Characterization of a sensory complex involved in antimicrobial peptide resistance

In their habitats, microorganisms are often in competition for limited nutrients. In order to succeed, many Gram-positive bacteria resort to production of peptide antibiotics. Therefore, resistance mechanisms against these compounds are essential. The first step of ensuring survival is the perception of the harmful drugs and mediation of resistance against it. In recent years, a group of ABC-transporters have been recognized as important resistance determinate against antimicrobial peptides. The expression of these transporters is generally regulated by a two-component system, which in most cases is encoded next to the transporter. Together they are described as detoxification modules. The permeases of the transporters are characterized by a large extracellular domain, while the histidine kinases lack an obvious input domain. One of the best understood examples is the BceRS-BceAB system of Bacillus subtilis, which mediates resistance against bacitracin, mersacidin and actagardine. For this system it was shown that the histidine kinase is not able to detect the substrate directly and instead has an absolute requirement for the transporter in stimulus perception. This describes a novel mode of signal transduction in which the transporter is the actual sensor and therefore regulates its own expression. To date, mechanistic details for this unique mode of signal transduction remain unknown. Several other examples have been described for transport proteins that have acquired additional sensing or regulatory functions beyond solute transport, and these have been designated trigger transporters. For these bifunctional transporters a direct protein-protein interaction with membrane-integrated or soluble components of signal transduction relays has been postulated. However, for most sensor/co-sensor pairs, conclusive proof of such an interaction is lacking, and so far little is known about the sites that might mediate contacts between the putative protein interfaces and how communication is achieved. Based on sequence and architectural similarities, we identified over 250 BceAB-like transporters in the protein database, which occurred almost exclusively in Firmicutes bacteria. To whether the regulatory interplay between the ABC transporter and the two-component system was a common theme in these antimicrobial peptide resistance modules, we carried out a phylogenetic study of these identified systems. We identified a clear coevolutionary relationship between transport permeases and histidine kinases. Furthermore, we identified conserved putative response regulator binding sites in the promoter regions of the transporter operons. Additionally, we were able to provide a tool to identify TCSs for transporters lacking a regulatory system in their genomic neighbourhood, which was based on the coclustering of histidine kinases and transporter permeases. These findings also suggested the existence of a sensory complex between BceAB-like transporters and BceS-like histidine kinases. To further investigate the signaling mechanism, we performed a random mutagenesis of the transport permease BceB with the aim to identify regions or residues within the transporter that are involved in signaling and/or resistance. With this approach we were able to identify mutations that affected either the ability for signaling or mediation of resistance. This showed a partial genetic separation of the two qualities, which could be achieved by single amino acid replacements. These results provide first insights into the signaling mechanism of the Bce system. In order to analyse the proposed communication between two-component system and ABC transporter, we further characterized their interactions by in vivo and in vitro approaches. We could demonstrate that the transporter BceAB is indeed able to interact directly with the histidine kinase. Because it was unknown how the signal perception by BceAB-type transporters occurs, we next analyzed substrate binding by the transporter permease BceB and could show direct binding of bacitracin by BceB. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the activity of the histidine kinase. In summary this thesis clearly shows the existence of a sensory complex comprised of BceRS-like two-component systems and BceAB-like ABC transporters and provides first functional insights into the mechanism of stimulus perception, signal transduction and antimicrobial resistance mechanism employed by these wide spread detoxification systems against antimicrobial peptides.Um sich in solch hart umkämpften Habitaten wie dem Boden zu behaupten sind Bakterien dazu übergegangen Antibiotika zu produzieren, um das Wachstum der Konkurrenz einzudämmen. Eine Gruppe solcher Substanzen sind antimikrobielle Peptide, die von Gram-positiven Bakterien produziert werden. Zum Schutz vor Peptidantibiotika haben Gram-positive Bakterien eine Vielzahl verschiedener Resistenzmechanismen entwickelt. Den effizientesten Resistenzmechanismus gegen Peptidantibiotika stellt eine Gruppe ATP-abhängiger ABC-Transporter dar. Diese Transporter weisen einen besonderen Transmembranaufbau auf. Sie bestehen aus zehn Transmembranhelices und einer großen extrazellulären Domäne. Die Expression dieser Transportergruppe wird durch ein Zweikomponentensystem reguliert. Die Histidinkinase besitzt ebenfalls einen ungewöhnlichen Transmembranaufbau, da sie keine offensichtliche Bindedomäne besitzt. Zusammen bilden der Transporter und die Histidinkinase ein Resistenzmodul gegen Peptidantibiotika, das in Firmicutes weit verbreitet ist. Eines der am besten verstandenen Systeme ist das BceRS-BceAB System in Bacillus subtilis. Dieses System vermittelt Resistenz gegen Bacitracin, Actagardin und Mersacidin. Für dieses System konnte gezeigt werden, dass die Histidinkinase BceS alleine nicht in der Lage ist, auf Bacitracin zu reagieren, sondern stattdessen für die Reizwahrnehmung und die Vermittlung der Resistenz auf den Transporter BceAB angewiesen ist. Der Transporter reguliert somit eine eigene Produktion. Wie der Resistenzmechanismus in diesem System genau funktioniert konnte bisher aber noch nicht hinreichend geklärt werden. Dass Transporter neben ihrer Funktion Substrate über eine Zellmembran zu transportieren auch an der Reizwahrnehmung und der Antwortregulation beteiligt sein können, ist in unterschiedlichsten Beispielen beschrieben worden. Um die Signalweiterleitung an membranständige oder zytoplasmatische Komponenten des Signalwegs gewährleisten zu können, müssen diese miteinander interagieren, zum Beispiel durch direkte Protein-Protein Interaktionen. Bisher konnte jedoch für viele solcher Sensorkomplexe keine endgültige Erklärung für solch eine Interaktion dargestellt werden. Basierend auf einer Datenbankanalyse konnten über 250 BceAB-artige Transporter identifiziert und ein Großteil davon einer BceS-artigen Histidinkinase zugeordnet werden. Durch eine phylogenetische Studie konnte weiterhin gezeigte werden, dass BceRS-artige Zweikomponentensysteme und BceAB-artige Transporter in Firmicutes Bakterien weit verbreitet sind und sich über Ko-Evolution gemeinsam zu Resistenzmodulen gegen Peptidantibiotika entwickelt haben. Dazu konnte eine konservierte Antwortregulator-Bindestelle in den Promoter Regionen der Transporteroperons bestimmt werden. Zudem war es möglich aufgrund dieser Klassifizierung für diejenigen Permeasen ohne ein benachbartes Zweikomponentensystem anhand der Genomsequenz ein mögliches Regulationssystem zuzuordnen. Diese Erkenntnisse unterstützten die Vermutung über einen sensorischen Komplex zwischen BceS-ähnlichen Histidinkinasen und BceAB-ähnlichen ABC Transportern. In einer weiteren Studie konnten mittels zufälliger Mutagenese der Transporterpermease BceB Aminosäurereste identifizierte werden, die an der Signalweiterleitung und/oder Resistenzvermittlung beteiligt waren. Durch einige der eingefügten Mutationen wurde nur die Signalweiterleitung bzw. nur die Resistenz beeinträchtigt. Dies spricht dafür, dass eine partielle genetische Trennung der Aufgaben des Transporters möglich ist. Hierdurch konnten erste wichtige Einblicke in den Signalweiterleitungsmechanismus des Bce-Systems gewonnen werden. Um die vorgeschlagene Kommunikation zwischen Zweikomponentensystem und ABCTransporter weiterführend zu untersuchen, wurden Interaktionsstudien durchgeführt. Die auf in vitro und in vivo Studien basierenden Ergebnisse konnten eine direkte Interaktion zwischen BceS und BceAB darstellen. Darüber hinaus konnten wir in dieser Arbeit durch eine Oberflächenresonanz- Spektroskopie zum ersten Mal zeigen, dass die Transporterpermease Bacitracin direkt und spezifisch bindet. Außerdem konnte durch eine in vitro Rekonstruktion des Signalwegs im Bce-System gezeigt werden, dass die Aktivität der Histidinkinase durch die Anwesenheit des Transporters beeinflusst wird. Zusammenfassend zeigt die vorliegende Arbeit direkte Hinweise, dass BceRS-artige Zweikomponentensysteme und BceAB-artige ABC-Transporter zusammen einen sensorischen Komplex für Peptidantibiotika bilden. Dies wird unterstützt durch erste funktionelle Einblicke in die Mechanismen der Reizwahrnehmung und Signalweiterleitung in diesen in Firmicutes Bakterien weit verbreiteten Resistenzsystemen

Characterization of a sensory complex involved in antimicrobial peptide resistance

In their habitats, microorganisms are often in competition for limited nutrients. In order to succeed, many Gram-positive bacteria resort to production of peptide antibiotics. Therefore, resistance mechanisms against these compounds are essential. The first step of ensuring survival is the perception of the harmful drugs and mediation of resistance against it. In recent years, a group of ABC-transporters have been recognized as important resistance determinate against antimicrobial peptides. The expression of these transporters is generally regulated by a two-component system, which in most cases is encoded next to the transporter. Together they are described as detoxification modules. The permeases of the transporters are characterized by a large extracellular domain, while the histidine kinases lack an obvious input domain. One of the best understood examples is the BceRS-BceAB system of Bacillus subtilis, which mediates resistance against bacitracin, mersacidin and actagardine. For this system it was shown that the histidine kinase is not able to detect the substrate directly and instead has an absolute requirement for the transporter in stimulus perception. This describes a novel mode of signal transduction in which the transporter is the actual sensor and therefore regulates its own expression. To date, mechanistic details for this unique mode of signal transduction remain unknown. Several other examples have been described for transport proteins that have acquired additional sensing or regulatory functions beyond solute transport, and these have been designated trigger transporters. For these bifunctional transporters a direct protein-protein interaction with membrane-integrated or soluble components of signal transduction relays has been postulated. However, for most sensor/co-sensor pairs, conclusive proof of such an interaction is lacking, and so far little is known about the sites that might mediate contacts between the putative protein interfaces and how communication is achieved. Based on sequence and architectural similarities, we identified over 250 BceAB-like transporters in the protein database, which occurred almost exclusively in Firmicutes bacteria. To whether the regulatory interplay between the ABC transporter and the two-component system was a common theme in these antimicrobial peptide resistance modules, we carried out a phylogenetic study of these identified systems. We identified a clear coevolutionary relationship between transport permeases and histidine kinases. Furthermore, we identified conserved putative response regulator binding sites in the promoter regions of the transporter operons. Additionally, we were able to provide a tool to identify TCSs for transporters lacking a regulatory system in their genomic neighbourhood, which was based on the coclustering of histidine kinases and transporter permeases. These findings also suggested the existence of a sensory complex between BceAB-like transporters and BceS-like histidine kinases. To further investigate the signaling mechanism, we performed a random mutagenesis of the transport permease BceB with the aim to identify regions or residues within the transporter that are involved in signaling and/or resistance. With this approach we were able to identify mutations that affected either the ability for signaling or mediation of resistance. This showed a partial genetic separation of the two qualities, which could be achieved by single amino acid replacements. These results provide first insights into the signaling mechanism of the Bce system. In order to analyse the proposed communication between two-component system and ABC transporter, we further characterized their interactions by in vivo and in vitro approaches. We could demonstrate that the transporter BceAB is indeed able to interact directly with the histidine kinase. Because it was unknown how the signal perception by BceAB-type transporters occurs, we next analyzed substrate binding by the transporter permease BceB and could show direct binding of bacitracin by BceB. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the activity of the histidine kinase. In summary this thesis clearly shows the existence of a sensory complex comprised of BceRS-like two-component systems and BceAB-like ABC transporters and provides first functional insights into the mechanism of stimulus perception, signal transduction and antimicrobial resistance mechanism employed by these wide spread detoxification systems against antimicrobial peptides.Um sich in solch hart umkämpften Habitaten wie dem Boden zu behaupten sind Bakterien dazu übergegangen Antibiotika zu produzieren, um das Wachstum der Konkurrenz einzudämmen. Eine Gruppe solcher Substanzen sind antimikrobielle Peptide, die von Gram-positiven Bakterien produziert werden. Zum Schutz vor Peptidantibiotika haben Gram-positive Bakterien eine Vielzahl verschiedener Resistenzmechanismen entwickelt. Den effizientesten Resistenzmechanismus gegen Peptidantibiotika stellt eine Gruppe ATP-abhängiger ABC-Transporter dar. Diese Transporter weisen einen besonderen Transmembranaufbau auf. Sie bestehen aus zehn Transmembranhelices und einer großen extrazellulären Domäne. Die Expression dieser Transportergruppe wird durch ein Zweikomponentensystem reguliert. Die Histidinkinase besitzt ebenfalls einen ungewöhnlichen Transmembranaufbau, da sie keine offensichtliche Bindedomäne besitzt. Zusammen bilden der Transporter und die Histidinkinase ein Resistenzmodul gegen Peptidantibiotika, das in Firmicutes weit verbreitet ist. Eines der am besten verstandenen Systeme ist das BceRS-BceAB System in Bacillus subtilis. Dieses System vermittelt Resistenz gegen Bacitracin, Actagardin und Mersacidin. Für dieses System konnte gezeigt werden, dass die Histidinkinase BceS alleine nicht in der Lage ist, auf Bacitracin zu reagieren, sondern stattdessen für die Reizwahrnehmung und die Vermittlung der Resistenz auf den Transporter BceAB angewiesen ist. Der Transporter reguliert somit eine eigene Produktion. Wie der Resistenzmechanismus in diesem System genau funktioniert konnte bisher aber noch nicht hinreichend geklärt werden. Dass Transporter neben ihrer Funktion Substrate über eine Zellmembran zu transportieren auch an der Reizwahrnehmung und der Antwortregulation beteiligt sein können, ist in unterschiedlichsten Beispielen beschrieben worden. Um die Signalweiterleitung an membranständige oder zytoplasmatische Komponenten des Signalwegs gewährleisten zu können, müssen diese miteinander interagieren, zum Beispiel durch direkte Protein-Protein Interaktionen. Bisher konnte jedoch für viele solcher Sensorkomplexe keine endgültige Erklärung für solch eine Interaktion dargestellt werden. Basierend auf einer Datenbankanalyse konnten über 250 BceAB-artige Transporter identifiziert und ein Großteil davon einer BceS-artigen Histidinkinase zugeordnet werden. Durch eine phylogenetische Studie konnte weiterhin gezeigte werden, dass BceRS-artige Zweikomponentensysteme und BceAB-artige Transporter in Firmicutes Bakterien weit verbreitet sind und sich über Ko-Evolution gemeinsam zu Resistenzmodulen gegen Peptidantibiotika entwickelt haben. Dazu konnte eine konservierte Antwortregulator-Bindestelle in den Promoter Regionen der Transporteroperons bestimmt werden. Zudem war es möglich aufgrund dieser Klassifizierung für diejenigen Permeasen ohne ein benachbartes Zweikomponentensystem anhand der Genomsequenz ein mögliches Regulationssystem zuzuordnen. Diese Erkenntnisse unterstützten die Vermutung über einen sensorischen Komplex zwischen BceS-ähnlichen Histidinkinasen und BceAB-ähnlichen ABC Transportern. In einer weiteren Studie konnten mittels zufälliger Mutagenese der Transporterpermease BceB Aminosäurereste identifizierte werden, die an der Signalweiterleitung und/oder Resistenzvermittlung beteiligt waren. Durch einige der eingefügten Mutationen wurde nur die Signalweiterleitung bzw. nur die Resistenz beeinträchtigt. Dies spricht dafür, dass eine partielle genetische Trennung der Aufgaben des Transporters möglich ist. Hierdurch konnten erste wichtige Einblicke in den Signalweiterleitungsmechanismus des Bce-Systems gewonnen werden. Um die vorgeschlagene Kommunikation zwischen Zweikomponentensystem und ABCTransporter weiterführend zu untersuchen, wurden Interaktionsstudien durchgeführt. Die auf in vitro und in vivo Studien basierenden Ergebnisse konnten eine direkte Interaktion zwischen BceS und BceAB darstellen. Darüber hinaus konnten wir in dieser Arbeit durch eine Oberflächenresonanz- Spektroskopie zum ersten Mal zeigen, dass die Transporterpermease Bacitracin direkt und spezifisch bindet. Außerdem konnte durch eine in vitro Rekonstruktion des Signalwegs im Bce-System gezeigt werden, dass die Aktivität der Histidinkinase durch die Anwesenheit des Transporters beeinflusst wird. Zusammenfassend zeigt die vorliegende Arbeit direkte Hinweise, dass BceRS-artige Zweikomponentensysteme und BceAB-artige ABC-Transporter zusammen einen sensorischen Komplex für Peptidantibiotika bilden. Dies wird unterstützt durch erste funktionelle Einblicke in die Mechanismen der Reizwahrnehmung und Signalweiterleitung in diesen in Firmicutes Bakterien weit verbreiteten Resistenzsystemen

Concurrent loss of MLH1, PMS2 and MSH6 immunoexpression in digestive system cancers indicating a widespread dysregulation in DNA repair processes

Immunohistochemical analysis of mismatch repair (MMR) protein expression is widely used to identify tumors with a deficient MMR (dMMR). MMR proteins (MLH1/PMS2 and MSH2/MSH6) work as functional heterodimers, which usually leads to the loss of expression in only one functional MMR heterodimer. Recently, there have been studies showing the simultaneous loss of immunoexpression in proteins of both heterodimers. Yet, this phenomenon has been rarely investigated. In this study, we retrospectively considered cases of different digestive system cancers (gastric cancer, ampullary cancer, small bowel cancer, colorectal cancer), which were immunohistochemically tested for dMMR within a 4-year period at our university hospital (n=352). Of the 103 cases showing dMMR, 5 cases (1.4% of all, 5.1% of dMMR cases) showed a concurrent loss of MLH1, PMS2 and MSH6 immunoexpression, whereas in the other 98 dMMR cases only one MMR heterodimer was affected. MLH1-/PMS2-/MSH6- cancer cases almost arose throughout the entire digestive tract: from the gastric antrum to the left colic flexur. To provide a comprehensive molecular characterization of this MLH1-/PMS2-/MSH6- immunophenotype, tumors were analyzed for microsatellite instability, MLH1 promotor hypermethylation and BRAF exon 15 status. Furthermore, we performed next-generation sequencing focusing on genes related to DNA repair. Here, we could detect pathogenic germline variants as well as multiple sporadic mutations in different genes involved in MMR and homologous recombination repair (HRR) respectively. The affected MMR/HRR-related genes were: ATM, BARD1, BRCA1, CDK12, CHEK1, CHEK2, FANCA, MLH1, MSH6, PALB2, TP53. Considering the biologic function of HRR/MMR proteins as potential drug targets and the low frequency of most of these mutations in digestive system cancers in general, their common occurrence in our MLH1-/PMS2-/MSH6- cases seems to be even more noteworthy, highlighting the need for recognition, awareness and further investigation of this unusual IHC staining pattern

A New Way of Sensing: Need-Based Activation of Antibiotic Resistance by a Flux-Sensing Mechanism

Sensing of and responding to environmental changes are of vital importance for microbial cells. Consequently, bacteria have evolved a plethora of signaling systems that usually sense biochemical cues either via direct ligand binding, thereby acting as "concentration sensors," or by responding to downstream effects on bacterial physiology, such as structural damage to the cell. Here, we describe a novel, alternative signaling mechanism that effectively implements a " flux sensor" to regulate antibiotic resistance. It relies on a sensory complex consisting of a histidine kinase and an ABC transporter, in which the transporter fulfills the dual role of both the sensor of the antibiotic and the mediator of resistance against it. Combining systems biological modeling with in vivo experimentation, we show that these systems in fact respond to changes in activity of individual resistance transporters rather than to changes in the antibiotic concentration. Our model shows that the cell thereby adjusts the rate of de novo transporter synthesis to precisely the level needed for protection. Such a flux-sensing mechanism may serve as a cost-efficient produce-to-demand strategy, controlling a widely conserved class of antibiotic resistance systems. IMPORTANCE Bacteria have to be able to accurately perceive their environment to allow adaptation to changing conditions. This is usually accomplished by sensing the concentrations of beneficial or harmful substances or by measuring the effect of the prevailing conditions on the cell. Here we show the existence of a new way of sensing the environment, where the bacteria monitor the activity of an antibiotic resistance transporter. Such a "flux-sensing" mechanism allows the cell to detect its current capacity to deal with the antibiotic challenge and thus precisely respond to the need for more transporters. We propose that this is a cost-efficient way of regulating antibiotic resistance on demand

All-Body-Cavity (ABC)-scopy: an approach for a feasible method of minimally invasive autopsy to allow for postmortem tissue sampling in cases where a conventional autopsy is denied

Objectives The decreasing autopsy numbers in many western countries have been partially attributed to the invasiveness of the autopsy, which causes relatives to decline postmortem examination. This issue has been addressed by developing methods of minimally or non-invasive autopsy, which could be shown to increase acceptance for autopsies. The aim of this study is to compare the All-Body-Cavity-scopy (ABC-scopy) to conventional autopsies for diagnostic accuracy. Methods The ABC-scopy is an endoscopic approach for minimally invasive autopsy involving laparoscopic and thoracoscopic evaluation of the accessible organs, followed by excision biopsies of relevant organs and conspicuous findings. The method was performed in 10 cases on deceased patients scheduled for autopsy, each followed by a conventional autopsy. Results The results gathered from ABC-scopy through observation and histopathological evaluation provided an acceptable diagnostic accuracy in 9 out of 10 autopsies when compared to those of the conventional autopsy for diagnostic findings. Conclusions The ABC-scopy is a feasible approach for minimally invasive autopsy that provides acceptable diagnostic value. Despite its minimally invasive nature, the procedure enables representative histology through providing large size excision biopsies from intraabdominal and thoracic organs, which is especially useful for examining disseminated diseases such as metastasized tumors

ALK, NUT, and TRK do not play relevant roles in gastric cancer — results of an immunohistochemical study in a large series

ALK, NUT, and TRK are rare molecular aberrations that are pathognomonic for specific rare tumors. In low frequencies, however, they are found in a wide range of other tumor entities. This study aimed to investigate the frequency, association with clinicopathological characteristics, and prognosis of the immunohistochemical expressions of ALK, NUT, and TRK in 477 adenocarcinomas of the stomach and gastroesophageal junction. Seven cases (1.5%) showed an expression of TRK. In NGS, no NTRK fusion was confirmed. No case with ALK or NUT expression was detected. ALK, NUT, and NTRK expression does not seem to play an important role in gastric carcinomas

A Sensory Complex Consisting of an ATP-Binding-Cassette Transporter and a Two-Component Regulatory System Controls Bacitracin Resistance in <em>Bacillus subtilis</em>

Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems

Comprehensive immunohistochemical study of the SWI/SNF complex expression status in gastric cancer reveals an adverse prognosis of SWI/SNF deficiency in genomically stable gastric carcinomas

SIMPLE SUMMARY: This study aimed to investigate the clinical relevance of immunohistochemical expression of proteins of the SWI/SNF complex, SMARCA2, SMARCA4 SMARCB1, ARID1A, ARID1B, and PBRM1 in 477 adenocarcinomas of the stomach and gastroesophageal junction. Additionally, the tumors were classified immunohistochemically in analogy to The Cancer Genome Atlas (TCGA) classification. Overall, 32% of cases demonstrated aberrant expression of the SWI/SNF complex. SWI/SNF aberration emerged as an independent negative prognostic factor for overall survival in all patients and in genomically stable patients in analogy to TCGA. In conclusion, determination of SWI/SNF status could be suggested in routine diagnostics in genomically stable tumors to identify patients who might benefit from new therapeutic options. ABSTRACT: The SWI/SNF complex has important functions in the mobilization of nucleosomes and consequently influences gene expression. Numerous studies have demonstrated that mutations or deficiency of one or more subunits can have an oncogenic effect and influence the development, progression, and eventual therapy resistance of tumor diseases. Genes encoding subunits of the SWI/SNF complex are mutated in approximately 20% of all human tumors. This study aimed to investigate the frequency, association with clinicopathological characteristics, and prognosis of immunohistochemical expression of proteins of the SWI/SNF complexes, SMARCA2, SMARCA4 SMARCB1, ARID1A, ARID1B, and PBRM1 in 477 adenocarcinomas of the stomach and gastroesophageal junction. Additionally, the tumors were classified immunohistochemically in analogy to The Cancer Genome Atlas (TCGA) classification. Overall, 32% of cases demonstrated aberrant expression of the SWI/SNF complex. Complete loss of SMARCA4 was detected in three cases (0.6%) and was associated with adverse clinical characteristics. SWI/SNF aberration emerged as an independent negative prognostic factor for overall survival in genomically stable patients in analogy to TCGA. In conclusion, determination of SWI/SNF status could be suggested in routine diagnostics in genomically stable tumors to identify patients who might benefit from new therapeutic options

Individualized targeted treatment in a case of a rare TFG::ROS1 fusion positive inflammatory myofibroblastic tumor (IMT)

Background Inflammatory myofibroblastic tumor (IMTs) are rare mesenchymal neoplasms with slow growth. Resection is considered as therapeutic standard, with chemotherapy being insufficiently effective in advanced disease. ALK translocations are present in 50% of cases, ROS1 fusions (YWHAE::ROS1, TFG::ROS1) are extremely rare. Here, we present a case with TFG::ROS1 fusion and highlight the significance of molecular tumor boards (MTBs) in clinical precision oncology for post-last-line therapy. Case Presentation A 32-year-old woman presented with IMT diagnosed at age 27 for biopsy and treatment evaluation. Previous treatments included multiple resections and systemic therapy with vinblastine, cyclophosphamide, and methotrexate. A computed tomography scan showed extensive tumor infiltration of the psoas muscles and the posterior abdomen. Next generation sequencing revealed an actionable ROS1 fusion (TFG::ROS1) with breakpoints at exon 4/35 including the kinase domain and activating the RAS-pathway. TFG, the Trk-fused gene, exerts functions such as intracellular trafficking and exhibits high sequence homology between species. Based on single reports about efficacy of ROS1-targeting in ROS1 translocation positive IMTs the patient was started on crizotinib, an ATP-competitive small molecule c-MET, ALK and ROS1-inhibitor. With a follow-up of more than 9 months, the patient continues to show a profound response with major tumor regression, improved quality of life and no evidence for severe adverse events. Conclusion This case underscores the importance of the availability of modern molecular diagnostics and interdisciplinarity in precision oncology to identify rare, disease-defining genotypes that make an otherwise difficult-to-treat disease targetable