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Introduction: Whole Exome Sequencing (WES) has emerged as an efficient tool in clinical cancer diagnostics to broaden the scope from panel-based diagnostics to screening of all genes and enabling robust determination of complex biomarkers in a single analysis., Methods: To assess concordance, six formalin-fixed paraffin-embedded (FFPE) tissue specimens and four commercial reference standards were analyzed by WES as matched tumor-normal DNA at 21 NGS centers in Germany, each employing local wet-lab and bioinformatics. Somatic and germline variants, copy-number alterations (CNAs), and complex biomarkers were investigated. Somatic variant calling was performed in 494 diagnostically relevant cancer genes. The raw data were collected and re-analyzed with a central bioinformatic pipeline to separate wet- and dry-lab variability., Results: The mean positive percentage agreement (PPA) of somatic variant calling was 76 % while the positive predictive value (PPV) was 89 % in relation to a consensus list of variants found by at least five centers. Variant filtering was identified as the main cause for divergent variant calls. Adjusting filter criteria and re-analysis increased the PPA to 88 % for all and 97 % for the clinically relevant variants. CNA calls were concordant for 82 % of genomic regions. Homologous recombination deficiency (HRD), tumor mutational burden (TMB), and microsatellite instability (MSI) status were concordant for 94 %, 93 %, and 93 % of calls, respectively. Variability of CNAs and complex biomarkers did not decrease considerably after harmonization of the bioinformatic processing and was hence attributed mainly to wet-lab differences., Conclusion: Continuous optimization of bioinformatic workflows and participating in round robin tests are recommended., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: MMT reports speaker and travel Expenses from Twist. JS reports speaker honoraria from DLS, Molecular Health, AstraZeneca and Biocartis, outside the submitted work. UL reports speaker fees from AstraZeneca, GSK, Novartis, Menarini, advisory board from AstraZeneca and Novartis. DH reports speaker honorary AstraZeneca, adboard BMS, WD speaker honoraries BMS & Novartis. SMB reports speaker honoraria, advisory board fees and research grants from AstraZeneca, Daiichi, Menarini, Novartis, Roche, BMS, Pfizer, Bayer, MSD, Merck, Amgen, Molecular Health, Targos, DLS, Janssen, GSK, QuIP, outside the submitted work. SL reports research grant from BMS, advisory board/speaker invitation from AstraZeneca, Eli Lilly, Roche and Takeda outside of this work. NTG reports research support from Janssen-Cilag and Advisory Boards from Janssen-Cilag, AstraZeneca, Daiichi-Sankyo and BMS outside the submitted work. WW reports research grants from Roche, MSD, BMS and AstraZeneca. Advisory board, lectures and speaker bureau fees from Roche, MSD, BMS, AstraZeneca, Pfizer, Merck, Lilly, Boehringer, Novartis, Takeda, Bayer, Janssen, Amgen, Astellas, Illumina, Eisai, Siemens, Agilent, ADC, GSK und Molecular Health. SO received reimbursement for travel expenses and payment for conference presentations from Illumina Inc. and Oxford Nanopore Technologies. CS reports research funding from BMS Stiftung Immunonkologie and institutional grants from Illumina outside the submitted work. CPS reports an investigator-initiated grant from Illumnia outside of the submitted work. PS reports grants from Inctye, BMS, Gilead, Falk, speakers bureau/advisory board from MSD, BMS, AstraZeneca, Incyte, Astellas, Janssen, Eisai, Amgen, Boehringer Ingelheim. DK reports personal fees for speaker honoraria from AstraZeneca, and Pfizer, personal fees for Advisory Board from Bristol-Myers Squibb, outside the submitted work. NP reports speaker fees from Novartis, Bayer, Roche, AstraZeneca, Illumina, BMS, MSD, PGDX/Labcorp, advisory board from Novartis, Lilly, Roche, Janssen, travel expenses from Novartis, AstraZeneca, Illumina, BMS, MSD, PGDX/Labcorp, Research grants from Illumina. JB reports grants from German Cancer Aid and consulting from MSD, outside the submitted work. AS reports participation in Advisory Board/Speaker’s Bureau for Astra Zeneca, AGCT, Bayer, Bristol-Myers Squibb, Eli Lilly, Illumina, Janssen, MSD, Novartis, Pfizer, Roche, Seattle Genetics, Takeda, and Thermo Fisher, grants from Bayer, Bristol-Myers Squibb, and Chugai, outside the submitted work. All other authors report no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Undifferentiated embryonal sarcoma of the liver is a rare mesenchymal malignancy that predominantly occurs in children. The relationship between this tumor entity and germline pathogenic variants (PVs) remains undefined. Here, we present the clinical case of a male patient diagnosed with undifferentiated embryonal sarcoma of the liver. Both germline and tumor samples were analyzed using next-generation sequencing. In the tumor tissue, PVs in TP53 (NM_000546.5):c.532del p.(His178Thrfs*69) and CHEK2 (NM_007194.4):c.85C>T p.(Gln29*) were identified, with both confirmed to be of germline origin. Copy number analyses indicated a loss of the wildtype TP53 allele in the tumor, consistent with a second hit, while it was the variant CHEK2 allele that was lost in the tumor. Our data indicate that the germline TP53 PV acts as a driver of tumorigenesis in the reported case and support a complex interaction between the germline TP53 and CHEK2 PVs. This case highlights the dynamic interplays of genetic alterations in tumorigenesis and emphasizes the need for continued investigation into the complex interactions between TP53 and CHEK2 PVs and into the association of undifferentiated embryonal sarcoma of the liver and Li-Fraumeni syndrome.

Purpose: Providing patient access to precision oncology (PO) is a major challenge of clinical oncologists. Here, we provide an easily transferable model from strategic management science to assess the outreach of a cancer center., Methods: As members of the German WERA alliance, the cancer centers in Würzburg, Erlangen, Regensburg and Augsburg merged care data regarding their geographical impact. Specifically, we examined the provenance of patients from WERA´s molecular tumor boards (MTBs) between 2020 and 2022 (n = 2243). As second dimension, we added the provenance of patients receiving general cancer care by WERA. Clustering our catchment area along these two dimensions set up a four-quadrant matrix consisting of postal code areas with referrals towards WERA. These areas were re-identified on a map of the Federal State of Bavaria., Results: The WERA matrix overlooked an active screening area of 821 postal code areas - representing about 50 % of Bavaria´s spatial expansion and more than six million inhabitants. The WERA matrix identified regions successfully connected to our outreach structures in terms of subsidiarity - with general cancer care mainly performed locally but PO performed in collaboration with WERA. We also detected postal code areas with a potential PO backlog - characterized by high levels of cancer care performed by WERA and low levels or no MTB representation., Conclusions: The WERA matrix provided a transparent portfolio of postal code areas, which helped assessing the geographical impact of our PO program. We believe that its intuitive principle can easily be transferred to other cancer centers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The efficacy of checkpoint inhibitors for non-small cell lung cancer (NSCLC) with MET exon 14 skipping (METΔ14ex) remains controversial., Materials and Methods: 110 consecutive METΔ14ex NSCLC patients receiving first-line chemotherapy (CHT) and/or immunotherapy (IO) in 10 German centers between 2016-2022 were analyzed., Results: Combined CHT-IO was given to 35/110 (32%) patients, IO alone to 43/110 (39%), and CHT to 32/110 (29%) upfront. Compared to CHT, CHT-IO showed longer progression-free survival (median PFS 6 vs. 2.5 months, p = 0.004), more objective responses (ORR 49% vs. 28%, p = 0.086) and numerically longer overall survival (OS 16 vs. 10 months, p = 0.240). For IO monotherapy, OS (14 vs. 16 months) and duration of response (26 vs. 22 months) were comparable to those of CHT-IO. Primary progressive disease (PD) was more frequent with IO compared to CHT-IO (13/43 vs. 3/35, p = 0.018), particularly for never-smokers (p = 0.041). Higher PD-L1 TPS were not associated with better IO outcomes, but TP53 mutated tumors showed numerically improved ORR (56% vs. 32%, p = 0.088) and PFS (6 vs. 3 months, p = 0.160), as well as longer OS in multivariable analysis (HR=0.54, p = 0.034) compared to their wild-type counterparts. Any second-line treatment was administered to 35/75 (47%) patients, with longer survival for capmatinib or tepotinib compared to crizotinib (PFS 10 vs. 3 months, p = 0.013; OS 16 vs. 13 months, p = 0.270)., Conclusion: CHT-IO is superior to CHT, and IO alone also effective for METΔ14ex NSCLC, especially in the presence of TP53 mutations and independent of PD-L1 expression, but never-smokers are at higher risk of primary PD., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: JK: speaker’s honoraria from BMS, AstraZeneca and Pfizer, travel grants from Takeda, advisory board honoraria from Takeda, Roche and AstraZeneca. DM: advisory board/lecture fees from AstraZeneca, BMS, Boehringer Ingelheim, Lilly, MSD, Novartis, Roche, Sanofi and Takeda. DiKa: advisory boards/speaker´s honoraria from BMS, Boehringer-Ingelheim, MSD, Roche, Janssen, Pfizer, AstraZeneca; support for attending meetings from Novartis, Boehringer-Ingelheim. MH: advisory board, speaker’s honoraria and travel grants from Boehringer-Ingelheim. BH: advisory board/lecture fees from AstraZeneca, Boehringer Ingelheim, BMS, MSD, Roche, Pfizer. MF: grants from AstraZeneca, BMS, MSD, and Roche; consulting fees from AstraZeneca, MSD, Roche, BMS; speaker´s honoraria from AstraZeneca, MSD, Roche, BMS. MK: speaker’s honoraria and travel grants from Veracyte Inc. AV: speaker’s honoraria from AstraZeneca. MA: speaker’s honoraria from Boehringer Ingelheim. CG: advisory board/lecture fees from Amgen, AstraZeneca, BMS, Boehringer Ingelheim, Eli Lilly, Takeda, MSD, Novartis, Pfizer, Roche, AbbVie, Tesaro/GSK and Blueprints Medicines. AT: consulting fees from Boehringer Ingelheim, Daiichi, Astra Zeneca, Roche, Pfizer, BMS, MSD, Sanofi, Lilly, Novartis; speaker’s honoraria from Boehringer Ingelheim, Daiichi, Astra Zeneca, Roche, Pfizer, BMS, MSD, Sanofi, Lilly, Novartis; travel grants from Sanofi, Janssen, Daiichi; BMS, MSD, AstraZeneca. MR: advisory board/lecture fees from Amgen, AstraZeneca, BMS, Boehringer, Lilly, Merck, MSD, Novartis, Pfizer, Roche, Samsung. NF: grants from Roche, consulting fees from AbbVie, Amgen, AstraZeneca, BeiGene, Berlinchemie, Boehringer Ingelheim, Bristol Myers&Squibb, Lilly, Merck Sharp&Dohme, Merck, Novartis, Pfizer, Roche, Sanofi, Takeda; support for attending meetings from Amgen, AstraZeneca, BMS, Janssen, Lilly, Takeda. AS: advisory board honoraria from BMS, AstraZeneca, ThermoFisher, Novartis, speaker’s honoraria from BMS, Illumina, AstraZeneca, Novartis, ThermoFisher, MSD, Roche, and research funding from Chugai and BMS. MT: research funding from AstraZeneca, BMS, Merck, Roche, Takeda; speaker’s honoraria from AstraZeneca, Beigene, Novartis, Eli Lilly, BMS, MSD, Roche, Celgene, Takeda, AbbVie, Boehringer Ingelheim, Pfizer, Eli Lilly, MSD, Takeda, Pfizer, Chugai, Daiichi Sankyo, GlaxoSmithKline, Janssen Oncology, Merck, Sanofi and travel grants from AstraZeneca, BMS, MSD, Novartis, Daiichi Sankyo, Janssen Oncology, Lilly, Merck, Pfizer, Roche, Sanofi, Takeda, Boehringer Ingelheim. PC: research funding from AstraZeneca, Amgen, Boehringer Ingelheim, Novartis, Roche, and Takeda, speaker’s honoraria from AstraZeneca, Janssen, Novartis, Roche, Pfizer, Thermo Fisher, Takeda, support for attending meetings from AstraZeneca, Eli Lilly, Daiichi Sankyo, Gilead, Novartis, Pfizer, Takeda, and personal fees for participating to advisory boards from AstraZeneca, Boehringer Ingelheim, Chugai, Pfizer, Novartis, MSD, Takeda and Roche, all outside the submitted work. All other authors have no conflicts of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The diagnosis of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), originally based on morphological assessment alone, has to bring together more and more disciplines. Today, modern AML/MDS diagnostics rely on cytomorphology, cytochemistry, immunophenotyping, cytogenetics, and molecular genetics. Only the integration of all these methods allows a comprehensive and complementary characterization of each case, which is a prerequisite for optimal AML/MDS diagnosis and treatment. In the following, we present why multidisciplinary and local diagnosis is essential today and will become even more important in the future, especially in the context of precision medicine. We present our idea and strategy implemented at Augsburg University Hospital, which has realized multidisciplinary diagnostics in AML/MDS in an interdisciplinary and decentralized approach. In particular, this includes the recent technical advances that molecular genetics provides with modern methods. The enormous amount of data generated by these techniques represents a major challenge, but also a unique opportunity. We will reflect on how this increase in knowledge can be integrated into routine practice to lead the way for personalized medicine in AML/MDS to improve patient care., (© 2023. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

SARS-CoV-2 may directly and indirectly damage lung tissue and other host organs, but there are few system-wide, untargeted studies of these effects on the human body. Here, we developed a parallelized mass spectrometry (MS) proteomics workflow enabling the rapid, quantitative analysis of hundreds of virus-infected FFPE tissues. The first layer of response to SARS-CoV-2 in all tissues was dominated by circulating inflammatory molecules. Beyond systemic inflammation, we differentiated between systemic and true tissue-specific effects to reflect distinct COVID-19-associated damage patterns. Proteomic changes in the lungs resembled those of diffuse alveolar damage (DAD) in non-COVID-19 patients. Extensive organ-specific changes were also evident in the kidneys, liver, and lymphatic and vascular systems. Secondary inflammatory effects in the brain were related to rearrangements in neurotransmitter receptors and myelin degradation. These MS-proteomics-derived results contribute substantially to our understanding of COVID-19 pathomechanisms and suggest strategies for organ-specific therapeutic interventions., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Objectives: Omicron lineages BA.1/2 are considered to cause mild clinical courses. Nevertheless, fatal cases after those infections are recognized but little is known about risk factors., Methods: A total of 23 full and three partial autopsies in deceased with known Omicron BA.1/2 infections have been consecutively performed. The investigations included histology, blood analyses, and molecular virus detection., Results: COVID-19-associated diffuse alveolar damage was found in only eight cases (31%). This rate is significantly lower compared with previous studies, including non-Omicron variants, where rates between 69% and 92% were observed. Neither vaccination nor known risk factors were significantly associated with a direct cause of death by COVID-19. Only those patients who were admitted to the clinic because of COVID-19 but not for other reasons had a significant association with a direct COVID-19 -caused death (P >0.001)., Conclusion: Diffuse alveolar damage still occurred in the Omicron BA.1/BA.2 era but at a considerably lower frequency than seen with previous variants of concern. None of the known risk factors discriminated the cases with COVID-19-caused death from those that died because of a different disease. Therefore, the host's genomics might play a key role in this regard. Further studies should elucidate the existence of such a genomic risk factor., Competing Interests: Declaration of competing interest The authors have no competinng interests to declare., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Recently, our group introduced Stroma AReactive Invasion Front Areas (SARIFA) as an independent prognostic predictor for a poorer outcome in colon cancer patients, which is probably based on immunologic alterations combined with a direct tumor-adipocyte interaction: the two together reflecting a distinct tumor biology. Considering it is already known that peripheral immune cells are altered in colorectal cancer (CRC) patients, this study aims to investigate the changes in lymphocyte subsets in SARIFA-positive cases and correlate these changes with the local immune response., Methods: Flow cytometry was performed to analyze B, T, and natural killer (NK) cells in the peripheral blood (PB) of 45 CRC patients. Consecutively, lymphocytes in PB, tumor-infiltrating lymphocytes (TILs), and CD56+ and CD57+ lymphocytes at the invasion front and the tumor center were compared between patients with SARIFA-positive and SARIFA-negative CRCs., Results: Whereas no differences could be observed regarding most PB lymphocyte populations as well as TILs, NK cells were dramatically reduced in the PB of SARIFA-positive cases. Moreover, CD56 and CD57 immunohistochemistry suggested SARIFA-status-dependent changes regarding NK cells and NK-like lymphocytes in the tumor microenvironment., Conclusion: This study proves that our newly introduced biomarker, SARIFA, comes along with distinct immunologic alterations, especially regarding NK cells.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Declarations of interest none, no external funding was received., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Reitsam, Märkl, Dintner, Waidhauser, Vlasenko and Grosser.)

(1) Background: molecular tumor boards (MTBs) are crucial instruments for discussing and allocating targeted therapies to suitable cancer patients based on genetic findings. Currently, limited evidence is available regarding the regional impact and the outreach component of MTBs; (2) Methods: we analyzed MTB patient data from four neighboring Bavarian tertiary care oncology centers in Würzburg, Erlangen, Regensburg, and Augsburg, together constituting the WERA Alliance. Absolute patient numbers and regional distribution across the WERA-wide catchment area were weighted with local population densities; (3) Results: the highest MTB patient numbers were found close to the four cancer centers. However, peaks in absolute patient numbers were also detected in more distant and rural areas. Moreover, weighting absolute numbers with local population density allowed for identifying so-called white spots-regions within our catchment that were relatively underrepresented in WERA MTBs; (4) Conclusions: investigating patient data from four neighboring cancer centers, we comprehensively assessed the regional impact of our MTBs. The results confirmed the success of existing collaborative structures with our regional partners. Additionally, our results help identifying potential white spots in providing precision oncology and help establishing a joint WERA-wide outreach strategy., Competing Interests: The authors declare no conflict of interest.

The rate of SARS-CoV-2 infections in vaccinees has become a relevant serious issue. This study aimed to determine the causes of death, histological organ alteration, and viral spread in relation to demographic, clinical-pathological, viral variants, and vaccine types for deceased individuals with proven SARS-CoV-2 infection after vaccination who died between January and November 2021. Twenty-nine consecutively collected cases were analyzed and compared to 141 nonvaccinated control cases. Autopsies were performed on 16 partially and 13 fully vaccinated individuals. Most patients were elderly and suffered from several relevant comorbidities. Real-time RT-PCR (RT-qPCR) identified a significantly increased rate of generalized viral dissemination within organ systems in vaccinated cases versus nonvaccinated cases (45% vs. 16%, respectively; P = 0.008) mainly with Ct-values of higher than 25 in non-respiratory samples. However, vaccinated cases also showed high viral loads, reaching Ct-values below 10, especially in the upper airways and lungs. This was accompanied by high rates of pulmonal bacterial or mycotic superinfections and the occurrence of immunocompromising factors, such as malignancies, immunosuppressive drug intake, or decreased immunoglobulin levels. All these findings were particularly accentuated in partially vaccinated patients compared to fully vaccinated individuals. The virus dissemination observed in our case study may indicate that patients with an impaired immune system have a decreased ability to eliminate the virus. However, the potential role of antibody-dependent enhancement must also be ruled out in future studies. Fatal cases of COVID-19 in vaccinees were rare and often associated with severe comorbidities or other immunosuppressive conditions., (© 2022. The Author(s).)

Hyper-IgE syndromes (HIES) are a group of inborn errors of immunity (IEI) caused by monogenic defects such as in the gene STAT3 (STAT3-HIES). Patients suffering from HIES show an increased susceptibility to Staphylococcus aureus (S. aureus) including skin abscesses and pulmonary infections. To assess if the underlying immune defect of STAT3-HIES patients influences the resistance patterns, pathogenicity factors or strain types of S. aureus. We characterized eleven S. aureus strains isolated from STAT3-HIES patients (n = 4) by whole genome sequencing (WGS) to determine presence of resistance and virulence genes. Additionally, we used multi-locus sequence typing (MLST) and protein A (spa) typing to classify these isolates. Bacterial isolates collected from this cohort of STAT3-HIES patients were identified as common spa types in Germany. Only one of the isolates was classified as methicillin-resistant S. aureus (MRSA). For one STAT3 patient WGS illustrated that infection and colonization occurred with different S. aureus isolates rather than one particular clone. The identified S. aureus carriage profile on a molecular level suggests that S. aureus strain type in STAT3-HIES patients is determined by local epidemiology rather than the underlying immune defect highlighting the importance of microbiological assessment prior to antibiotic treatment., (© 2022. The Author(s).)

Background: EGFR exon20 insertions (ex20ins) are targeted by novel compounds in non-small-cell lung cancer (NSCLC). However, data about outcome under conventional therapies and the influence of molecular features are scarce., Patients and Methods: We retrospectively analysed 118 patients with evaluation of radiologic response based on RECIST v1.1. TP53 status was available for 88 cases., Results: Platinum doublets and chemoimmunotherapy showed similar response rates (20-25%), disease control rates (80%) and median progression-free survival (mPFS, ≈7 months), which were longer compared to monochemotherapy (9%, 59%, 4.1 months), EGFR inhibitors (0%, 46%, 3.0) and PD-(L)1 inhibitors (0%, 30%, 2.1; p < 0.05). Overall survival (OS) was not dependent on the choice of first-line treatment, but related to more lines of systemic therapy (p < 0.05). TP53 mutations and brain metastases were associated with shorter PFS under platinum doublets and EGFR inhibitors (HR 3.3-6.1, p < 0.01), and shorter OS for patients receiving both treatments (p < 0.05). More tumour CD8 + and less Th1 cells were associated with longer OS independent of brain and TP53 status (p < 0.01). No difference in outcome was noted according to the ex20ins site and use of pemetrexed (vs. other cytotoxics) or bevacizumab. Long-lasting responses (>1 year) occasionally occurred under EGFR inhibitors for both 'near-' and 'far-loop' variants., Conclusions: Platinum doublets and chemoimmunotherapy have the highest activity with ORR of 20-25% and mPFS of approximately 7 months, regardless of the cytotoxic partner, while PD-(L)1 inhibitors show limited efficacy. TP53 mutations, brain metastases and a lower tumour CD8/Th1-cell ratio are independently associated with shorter survival., Competing Interests: Conflict of interest statement PC: research funding from Amgen, AstraZeneca, Boehringer Ingelheim, Novartis, Roche, Takeda, and advisory board/lecture fees from AstraZeneca, Boehringer Ingelheim, Chugai, Daiichi Sankyo, Gilead, Novartis, Pfizer, Roche, Takeda. JR: lecture fees from AstraZeneca, Boehringer Ingelheim. FCS: research funding from Roche; non-financial support from Lilly; personal fees from Takeda, and Pfizer, outside the submitted work. BH: advisory board/lecture fees from AstraZeneca, Boehringer Ingelheim, BMS, MSD, Roche, Pfizer. JK: research funding from AstraZeneca and Celgene. MJ: speaker's honoraria from Roche, Boehringer, and travel grants from Daiichi Sankyo. DK: advisory boards/speakers honoraria from AstraZeneca, BMS, Boehringer Ingelheim, GSK, MSD, Novartis, Pfizer, Roche, Takeda. FE: speaker's honoraria from Roche. FB: research funding from AstraZeneca, BMS and Roche, and travel grants from BMS and MSD. RS: research funding from BMS, and speaker's honoraria from AstraZeneca and Roche. AT: research funding from BMS. MW: research funding from Roche; Personal fees from Roche, AstraZeneca, Boehringer, Kite, Novartis, Merck, BMS, Heidelberg Pharma; Non-financial support from AstraZeneca, BMS, Glenmark; outside the submitted work. SL: advisory board, speaker's honoraria and travel support from BerGenBio, Novartis, Lilly, BMS, MSD, Roche, Celgene, Takeda, AstraZeneca, Sanofi, as well as research funding from Roche, BMS, BerGenBio. WB: consulting fees from AstraZeneca, Boehringer Ingelheim, BMS, Lilly, MSD, Pfizer, Roche, Sanofi, honoraria for lectures from AstraZeneca, Boehringer Ingelheim, BMS, Lilly, MSD, Pfizer, Roche, Sanofi. Travel grants from AstraZeneca, Boehringer Ingelheim, MSD, Roche. CS: advisory board honoraria from AstraZeneca, Boehringer Ingelheim, BMS, MSD, Novartis, Roche, Pfizer, Takeda. Speaker's honoraria from AstraZeneca, Boehringer, Lilly, Roche, MSD, Takeda. DM: advisory board/lecture fees from AstraZeneca, BMS, Boehringer Ingelheim, Lilly, MSD, Novartis, Roche, Sanofi and Takeda (no personal honoraria) NF: advisory board/lecture fees from AbbVie, AstraZeneca, BMS, Boehringer Ingelheim, Pfizer, Roche, MSD, Takeda. JK: advisory board member without receiving any personal fees for Roche Pharma, Boehringer Ingelheim, BMS, MSD, Amgen, Lilly and Takeda. MR: advisory board/lecture fees from Amgen, AstraZeneca, BMS, Boehringer, Lilly, Merck, MSD, Novartis, Pfizer, Roche, Samsung. FG: grants and personal fees from AstraZeneca, Boehringer Ingelheim, BMS, Eli Lilly, MSD, Novartis, Pfizer, Roche, Takeda, as well as personal fees from AbbVie, Tesaro/GSK, Blueprint Medicines, Amgen. CG: advisory board/lecture fees from Amgen, AstraZeneca, BMS, Boehringer Ingelheim, Eli Lilly, Takeda, MSD, Novartis, Pfizer, Roche, AbbVie, Tesaro/GSK and Blueprints Medicines. AS: advisory board honoraria from BMS, AstraZeneca, ThermoFisher, Novartis, speaker's honoraria from BMS, Illumina, AstraZeneca, Novartis, ThermoFisher, MSD, Roche, and research funding from Chugai and BMS. MT: advisory board honoraria from Novartis, Eli Lilly, BMS, MSD, Roche, Celgene, Takeda, AbbVie, Boehringer Ingelheim, Pfizer, speaker's honoraria from Eli Lilly, MSD, Takeda, Pfizer, research funding from AstraZeneca, BMS, Celgene, Novartis, Roche, Takeda, and travel grants from BMS, MSD, Novartis, Boehringer. All remaining authors have declared no conflicts of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Confronted with an emerging infectious disease at the beginning of the COVID-19 pandemic, the medical community faced concerns regarding the safety of autopsies on those who died of the disease. This attitude has changed, and autopsies are now recognized as indispensable tools for understanding COVID-19, but the true risk of infection to autopsy staff is nevertheless still debated. To clarify the rate of SARS-CoV-2 contamination in personal protective equipment (PPE), swabs were taken at nine points in the PPE of one physician and one assistant after each of 11 full autopsies performed at four centers. Swabs were also obtained from three minimally invasive autopsies (MIAs) conducted at a fifth center. Lung/bronchus swabs of the deceased served as positive controls, and SARS-CoV-2 RNA was detected by real-time RT-PCR. In 9 of 11 full autopsies, PPE samples tested RNA positive through PCR, accounting for 41 of the 198 PPE samples taken (21%). The main contaminated items of the PPE were gloves (64% positive), aprons (50% positive), and the tops of shoes (36% positive) while the fronts of safety goggles, for example, were positive in only 4.5% of the samples, and all the face masks were negative. In MIAs, viral RNA was observed in one sample from a glove but not in other swabs. Infectious virus isolation in cell culture was performed on RNA-positive swabs from the full autopsies. Of all the RNA-positive PPE samples, 21% of the glove samples, taken in 3 of 11 full autopsies, tested positive for infectious virus. In conclusion, PPE was contaminated with viral RNA in 82% of autopsies. In 27% of autopsies, PPE was found to be contaminated even with infectious virus, representing a potential risk of infection to autopsy staff. Adequate PPE and hygiene measures, including appropriate waste deposition, are therefore essential to ensure a safe work environment., (© 2022. The Author(s).)

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.

Compared to other malignancies, there is a lack of easy-to-evaluate biomarkers for gastric cancer, which is associated with an adverse clinical outcome in many cases. Here, we present Stroma AReactive Invasion Front Areas (SARIFA) as a new histological prognostic marker. We defined SARIFA as the direct contact between a cluster of tumor glands/cells comprising at least five tumor cells and inconspicuous surrounding adipose tissue at the invasion front. A total of 480 adenocarcinomas of the stomach and the gastroesophageal junction from two different collections were classified according to SARIFA. To understand the potential underlying mechanisms, a transcriptome analysis was conducted using digital spatial profiling (DSP). It was found that 20% of the tumors were SARIFA-positive. Kappa values between the three pathologists were good in both collections: 0.74 and 0.78. Patients who presented SARIFA-positive tumors had a significantly lower overall survival in Collections A (median: 20.0 versus 44.0 months; p = 0.014, n = 160) and B (median: 15.0 versus 41.0 months; p < 0.0001, n = 320). SARIFA positivity emerged as a negative independent prognostic factor for overall survival (HR 1.638, 95% CI 1.153-2.326, p = 0.006). Using DSP, the most upregulated genes in SARIFA-positive cases were those associated with triglyceride catabolism and endogenous sterols. COL15A1, FABP2, and FABP4 were differentially expressed in positive cases. At the protein level, the expression of proteins related to lipid metabolism was confirmed. SARIFA combines low inter-observer variability, minimal effort, and high prognostic relevance, and is therefore an extremely promising biomarker related to tumor-promoting adipocytes in gastric cancer. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland., (© 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.)

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.

Background: COVID-19 is only partly understood, and the level of evidence available in terms of pathophysiology, epidemiology, therapy, and long-term outcome remains limited. During the early phase of the pandemic, it was necessary to effectively investigate all aspects of this new disease. Autopsy can be a valuable procedure to investigate the internal organs with special techniques to obtain information on the disease, especially the distribution and type of organ involvement., Methods: During the first wave of COVID-19 in Germany, autopsies of 19 deceased patients were performed. Besides gross examination, the organs were analyzed with standard histology and polymerase-chain-reaction for SARS-CoV-2. Polymerase chain reaction positive localizations were further analyzed with immunohistochemistry and RNA-in situ hybridization for SARS-CoV-2., Results: Eighteen of 19 patients were found to have died due to COVID-19. Clinically relevant histological changes were only observed in the lungs. Diffuse alveolar damage in considerably different degrees was noted in 18 cases. Other organs, including the central nervous system, did not show specific micromorphological alterations. In terms of SARS-CoV-2 detection, the focus remains on the upper airways and lungs. This is true for both the number of positive samples and the viral load. A highly significant inverse correlation between the stage of diffuse alveolar damage and viral load was found on a case and a sample basis. Mediastinal lymph nodes and fat were also affected by the virus at high frequencies. By contrast, other organs rarely exhibited a viral infection. Moderate to strong correlations between the methods for detecting SARS-CoV-2 were observed for the lungs and for other organs., Conclusions: The lung is the most affected organ in gross examination, histology and polymerase chain reaction. SARS-CoV-2 detection in other organs did not reveal relevant or specific histological changes. Moreover, we did not find CNS involvement., Competing Interests: The authors have declared that no competing interests exist.

Before initiating treatment of advanced non-small-cell lung cancer with tyrosine kinase inhibitors (eg, erlotinib, gefitinib, osimertinib, and afatinib), which inhibit the catalytic activity of epidermal growth factor receptor (EGFR), clinical guidelines require determining the EGFR mutational status for activating (EGFR exons 18, 19, 20, or 21) and resistance (EGFR exon 20) mutations. The EGFR resistance mutation T790M should be monitored at cancer progression. The Idylla EGFR Mutation Assay, performed on the Idylla molecular diagnostics platform, is a fully automated (<2.5 hours turnaround time) sample-to-result molecular test to qualitatively detect 51 EGFR oncogene point mutations, deletions, or insertions. In a 15-center evaluation, Idylla results on 449 archived formalin-fixed, paraffin-embedded tissue sections, originating from non-small-cell lung cancer biopsies and resection specimens, were compared with data obtained earlier with routine reference methods, including next-generation sequencing, Sanger sequencing, pyrosequencing, mass spectrometry, and PCR-based assays. When results were discordant, a third method of analysis was performed, when possible, to confirm test results. After confirmation testing and excluding invalids/errors and discordant results by design, a concordance of 97.6% was obtained between Idylla and routine test results. Even with <10 mm 2 of tissue area, a valid Idylla result was obtained in 98.9% of the cases. The Idylla EGFR Mutation Assay enables sensitive detection of most relevant EGFR mutations in concordance with current guidelines, with minimal molecular expertise or infrastructure., (Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose: Gastrointestinal stromal tumors (GISTs) are the most common soft tissue tumors of the GI tract. Studies have been published reporting additional neoplasms in GIST patients. This study aimed to evaluate possible associations of mutation type, morphology, and clinical aspects of GISTs., Methods: All cases of GIST were identified from our pathology files. Coding exons of KIT and PDGFRA in GISTs with additional malignancies were sequenced., Results: A total of 70 of 188 (37%) retrieved patients with confirmed diagnosis of GIST showed at least one additional malignant neoplasm. Fifty of these GISTs were located in the stomach (71%), 8 in the small intestine (11%), 5 in the colon/rectum (7%), and 7 cases (6.2%) were of undetermined sites of origin. The distribution of identified mutations was similar to that described in GISTs without secondary malignancies. A total of 37 of 57 cases (65%) showed mutations in the KIT gene exon 11, 3 (5%) cases in exon 9, and 1 (2%) case in exon 13. Nine tumors (16%) had mutations of the PDGFRA gene. KIT and PDGFRA wild-type status were found in seven cases (12%). Most of the secondary neoplasms were located within the GI tract (34%), in the urogenital system (24%), or the breast/female genital tract (20%)., Conclusion: This study confirms the high rate of additional malignant tumors in GIST patients. GIST features in these cases are very similar to those with sole GIST.

Two-component systems constitute phosphotransfer signaling pathways and enable adaptation to environmental changes, an essential feature for bacterial survival. The general stress response (GSR) in the plant-protecting alphaproteobacterium Sphingomonas melonis Fr1 involves a two-component system consisting of multiple stress-sensing histidine kinases (Paks) and the response regulator PhyR; PhyR in turn regulates the alternative sigma factor EcfG, which controls expression of the GSR regulon. While Paks had been shown to phosphorylate PhyR in vitro, it remained unclear if and under which conditions direct phosphorylation happens in the cell, as Paks also phosphorylate the single domain response regulator SdrG, an essential yet enigmatic component of the GSR signaling pathway. Here, we analyze the role of SdrG and investigate an alternative function of the membrane-bound PhyP (here re-designated PhyT), previously assumed to act as a PhyR phosphatase. In vitro assays show that PhyT transfers a phosphoryl group from SdrG to PhyR via phosphoryl transfer on a conserved His residue. This finding, as well as complementary GSR reporter assays, indicate the participation of SdrG and PhyT in a Pak-SdrG-PhyT-PhyR phosphorelay. Furthermore, we demonstrate complex formation between PhyT and PhyR. This finding is substantiated by PhyT-dependent membrane association of PhyR in unstressed cells, while the response regulator is released from the membrane upon stress induction. Our data support a model in which PhyT sequesters PhyR, thereby favoring Pak-dependent phosphorylation of SdrG. In addition, PhyT assumes the role of the SdrG-phosphotransferase to activate PhyR. Our results place SdrG into the GSR signaling cascade and uncover a dual role of PhyT in the GSR.

Two-component systems are major signal transduction pathways, which consist of histidine kinases and response regulators that communicate through phosphorylation. Here, we highlight a distinct class of single-domain response regulators containing the PFXFATG[G/Y] motif that are activated by a mechanism distinct from the Y-T coupling described for prototypical receiver domains. We first solved the structures of inactive and active SdrG, a representative of the FAT GUY family, and then biochemically and genetically characterized variants in which residues in this motif were mutated. Our results support a model of activation mainly driven by a conserved lysine and reveal that the rotation of the threonine induces the reorganization of several aromatic residues in and around the PFXFATG[G/Y] motif to generate intermediates resembling those occurring during classical Y-T coupling. Overall, this helps define a new subfamily of response regulators that emerge as important players in physiological adaptation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Unlabelled: 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., (Copyright © 2015 Fritz et al.)

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., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

In Firmicutes bacteria, ATP-binding cassette (ABC) transporters have been recognized as important resistance determinants against antimicrobial peptides. Together with neighboring two-component systems (TCSs), which regulate their expression, they form specific detoxification modules. Both the transport permease and sensor kinase components show unusual domain architecture: the permeases contain a large extracellular domain, while the sensor kinases lack an obvious input domain. One of the best-characterized examples is the bacitracin resistance module BceRS-BceAB of Bacillus subtilis. Strikingly, in this system, the ABC transporter and TCS have an absolute mutual requirement for each other in both sensing of and resistance to bacitracin, suggesting a novel mode of signal transduction in which the transporter constitutes the actual sensor. We identified over 250 such BceAB-like ABC transporters in the current databases. They occurred almost exclusively in Firmicutes bacteria, and 80% of the transporters were associated with a BceRS-like TCS. Phylogenetic analyses of the permease and sensor kinase components revealed a tight evolutionary correlation. Our findings suggest a direct regulatory interaction between the ABC transporters and TCSs, mediating communication between both components. Based on their observed coclustering and conservation of response regulator binding sites, we could identify putative corresponding two-component systems for transporters lacking a regulatory system in their immediate neighborhood. Taken together, our results show that these types of ABC transporters and TCSs have coevolved to form self-sufficient detoxification modules against antimicrobial peptides, widely distributed among Firmicutes bacteria.