoing esophageal resection for adenocarcinoma between 2005 and 2009 were included. Patients were assigned using the preoperative risk score according to Schröder et al. [Langenbecks Arch Surg 2006;391:455-460] and the Dindo classification regarding the severity of complications. RESULTS: Of all the patients, 12% had a 'normal', 54% a 'moderate' and 34% a 'high' preoperative risk score. Postoperative complications occurred in 79%. Furthermore, 36 or 21 or 14 or 7% of patients experienced complications of category I/II or III or IV or V, respectively. There was a significant association between preoperative risk score and overall morbidity (p = 0.010), mortality (p = 0.035) and anastomotic insufficiency (p = 0.023). Furthermore, higher preoperative risk score was significant related to increasing severity of postoperative complications (grade IV according to the Dindo classification: p = 0.018, Dindo grade V: p = 0.035). Neoadjuvant therapy consisting of cisplatin and 5-fluorouracil had no influence. CONCLUSION: As we demonstrated, a significant association between preoperative risk score and occurrence and severity of postoperative complications after open Ivor-Lewis esophagectomy, standardized, organ-specific pre- and postoperative categorizations might be useful for individual clinical decision making in this group of patients.

Association of RHAMM with E2F1 promotes tumour cell extravasation by transcriptional up-regulation of fibronectin.

Dissemination of cancer cells from primary to distant sites is a complex process; little is known about the genesis of metastatic changes during disease development. Here we show that the metastatic potential of E2F1-dependent circulating tumour cells (CTCs) relies on a novel function of the hyaluronan-mediated motility receptor RHAMM. E2F1 directly up-regulates RHAMM, which in turn acts as a co-activator of E2F1 to stimulate expression of the extracellular matrix protein fibronectin. Enhanced fibronectin secretion links E2F1/RHAMM transcriptional activity to integrin-ß1-FAK signalling associated with cytoskeletal remodelling and enhanced tumour cell motility. RHAMM depletion abolishes fibronectin expression and cell transmigration across the endothelial layer in E2F1-activated cells. In a xenograft model, knock-down of E2F1 or RHAMM in metastatic cells protects the liver parenchyma of mice against extravasation of CTCs, whereas the number of transmigrated cells increases in response to E2F1 induction. Expression data from clinical tissue samples reveals high E2F1 and RHAMM levels that closely correlate with malignant progression. These findings suggest a requirement for RHAMM in late-stage metastasis by a mechanism involving cooperative stimulation of fibronectin, with a resultant tumourigenic microenvironment important for enhanced extravasation and distant organ colonization. Therefore, stimulation of the E2F1-RHAMM axis in aggressive cancer cells is of high clinical significance. Targeting RHAMM may represent a promising approach to avoid E2F1-mediated metastatic dissemination.

MALDI mass spectrometry imaging of bioactive lipids in mouse brain with a Synapt G2-S mass spectrometer operated at elevated pressure: improving the analytical sensitivity and the lateral resolution to ten micrometers.

Mass spectrometers from the Synapt-G1/G2 family (Waters) are widely employed for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). A lateral resolution of about 50 µm is typically achieved with these instruments, that is, however, below the often desired cellular resolution. Here, we show the first MALDI-MSI examples demonstrating a lateral resolution of about ten micrometers obtained with a Synapt G2-S HDMS mass spectrometer without oversampling. This improvement became possible by laser beam shaping using a 4:1 beam expander and a circular aperture for spatial mode filtering and by replacement of the default focusing lens. We used dithranol as an effective matrix for imaging of acidic lipids such as sulfatides, gangliosides, and phosphatidylinositols in the negative ion mode. At the same time, the matrix enables MS imaging of more basic lipids in the positive ion mode. Uniform matrix coatings with crystals having average dimensions between 0.5 and 3 µm were obtained upon spraying a chloroform/methanol matrix solution. Increasing the cooling gas pressure in the MALDI ion source after adding an additional gas line was furthermore found to increase the ion abundances of labile lipids such as gangliosides. The combined characteristics are demonstrated with the MALDI-MSI analysis of fine structures in coronal mouse brain slices.

The microRNA-200 family--a potential diagnostic marker in hepatocellular carcinoma?

BACKGROUND: Hepatocellular carcinoma (HCC) represents the main cause of death among patients with cirrhotic liver disease, but little is known about mechanisms of cirrhosis associated carcinogenesis. We investigated the diagnostic impact of microRNA-200 (miR-200) family members as important epigenetic regulators of epithelial-mesenchymal transition (EMT) to differentiate between patients with HCC and liver cirrhosis. METHODS: Expression of the miR-200 family was investigated by qRT-PCR in specimens of HCC patients with and without cirrhosis. Benign specimens with and without cirrhosis served as controls. Expression of the EMT markers ZEB-1, E-cadherin and vimentin was examined using immunohistochemistry. RESULTS: MiR-200a and miR-200b were significantly downregulated in HCC (miR-200a: -40.1% (P = 0.0002); miR-200b: -52.3% (P = 0.0002)), and in HCC cirrhotic tissue (miR-200a: -40.2% (P = 0.004); miR-200b: -51.1% (P = 0.007)) compared to liver cirrhosis. Spearman's Rho analysis revealed a significant negative correlation of miR-200a and miR-200b to the expression of the mesenchymal markers Vimentin (P < 0.007) and ZEB-1 (P < 0.0005) and a significant positive correlation to the epithelial marker E-cadherin (P < 0.0002). CONCLUSIONS: MiR-200 family members and their targets are significantly deregulated in HCC and liver cirrhosis. The miR-200 family is able to distinguish between cirrhotic and HCC tissue and could serve as an early marker for cirrhosis-associated HCC.

The need for harmonized structured documentation and chances of secondary use - results of a systematic analysis with automated form comparison for prostate and breast cancer.

INTRODUCTION: Medical documentation is a time-consuming task and there is a growing number of documentation requirements. In order to improve documentation, harmonization and standardization based on existing forms and medical concepts are needed. Systematic analysis of forms can contribute to standardization building upon new methods for automated comparison of forms. Objectives of this research are quantification and comparison of data elements for breast and prostate cancer to discover similarities, differences and reuse potential between documentation sets. In addition, common data elements for each entity should be identified by automated comparison of forms. MATERIALS AND METHODS: A collection of 57 forms regarding prostate and breast cancer from quality management, registries, clinical documentation of two university hospitals (Erlangen, Münster), research datasets, certification requirements and trial documentation were transformed into the Operational Data Model (ODM). These ODM-files were semantically enriched with concept codes and analyzed with the compareODM algorithm. Comparison results were aggregated and lists of common concepts were generated. Grid images, dendrograms and spider charts were used for illustration. RESULTS: Overall, 1008 data elements for prostate cancer and 1232 data elements for breast cancer were analyzed. Average routine documentation consists of 390 data elements per disease entity and site. Comparisons of forms identified up to 20 comparable data elements in cancer conference forms from both hospitals. Urology forms contain up to 53 comparable data elements with quality management and up to 21 with registry forms. Urology documentation of both hospitals contains up to 34 comparable items with international common data elements. Clinical documentation sets share up to 24 comparable data elements with trial documentation. Within clinical documentation administrative items are most common comparable items. Selected common medical concepts are contained in up to 16 forms. DISCUSSION: The amount of documentation for cancer patients is enormous. There is an urgent need for standardized structured single source documentation. Semantic annotation is time-consuming, but enables automated comparison between different form types, hospital sites and even languages. This approach can help to identify common data elements in medical documentation. Standardization of forms and building up forms on the basis of coding systems is desirable. Several comparable data elements within the analyzed forms demonstrate the harmonization potential, which would enable better data reuse. CONCLUSION: Identifying common data elements in medical forms from different settings with systematic and automated form comparison is feasible.

Adherence to interdisciplinary tumor board recommendations as an expression of quality-assured patient care: results of a bicentric German analysis.

PURPOSE: Interdisciplinary tumor boards (ITBs) represent a central part of standard cancer care defining a guidelines-guided treatment plan adapted to the patient's capabilities, comorbidities and wishes in a multi-professional team. The implementation rate of ITB recommendations can be monitored by structured adherence analyses. But (inter)national definitions how to measure the level of implementation are missing. Here, we present results of 4 years of ITB adherence analyses in a bicentric German Comprehensive Cancer Center (CCC). METHODS: Between 2018 and 2021, for at least 1 month, the implementation rate of recommendations of 8 different ITBs of 2 CCC sites was evaluated manually according to harmonized criteria between both sites regarding the degree of implementation of ITB's recommendations. RESULTS: In total, 1104 cases were analyzed (65% male, 35% female). Mean distance from patient's home to the CCC was 57 km (range 0.8-560.6 km). For 949 cases (86%) with known follow-up, the adherence rate was 91.9% (95% CI 0.9; 0.935). In 8.1%, ITB decisions were not implemented due to medical reasons (45.4%), patient's wish (35.1%) and unknown reasons (19.5%). Logistic regression revealed neither age (OR = 0.998, p = 0.90), nor gender (OR = 0.98, p = 0.92) or the distance from patient's home to the CCC (OR = 1.001, p = 0.54) were significantly associated with ITB adherence. CONCLUSION: ITB adherences analyses can serve as a quality management tool to monitor the implementation rate of ITB recommendations and to stay in contact with practitioners, other hospitals and state cancer registries to share data and resources in accordance with data protection requirements for continuously improvement of quality management and patient care.

Shiga toxin glycosphingolipid receptor expression and toxin susceptibility of human pancreatic ductal adenocarcinomas of differing origin and differentiation.

Shiga toxins (Stxs) are composed of an enzymatically active A subunit (StxA) and a pentameric B subunit (StxB) that preferentially binds to the glycosphingolipid (GSL) globo\xadtriaosylceramide (Gb3Cer/CD77) and to a reduced extent to globotetraosylceramide (Gb4Cer). The identification of Gb3Cer as a tumor-associated GSL in human pancreatic cancer prompted us to investigate the expression of Gb3Cer and Gb4Cer in 15 human pancreatic ductal adenocarcinoma cell lines derived from primary tumors and liver, ascites, and lymph node metastases. Thin-layer chromatography overlay assays revealed the occurrence of Gb3Cer in all and of Gb4Cer in the majority of cell lines, which largely correlated with transcriptional expression analysis of Gb3Cer and Gb4Cer synthases. Prominent Gb3Cer and Gb4Cer lipoform heterogeneity was based on ceramides carrying predominantly C16:0 and C24:0/C24:1 fatty acids. Stx2-mediated cell injury ranged from extremely high sensitivity (CD(50) of 0.94 pg/ml) to high refractiveness (CD(50) of 5.8 µg/ml) and to virtual resistance portrayed by non-determinable CD(50) values even at the highest Stx2 concentration (10 µg/ml) applied. Importantly, Stx2-mediated cytotoxicity did not correlate with Gb3Cer expression (the preferential Stx receptor), suggesting that the GSL receptor content does not primarily determine cell sensitivity and that other, yet to be delineated, cellular factors might influence the responsiveness of cancer cells.

Trust in Healthcare during COVID-19 in Europe: vulnerable groups trust the least.

Aim: We examined predictors of trust in the healthcare system during the COVID-19 pandemic in 27 European countries. Subjects and methods: We used population-based data drawn from the Living, working and COVID-19 survey (N = 21,884, 52% female, ages 18 to 92 years) covering 27 European countries dated June and July 2020. Multilevel linear regression, linear regression, and regression-tree analyses were conducted. Results: We found that most participants tended to trust the healthcare system, although a substantial part could still be classified as distrusting (approx. 21%). Multiple variables, including being middle-aged or of older age, being female, lower levels of education, unemployment, worse general health status, having income difficulties, having unmet needs for healthcare, no healthcare contact during the COVID-19 pandemic, higher mental distress, and loneliness, were significantly associated with lower levels of trust. Among these variables mental distress, income difficulties, and unmet needs for healthcare emerged as especially important and, across European regions and countries, consistent predictors for lower trust in the healthcare system during the COVID-19 pandemic. Conclusions: Medically vulnerable subgroups, such as individuals with unmet healthcare needs, higher levels of mental distress, and older age, as well as people living in socially and economically vulnerable situations, such as higher levels of loneliness and financial difficulties, were the least trusting of the healthcare system during the COVID-19 pandemic. As these vulnerable subgroups are also at highest risk for contracting COVID-19 and experiencing negative COVID-19-related outcomes, more targeted prevention and intervention efforts should be implemented in these groups. Supplementary Information: The online version contains supplementary material available at 10.1007/s10389-022-01705-3.

Decision Conflicts in Clinical Care during COVID-19: A Patient Perspective.

(1) Background: Uncertainty is typical for a pandemic or similar healthcare crisis. This affects patients with resulting decisional conflicts and disturbed shared decision making during their treatment occurring to a very different extent. Sociodemographic factors and the individual perception of pandemic-related problems likely determine this decisional dilemma for patients and can characterize vulnerable groups with special susceptibility for decisional problems and related consequences. (2) Methods: Cross-sectional data from the OnCoVID questionnaire study were used involving 540 patients from 11 participating institutions covering all major regions in Germany. Participants were actively involved in clinical treatment in oncology or psychiatry during the COVID-19 pandemic. Questionnaires covered five decision dimensions (conflicts and uncertainty, resources, risk perception, perception of consequences for clinical processes, perception of consequences for patients) and very basic demographic data (age, gender, stage of treatment and educational background). Decision uncertainties and distress were operationalized using equidistant five-point scales. Data analysis was performed using descriptive and various multivariate approaches. (3) Results: A total of 11.5% of all patients described intensive uncertainty in their clinical decisions that was significantly correlated with anxiety, depression, loneliness and stress. Younger and female patients and those of higher educational status and treatment stage had the highest values for these stressors (p < 0.001). Only 15.3% of the patients (14.9% oncology, 16.2% psychiatry; p = 0.021) considered the additional risk of COVID-19 infections as very important for their disease-related decisions. Regression analysis identified determinants for patients at risk of a decisional dilemma, including information availability, educational level, age group and requirement of treatment decision making. (4) Conclusions: In patients, the COVID-19 pandemic induced specific decisional uncertainty and distress accompanied by intensified stress and psychological disturbances. Determinants of specific vulnerability were related to female sex, younger age, education level, disease stages and perception of pandemic-related treatment modifications, whereas availability of sufficient pandemic-related information prevented these problems. The most important decisional criteria for patients under these conditions were expected side effects/complications and treatment responses.

Differences in CD75s- and iso-CD75s-ganglioside content and altered mRNA expression of sialyltransferases ST6GAL1 and ST3GAL6 in human hepatocellular carcinomas and nontumoral liver tissues.

The sialic acid-specific cytotoxic lectin viscumin and its recombinant equivalent rViscumin specifically bind to CD75s-gangliosides with terminal Neu5Acα6Galß4GlcNAc sequence. We, therefore, comparatively analyzed the content of CD75s-gangliosides and closely related iso-CD75s-gangliosides (terminated by Neu5Acα3Galß4GlcNAc sequence) and the gene expression of associated ß-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) and ß-galactoside α-2,3-sialyltransferase 6 (ST3GAL6), respectively, in 35 hepatocellular carcinoma (HCC) patients. Ganglioside structures were identified in lipid extracts of matched pairs of malignant and nonmalignant liver tissues by thin-layer chromatography immunodetection coupled with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry. CD75s- and iso-CD75s-gangliosides were found to be deregulated in tumor tissues and showed an elevated occurrence in 35 and 41% of HCCs, respectively, compared with nontumoral liver tissues. Statistical analysis revealed a correlation between enhanced iso-CD75s-ganglioside amount and a poor histopathological differentiation (τ = 0.317, P = 0.045) and a significant association of CD75s- and iso-CD75s-ganglioside levels in nontumorous (τ = 0.392, P = 0.003) and in tumorous tissues (τ = 0.650, P < 0.001). Quantitative real-time polymerase chain reaction gene expression analysis of sialyltransferases exhibited no difference in ST6GAL1 expression in cancerous and adjacent noncancerous tissues. Interestingly, the ST3GAL6 expression was significantly diminished in HCCs (P = 0.003). The results indicate that the occurrence of CD75s- and iso-CD75s-gangliosides in tumor tissues is largely independent of the transcriptional expression of ST6GAL1 and ST3GAL6, respectively. Thus, further experiments are required to explore the rationale behind the differential ganglioside level and to validate the applicability of CD75s- and iso-CD75s-gangliosides as targets for individual HCC therapies.

[Psychooncological interventions - what do cancer patients aged 60 years or older wish for?]. / Psychoonkologische Angebote - was wunschen uber 60-jahrige Krebspatienten?

OBJECTIVES: To determine the acceptance of psychooncological interventions and predictors of subjective needs in cancer patients over 60 years of age. METHODS: We examined 292 in- and outpatients (51% female, 51.7% aged over 60 years) from the Interdisciplinary Cancer Center of the University Hospital of Münster with a questionnaire designed to assess their wish to participate in various psychooncological interventions (FIPA) and with the Hospital Anxiety and Depression Scale (HADS-D). RESULTS: 73.5 % of those over 60 years and 87.9 % of those under 60 years professed a wish for at least one specific psychooncological intervention. Higher age, lower levels of anxiety and cancer relapse were negative predictors of the readiness to attend psychooncological interventions (9 % variance explained). As to specific interventions, those aged 60 years or older showed lower acceptance only for relaxation techniques. CONCLUSIONS: Readiness to attend psychooncological interventions was high with only marginal differences between the age groups. Because objective features of the patient and the disease resulted only in a low predictability of the subjective need for psychooncological interventions, routine screening of cancer patients seems an important issue.

Patterns of Changes in Oncological Care due to COVID-19: Results of a Survey of Oncological Nurses and Physicians from the Region of Hanover, Germany.

BACKGROUND: Healthcare staff is confronted with intensive decisional conflicts during the pandemic. Due to the specific burden of this moral distress in oncology, the investigation aimed at quantification of these conflicts and identification of risk factors that determine the extent and severity of these conflicts. We examined the heterogeneity of changes in oncology care due to COVID-19. METHODS: We conducted a survey of oncological physicians and nurses in the region of Hanover, Germany in the second half of 2020. Overall, N = 200 respondents, 54% nurses, were included in the sample. Indicators of changes in oncology care were used to determine profiles of changes. To characterize these profiles, a diverse set of variables, including decision conflicts, uncertainty, age, gender, work experience, changes in communication with patients, psychological distress, work stress, process organization, and personnel resources, was obtained. Latent class analysis was conducted to determine these latent profiles. RESULTS: We found that three distinct profiles best described the overall changes in oncology care due to COVID-19 in our sample, with each profile being associated with specific characteristics: (1) "Few Changes in Oncology Care" profile with 33% of participants belonging to this profile, (2) "Medium Changes in Oncology Care" profile with 43% of participants, and (3) "Severe Changes in Oncology Care" profile (24%). Participants from these profiles significantly differed regarding their age, work experience, occupational group, the prevalence of decision conflicts, decision uncertainty, quality of communication with patients, and quality of process organization. CONCLUSIONS: Distinct profiles of change in oncology care due to COVID-19 can be identified. Most participants reported small to medium changes, while some participants also reported severe changes. Profiles also differed regarding their associated characteristics. As such, specific consequences for better pandemic preparedness can be derived based on the current study. Future studies should investigate the patterns of changes in routine care due to COVID-19.

Structural profiling of individual glycosphingolipids in a single thin-layer chromatogram by multiple sequential immunodetection matched with Direct IR-MALDI-o-TOF mass spectrometry.

The thin-layer chromatography (TLC) immunoenzyme overlay assay is a widely used tool for antibody-mediated identification of glycosphingolipids (GSLs) in mixtures. However, because the majority of GSLs is left unexamined in a chromatogram of a single assay, we developed a novel method that permits detection of various GSLs by sequential multiple immunostaining combined with individual coloring of GSLs in the same chromatogram. Specific staining was achieved by means of primary anti-GSL antibodies, directed against lactosylceramide, globotriaosylceramide, and globotetraosylceramide, in conjunction with alkaline phosphatase (AP)- or horseradish peroxidase (HRP)-conjugated secondary antibodies together with the appropriate chromogenic substrates. Triple coloring with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-AP, Fast Red-AP, and 3,3'-diaminobenzidine (DAB)-HRP resulted in blue, red, and black precipitates, respectively, following three sequential immunostaining rounds. Structures of antibody-detected GSLs were determined by direct coupling of TLC with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry. This combinatorial technique was used to demonstrate structural GSL profiling of crude lipid extracts from human hepatocellular cancer. This powerful technology allows efficient structural characterization of GSLs in small tissue samples and marks a further step forward in the emerging field of glycosphingolipidomics.

Tumor-associated CD75s- and iso-CD75s-gangliosides are potential targets for adjuvant therapy in pancreatic cancer.

Pancreatic adenocarcinoma confers one of the highest mortality rates in malignant human tumors with very poor prognosis. Because as yet no treatments are available that produce a substantial survival benefit for this fatal neoplasia, new therapeutic concepts are urgently required to support cancer standard treatment. In search of tumor-associated gangliosides with therapeutic background, we probed a random collection of cancerous and adjacent normal postoperative tissue samples from 38 patients for the expression of CD75s- and iso-CD75s-gangliosides. We exhaustively analyzed the expression of CD75s-1-ganglioside (IV(6)Neu5Ac-nLc4Cer) and structurally closely related iso-CD75s-1-ganglioside (IV(3)Neu5Ac-nLc4Cer) by means of immunohistology of cryosections and semiquantitative TLC of tissue lipid extracts combined with mass spectrometry. CD75s-1- and iso-CD75s-1-ganglioside showed an elevated expression in 42% and 66% of the tumors, respectively, indicating a significant association with neoplastic transformation (P = 0.001). Thus, increased expression of CD75s-1- and iso-CD75s-1-gangliosides renders these cell surface molecules promising candidates for oncologic applications. Further statistical analysis revealed a significant enhancement of CD75s-1-ganglioside in the group of less differentiated tumors (grade >2) suggesting this ganglioside as a potential marker for poor differentiation. The CD75s-specific antitumor drug rViscumin, which represents the recombinant counterpart of the ribosome-inactivating lectin viscumin, has successfully passed clinical phase I trials and provides an opportunity for treating pancreatic cancer. Consequently, if an enhanced expression is existent in malignant tissues, we propose the targeting of CD75s-gangliosides with rViscumin as a novel potential strategy in adjuvant treatment of pancreatic malignancies.