Identifikation von Neoantigenen als Zielstruktur Tumor-infiltrierender Lymphozyten bei Patienten mit Triple Negativem oder HER2+ Brustkrebs

During the last decade cancer-immunotherapy has become an encouraging new therapeutic option for patients with different malignancies. Especially targeting neoantigens with adoptive T-cell transfer or cancer vaccines appears to be a promising approach. Those neoantigens, which arise in the process of tumorigenesis, are tumor specific and lack of central tolerance. Consequently, they can elicit high avidity T-cell responses. Immunotherapeutic approaches, like checkpoint inhibitors also showed durable responses in breast cancer as well, but only in a small proportion of patients. In particular for the more aggressive subtypes of breast cancer such as HER2+ and Triple Negative Breast Cancer (TNBC), recent studies have demonstrated a strong association between the number of tumor infiltrating lymphocytes (TILs) and response to neoadjuvant chemotherapy. The targets of these TILs offer potential therapeutic targets, but they are largely unknown for breast cancer. Consequently, it is the aim of this study is to identify those. To determine the composition of the lymphocytes in breast cancer, single cell suspensions of lump biopsies were generated and analyzed by flow cytometry. We could observe that there are significantly more CD4+ than CD8+ T-cells in those biopsies. Additionaly, T-cells from breast cancer biopsies at the point of diagnosis were unspecifically expanded for 60 patients with HER2+ and 51 patients with TNBC. Based on whole genome sequencing of tumor and autologous blood (as reference), non-synonymous mutations were identified. The RNA expression of the encoding genes was used to determine potential neoantigens. Finally, binding analysis to patient’s specific HLA molecules was performed and potential neoantigens were synthesized. Those peptides were loaded onto autologous antigen presenting cells (APCs) and cocultured with expanded TILs. After expansion of all IFN-γ + or CD137 + T-cells, those T-cells were tested for neoantigen-specificity on clonal level. Thereby, we were able to identify neoantigen-reactive T-cells in two of three patients. For the TNBC patient 050 we could analyze a polyclonal CD8+ T-cell reactivity against the HLA-A*11:01 restricted neoantigen PNMAL1 P100R. Moreover, we could observe a CD4+ T-cell response against the HLA-DQ A1*02:01, B1*02:02 restricted neoantigen CARS2 Q171H for the same patient. Furthermore, we identified a polyclonal reactivity of CD4+ T-cells in a HER2+ patient 010 against RBMX T55I. It should be emphasized that this neoantigen is presented upon both of the patient’s HLA-DP alleles 02:01 and 04:01. Furthermore, we could identify another RBMX T55I reactive T-cell clone expanded from the resected tumor tissue after chemotherapy. Peptide titration showed an effective recognition of all neoantigens in a concentration as low as 10 ng/ml. While there was no recognition of the wildetyp variant for RBMX and PNMAL1, the CARS2 reactive T-cell clone showed some recognition of the non-mutated variant at higher peptide concentrations. Effective processing and presenting of all three neoantigens after transduction of the full-length mutated protein in APCs was demonstrated. Additionaly, processing and presentation of the MHC-class I neoantigen PNMAL1 P100R in the breast cancer cell-line MCF-7 was seen after transduction of the respective protein and HLA-molecule. Furthermore, after transduction of RBMX T55I into MCF-7, an IFN-y treatment lead to MHC-class-II upregulation and consequently to the presentation of the antigen on endogenous HLA. This indicates that inflammation may lead to direct presentation of this neoantigens. Therefore, both PNMAL1 P100R and RBMX T55I represent direct targets on breast cancer cells. Independent of the IFN-y induced MHC-II upregulation the indirect presentation on APCs of both MHC-II antigens RBMX and CARS2 was demonstrated after lysation of the transduced MCF-7. We could prove the presence of neoantigen-specific TILs in two out of three patients with TNBC and HER2+ BC in this study. On the one hand the neoantigens identified here prove the possibility to find those with the method established here, and, on the other hand, directly offer potential target structures for the patients.Während des letzten Jahrzehntes hat sich die Tumor-Immuntherapie als erfolgversprechende therapeutische Option für Patienten mit verschiedenen Malignitäten entwickelt. Hierbei scheinen insbesondere Therapien, wie beispielsweise der adoptive T-Zelltransfer oder Tumorvakzine, gerichtet gegen Neoantigene vielversprechend. Neoantigene entstehen im Prozess der Tumorgenese und sind folglich tumorspezifisch, unterliegen keiner zentralen Toleranz und vermögen somit eine hoch-avide T-Zell-Antwort hervorzurufen. Auch im Brustkrebs zeigten immuntherapeutische Ansätze, wie Checkpoint-Inhibitoren teils langfristiges Ansprechen, allerdings nur bei einem geringen Anteil der Patienten. Insbesondere für die beiden aggressiveren Subgruppen HER2+ und Triple Negativer Brustkrebs (TNBC, triple negative breast cancer) liegt jedoch eine starke Evidenz für eine positive Korrelation der Anzahl an Tumor infiltrierender Lymphozyten (TILs, tumor infiltrating lymphocytes) zum Ansprechen nach neoadjuvanter Chemotherapie vor. Da die Erkennungsstrukturen dieser TILs in Brustkrebs bislang weitgehend unbekannt sind, aber potenzielle therapeutische Zielstrukturen bieten, ist es das Ziel dieser Arbeit solche zu identifizieren. Um einen Einblick in die Zusammensetzung der Lymphozyten in Brustkrebs zu erhalten, wurden Einzelzellsuspensionen der Stanzbiopsien generiert und durchflusszytometrisch analysiert. In diesen sind signifikant mehr CD4+ als CD8+ T-Zellen vorhanden. Zudem wurden T-Zellen aus Brustkrebsbiopsien zum Zeitpunkt der Diagnose für 60 Patienten mit HER2+ und 51 Patienten mit TNBC unspezifisch expandiert. Auf Grundlage der Ganzgenomsequenzierung von Tumor und autologem Blut (als Referenz) wurden nicht-synonyme Mutationen identifiziert, deren Expression mittels RNA-Sequenzierung verifiziert und anschließend mittels Bindungsprognose zu den patientenspezifischen HLA-Molekülen potenzielle Neoantigene bestimmt und schließlich synthetisiert. Die potenziellen Neoepitope wurden als synthetische Peptide auf autologe Antigenpräsentierende Zellen (APCs, Antigen presenting cells) geladen und mit den expandierten TILs co-kultiviert. Nach Expansion aller IFN-y+ oder CD137+ Zellen wurden diese anschließend auf klonaler Ebene auf Neoantigen-Spezifität getestet. Hierbei konnten wir bei zwei von drei Patientinnen Neoantigen-reaktive T-Zellen identifizieren. Bei der TNBC Patientin 050 lag eine polyklonale Reaktivität CD8+ T-Zellen gegen das HLA-A*11:01 restringierte Neoantigen PNMAL1 P100R vor. Zudem enthielten die TILs dieser Patientin auch CD4+ T-Zellen gegen das HLA-DQ A1*02:01, B1*02:02 restringierte Neoantigen CARS2 Q171H. Bei der HER2+ Patientin 010 konnte wiederrum eine polyklonale Reaktivität CD4+ T-Zellen gegen RBMX T55I beobachtet werden. Hervorzuheben ist hierbei, dass dieses Neoantigen über beide HLA-DP Allele 04:01 und 02:01 der Patientin präsentiert wird. Zudem konnte in dem entnommen Tumorgewebe nach Chemotherapie ein weiterer RBMX-reaktiver T-Zell-Klon identifiziert werden. Eine effektive Erkennung aller Neoantigene lag bereits in einer Konzentration von 10 ng/ml vor. Während für RBMX und PNMAL1 keine Erkennung der wildtyp Variante beobachtet wurde, zeigte der CARS2 reaktive T-Zell-Klon stets eine gewisse Erkennung der nicht-mutierten Variante, insbesondere bei hohen Peptidkonzentrationen. Erfolgreiche Prozessierung und Präsentation aller Neoantigene konnten wir nach Transduktion des gesamten mutierten Proteins auf APCs nachweisen. Zudem wurde auch das MHC-Klasse-I Neoantigen PNMAL1 P100R in der Brustkrebszelllinie MCF-7 nach Transduktion des entsprechenden Proteins und des respektiven HLA-Moleküls erfolgreich präsentiert und prozessiert. Ferner konnte das Neoantigen RBMX T55I nach IFN-y induzierter Hochregulierung der MHC-Klasse-II-Moleküle über endogenes HLA-DP auf RBMX transduzierten MCF-7 präsentiert werden. Somit weisen unsere Daten darauf hin, dass dieses Antigen im Rahmen einer Inflammation auf Brustkrebszellen prozessiert und präsentiert werden könnte und ebenso wie PNMAL1 P100R ein direktes Zielantigen auf Brustkrebszellen darstellt. Unabhängig von der IFN-γ induzierten MHC-Klasse-II Hochregulation konnte eine indirekte Präsentation der beiden MHC-Klasse-II Antigene RBMX und CARS2 gezeigt werden. Hierzu wurden Zelllysate der RBMX, bzw. CARS2 transduzierter MCF-7 Zellen auf autologen APCs geladen, was zu einer T-Zell-Erkennung gegen die Neoantigene führte. Mit dieser Studie konnten wir das Vorhandensein Neoantigen-Spezifischer TILs in zwei von drei Patienten mit HER2+ und TNBC nachweisen. Die hier identifizierten Neoantigene beweisen einerseits die Möglichkeit mit der hier etablierten Methode solche zu finden und bieten zudem direkt potenzielle Zielstrukturen für die Patientinnen

South Dakota Parents’ Knowledge of Congenital Cytomegalovirus, Its Long-Term Health Effects, and Methods for Minimizing Exposure

Congenital CMV (cCMV) is acknowledged as one of the most common causes of nonhereditary sensorineural hearing loss and an important cause of neurodevelopmental delay in children. Despite the danger cCMV poses, many parents are unaware of the virus, its sequelae, mode of transmission, and preventative behaviors. The purpose of the study was to determine South Dakota parents’ knowledge of cCMV, its sequelae, and ways to minimize exposure. An electronic survey was utilized for data collection. Parents of children born in South Dakota from 2011 to 2018 were asked about their knowledge of CMV and cCMV, including common sequelae and ways to minimize exposure. Flyers were sent to randomly selected daycares and the link was posted on social media pages to advertise the electronic survey to South Dakota parents. After completing the survey, participants were directed to cCMV educational resources. Respondents were more knowledgeable regarding the sequelae of cCMV rather than its transmission process or ways in which viral exposure can be minimized. Results show that there remains a need for cCMV awareness in South Dakota, particularly with a large focus on preventative measures

Expected values for gastrointestinal and pancreatic hormone concentrations in healthy volunteers in the fasting and postprandial state.

BACKGROUND: Gastrointestinal hormones regulate intestinal transit, control digestion, influence appetite and promote satiety. Altered production or action of gut hormones, including glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and peptide YY (PYY), may contribute to the biological basis of obesity and altered glucose homeostasis. However, challenges in analytical methodology and lack of clarity on expected values for healthy individuals have limited progress in this field. The aim of this study was to describe expected concentrations of gastrointestinal and pancreatic hormones in healthy volunteers following a standardized meal test (SMT) or 75 g oral glucose tolerance test (OGTT). METHODS: A total of 28 healthy volunteers (12 men, 16 women; mean age 31.3 years; mean body mass index 24.9 kg/m2) were recruited to attend a hospital clinic on two occasions. Volunteers had blood sampling in the fasting state and were given, in randomized order, an oral glucose tolerance test (OGTT) and standardized mixed liquid meal test with venepuncture at timed intervals for 4 h after ingestion. Analytical methods for gut and pancreatic hormones were assessed and optimized. Concentrations of gut and pancreatic hormones were measured and used to compile ranges of expected values. RESULTS: Ranges of expected values were created for glucose, insulin, glucagon, GLP-1, GIP, PYY and free fatty acids in response to a standardized mixed liquid meal or OGTT. Intact proinsulin and C-peptide levels were also measured following the OGTT. CONCLUSIONS: These ranges of expected values can now be used to compare gut hormone concentrations between healthy individuals and patient groups

The effect of encapsulated glutamine on gut peptide secretion in human volunteers.

CONTEXT: Weight loss and improved blood glucose control after bariatric surgery have been attributed in part to increased ileal nutrient delivery with enhanced release of glucagon-like peptide 1 (GLP-1). Non-surgical strategies to manage obesity are required. The aim of the current study was to assess whether encapsulated glutamine, targeted to the ileum, could increase GLP-1 secretion, improve glucose tolerance or reduce meal size. METHODS: A single-center, randomised, double blind, placebo-controlled, cross-over study was performed in 24 healthy volunteers and 8 patients with type 2 diabetes. Fasting participants received a single dose of encapsulated ileal-release glutamine (3.6 or 6.0 g) or placebo per visit with blood sampling at baseline and for 4h thereafter. Glucose tolerance and meal size were studied using a 75 g oral glucose tolerance test and ad libitum meal respectively. RESULTS: In healthy volunteers, ingestion of 6.0 g glutamine was associated with increased GLP-1 concentrations after 90 min compared with placebo (mean 10.6 pg/ml vs 6.9 pg/ml, p=0.004), increased insulin concentrations after 90 min (mean 70.9 vs 48.5, p=0.048), and increased meal size at 120 min (mean 542 g eaten vs 481 g, p=0.008). Ingestion of 6.0 g glutamine was not associated with significant differences in GLP-1, glucose or insulin concentrations after a glucose tolerance test in healthy or type 2 diabetic participants. CONCLUSIONS: Single oral dosing of encapsulated glutamine did not provoke consistent increases in GLP-1 and insulin secretion and was not associated with beneficial metabolic effects in healthy volunteers or patients with type 2 diabetes.This project was supported by a project grant from the European Union Seventh Framework Programme (FP7/2007-2013; grant agreement n° 266,408) as part of a larger collaboration called Full4Health. Claire Meek receives salary funding from the Wellcome Trust Translational Medicine and Therapeutics Programme which is funded by the Wellcome Trust in association with Glaxo SmithKline. FMG and FR were funded by the Wellcome Trust (WT088357/Z/09/Z and WT084210/Z/07/Z.)This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.peptides.2015.10.00

Expected values for gastrointestinal and pancreatic hormone concentrations in healthy volunteers in the fasting and postprandial state

Background: Gastrointestinal hormones regulate intestinal transit, control digestion, influence appetite and promote satiety. Altered production or action of gut hormones, including glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and peptide YY (PYY), may contribute to the biological basis of obesity and altered glucose homeostasis. However, challenges in analytical methodology and lack of clarity on expected values for healthy individuals have limited progress in this field. The aim of this study was to describe expected concentrations of gastrointestinal and pancreatic hormones in healthy volunteers following a standardized meal test (SMT) or 75 g oral glucose tolerance test (OGTT). Methods: A total of 28 healthy volunteers (12 men, 16 women; mean age 31.3 years; mean body mass index 24.9 kg/m2) were recruited to attend a hospital clinic on two occasions. Volunteers had blood sampling in the fasting state and were given, in randomized order, an oral glucose tolerance test (OGTT) and standardized mixed liquid meal test with venepuncture at timed intervals for 4 h after ingestion. Analytical methods for gut and pancreatic hormones were assessed and optimized. Concentrations of gut and pancreatic hormones were measured and used to compile ranges of expected values. Results: Ranges of expected values were created for glucose, insulin, glucagon, GLP-1, GIP, PYY and free fatty acids in response to a standardized mixed liquid meal or OGTT. Intact proinsulin and C-peptide levels were also measured following the OGTT. Conclusions: These ranges of expected values can now be used to compare gut hormone concentrations between healthy individuals and patient groups

Network Compression as a Quality Measure for Protein Interaction Networks

With the advent of large-scale protein interaction studies, there is much debate about data quality. Can different noise levels in the measurements be assessed by analyzing network structure? Because proteomic regulation is inherently co-operative, modular and redundant, it is inherently compressible when represented as a network. Here we propose that network compression can be used to compare false positive and false negative noise levels in protein interaction networks. We validate this hypothesis by first confirming the detrimental effect of false positives and false negatives. Second, we show that gold standard networks are more compressible. Third, we show that compressibility correlates with co-expression, co-localization, and shared function. Fourth, we also observe correlation with better protein tagging methods, physiological expression in contrast to over-expression of tagged proteins, and smart pooling approaches for yeast two-hybrid screens. Overall, this new measure is a proxy for both sensitivity and specificity and gives complementary information to standard measures such as average degree and clustering coefficients