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T lymphocytes (T cells) represent one of the major cell populations of the immune system. Named by the place of their development, the thymus, several types can be distinguished as the αβ T cells, the γδ T cells, the mucosa-associated invariant T cells (MAIT), and the natural killer T (NKT) cells. The αβ lineages of CD4+ THelper and the CD8+ T cytotoxic cells with the T cell receptor (TCR) composed of α- and β-chain are major players of the adaptive immune system. In the thymus, CD4+ and CD8+ single positive (SP) αβ cells represent the ultimate result of positive and negative selection of CD4+CD8+ double positive (DP) thymocytes. The DP population derives from the double negative (DN) thymocytes that develop from bone marrow-derived progenitors through different stages (DN1-DN4) that are characterized by CD25 and CD44 surface expression.
NFATc1, a member of the Nuclear Factor of Activated T cells (NFAT) transcription factors family, is critically involved in the differentiation and function of T cells. During thymocyte development, the nuclear expression of NFATc1 reaches the highest level at the DN3 (CD44-CD25+) stage. The hematopoietic cell-specific ablation of NFATc1 activity results in an arrest of thymocyte differentiation at the DN1 (CD44+CD25-) stage. On the other hand, over-expression of a constitutively active version of NFATc1 results in an impaired transition of DN3 cells to the DN4 (CD44-CD25-) stage, suggesting that a certain threshold level of NFATc1 activity is critical at this point.
ChIP-seq and RNA-seq analysis allowed us the identification of NFATc1/A target genes involved in lineage development as the Tcra and Tcrb gene loci. Furthermore, we identified multiple NFATc1-regulated genes that are involved in γδ T cell development. In the mouse models, Rag1Cre-Nfatc1fl/fl and Rag1Cre-E2fl/fl, in which the activity of NFATc1 or inducible NFATc1 in the latter is impaired during the early stages of thymocyte development, we observed increased numbers of γδ T cells. These γδ T cells showed an unusual overexpression of CD4, a lack of CD24 expression, and overexpression of the anti-apoptotic gene Bcl2a1a.
We hypothesize that during the DN stages NFATc1 plays an important role in regulating crucial steps of αβ thymocyte development and when NFATc1 activity is missing this may disturb αβ development resulting in alternative cell fates like γδ T cells.
Aims
To investigate Epstein‐Barr virus (EBV) latency types in 19 cases of EBV‐positive nodular lymphocyte‐predominant Hodgkin lymphoma (NLPHL), as such information is currently incomplete.
Methods and results
Immunohistochemistry (IHC) for CD20, CD79a, PAX5, OCT2, CD30, CD15, CD3 and programmed cell death protein 1 was performed. For EBV detection, in‐situ hybridisation (ISH) for EBV‐encoded RNA (EBER) was employed combined with IHC for EBV‐encoded latent membrane protein (LMP)‐1, EBV‐encoded nuclear antigen (EBNA)‐2, and EBV‐encoded BZLF1. In 95% of the cases, neoplastic cells with features of Hodgkin and Reed–Sternberg (HRS) cells were present, mostly showing expression of CD30. In all cases, the B‐cell phenotype was largely intact, and delineation from classic Hodgkin lymphoma (CHL) was further supported by myocyte enhancer factor 2B (MEF2B) detection. All tumour cells were EBER‐positive except in two cases. EBV latency type II was most frequent (89%) and type I was rare. Cases with latency type I were CD30‐negative. Five cases contained some BZLF1‐positive and/or EBNA‐2‐positive bystander lymphocytes.
Conclusions
As HRS morphology of neoplastic cells and CD30 expression are frequent features of EBV‐positive NLPHL, preservation of the B‐cell transcription programme, MEF2B expression combined with NLPHL‐typical architecture and background composition facilitate distinction from CHL. EBER ISH is the method of choice to identify these cases. The majority present with EBV latency type II, and only rare cases present with latency type I, which can be associated with missing CD30 expression. The presence of occasional bystander lymphocytes expressing BZLF1 and/or EBNA‐2 and the partial EBV infection of neoplastic cells in some cases could indicate that EBV is either not primarily involved or is only a transient driver in the pathogenesis of EBV‐positive NLPHL.
The defense against invading pathogens is, amongst other things, mediated via the action of antibodies. Class-switched antibodies and antibodies of high affinity are produced by plasma cells descending from germinal center B (GCB) cells. GCB cells develop in the germinal center (GC), a specialized microstructure found in the B-cell follicle of secondary lymphoid organs. GCB-cell maturation and proliferation are supported by follicular T- helper (Tfh) cells. On the other hand, follicular regulatory T (Tfr) cells control this process in quantity and quality preventing, for instance, the formation of autoantibodies directed against endogenous structures. The development of GCB, Tfh and Tfr cells essentially depends on the migration into the GC, which is mediated via the expression of the chemokine receptor CXCR5.
One transcription factor highly expressed in follicular T cells, comprising Tfh and Tfr cells, is NFATc1. Tfr cells additionally express the transcriptional repressor Blimp-1, which is not expressed in Tfh cells. We found that NFATc1 is transactivating Cxcr5 via response elements in the promoter and enhancer in vitro. Blimp-1 binds to the same elements, transactivating Cxcr5 expression in cooperation with NFATc1, whilst mediating Cxcr5- repression on its own. In Tfr cells Blimp-1 suppresses CXCR5 expression in the absence of NFATc1. Blimp-1 itself is necessary to restrict Tfr-cell frequencies and to mediate Tfr- cell function as in mice with Blimp-1-ablated Tregs high frequencies of Tfr cells do not reduce GCB- or Tfh cell frequencies. NFATc1 and Blimp-1 double deficient Tfr cells show additional loss of function, which becomes visible in clearly expanded antibody titers.
To evaluate the function of NFATc1 in Tfr cells, we not only deleted it, but also overexpressed a constitutive active form of NFATc1/aA (caNFATc1/aA) in regulatory T cells (Tregs). The latter is leading to an upregulation of CXCR5 per cell, without changing Tfh or Tfr-cell frequencies. However, the high density of surface CXCR5 enhances the migration of Tfr cells deep into the GC, which results in a tighter control of the antigen- specific humoral immune response. Additionally, caNFATc1/aA increases the expression of genes coding for Tfr effector molecules like Il1rn, Il10, Tigit and Ctla4. Interestingly, this part of the transcriptional change is dependent on the presence of Blimp-1. Furthermore, Blimp-1 regulates the expression of multiple chemokine receptor genes on the background of caNFATc1/aA.
In contrast, when caNFATc1/aA is overexpressed in all T cells, the frequencies of Tfh- and GCB cells are dominantly reduced. This effect seems to stem from the conventional T- cell (Tcon) side, most probably originating from increased secretion of interleukin-2 (IL- 2) via the caNFATc1/aA overexpressing Tcons. IL-2 is known to hinder the germinal center reaction (GCR) and it might in its abundance not be neutralizable by Tfr cells.
Taken together, NFATc1 and Blimp-1 cooperate to control the migration of Tfr cells into the GC. Tfr cells in the GC depend on NFATc1 and Blimp-1 to perform their proper function. Overexpression of caNFATc1 in Tregs strengthens Tfr function in a Blimp-1-dependent manner, whilst overexpression of caNFATc1 in all T cells dominantly diminishes the GCR.
Squamous cell carcinomas (SCC) frequently have an exceptionally high mutational burden. As consequence, they rapidly develop resistance to platinum-based chemotherapy and overall survival is limited. Novel therapeutic strategies are therefore urgently required. SCC express ∆Np63, which regulates the Fanconi Anemia (FA) DNA-damage response in cancer cells, thereby contributing to chemotherapy-resistance. Here we report that the deubiquitylase USP28 is recruited to sites of DNA damage in cisplatin-treated cells. ATR phosphorylates USP28 and increases its enzymatic activity. This phosphorylation event is required to positively regulate the DNA damage repair in SCC by stabilizing ∆Np63. Knock-down or inhibition of USP28 by a specific inhibitor weakens the ability of SCC to cope with DNA damage during platin-based chemotherapy. Hence, our study presents a novel mechanism by which ∆Np63 expressing SCC can be targeted to overcome chemotherapy resistance. Limited treatment options and low response rates to chemotherapy are particularly common in patients with squamous cancer. The SCC specific transcription factor ∆Np63 enhances the expression of Fanconi Anemia genes, thereby contributing to recombinational DNA repair and Cisplatin resistance. Targeting the USP28-∆Np63 axis in SCC tones down this DNA damage response pathways, thereby sensitizing SCC cells to cisplatin treatment.
Glioblastoma leads to a fatal course within two years in more than two thirds of patients. An essential cornerstone of therapy is chemotherapy with temozolomide (TMZ). The effect of TMZ is counteracted by the cellular repair enzyme O\(^6\)-methylguanine-DNA methyltransferase (MGMT). The MGMT promoter methylation, the main regulator of MGMT expression, can change from primary tumor to recurrence, and TMZ may play a significant role in this process. To identify the potential mechanisms involved, three primary stem-like cell lines (one astrocytoma with the mutation of the isocitrate dehydrogenase (IDH), CNS WHO grade 4 (HGA)), and two glioblastoma (IDH-wildtype, CNS WHO grade 4) were treated with TMZ. The MGMT promoter methylation, migration, proliferation, and TMZ-response of the tumor cells were examined at different time points. The strong effects of TMZ treatment on the MGMT methylated cells were observed. Furthermore, TMZ led to a loss of the MGMT promoter hypermethylation and induced migratory rather than proliferative behavior. Cells with the unmethylated MGMT promoter showed more aggressive behavior after treatment, while HGA cells reacted heterogenously. Our study provides further evidence to consider the potential adverse effects of TMZ chemotherapy and a rationale for investigating potential relationships between TMZ treatment and change in the MGMT promoter methylation during relapse.
Targeting molecular alterations as an effective treatment for isocitrate dehydrogenase-wildtype glioblastoma (GBM) patients has not yet been established. Sterol-O-Acyl Transferase 1 (SOAT1), a key enzyme in the conversion of endoplasmic reticulum cholesterol to esters for storage in lipid droplets (LD), serves as a target for the orphan drug mitotane to treat adrenocortical carcinoma. Inhibition of SOAT1 also suppresses GBM growth. Here, we refined SOAT1-expression in GBM and IDH-mutant astrocytoma, CNS WHO grade 4 (HGA), and assessed the distribution of LD in these tumors. Twenty-seven GBM and three HGA specimens were evaluated by multiple GFAP, Iba1, IDH1 R132H, and SOAT1 immunofluorescence labeling as well as Oil Red O staining. To a small extent SOAT1 was expressed by tumor cells in both tumor entities. In contrast, strong expression was observed in glioma-associated macrophages. Triple immunofluorescence labeling revealed, for the first time, evidence for SOAT1 colocalization with Iba1 and IDH1 R132H, respectively. Furthermore, a notable difference in the amount of LD between GBM and HGA was observed. Therefore, SOAT1 suppression might be a therapeutic option to target GBM and HGA growth and invasiveness. In addition, the high expression in cells related to neuroinflammation could be beneficial for a concomitant suppression of protumoral microglia/macrophages.
Macroautophagy (hereafter referred to as autophagy) is a homeostatic process that preserves cellular integrity. In mice, autophagy regulates pancreatic ductal adenocarcinoma (PDAC) development in a manner dependent on the status of the tumor suppressor gene Trp53. Studies published so far have investigated the impact of autophagy blockage in tumors arising from Trp53-hemizygous or -homozygous tissue. In contrast, in human PDACs the tumor suppressor gene TP53 is mutated rather than allelically lost, and TP53 mutants retain pathobiological functions that differ from complete allelic loss. In order to better represent the patient situation, we have investigated PDAC development in a well-characterized genetically engineered mouse model (GEMM) of PDAC with mutant Trp53 (Trp53\(^{R172H}\)) and deletion of the essential autophagy gene Atg7. Autophagy blockage reduced PDAC incidence but had no impact on survival time in the subset of animals that formed a tumor. In the absence of Atg7, non-tumor-bearing mice reached a similar age as animals with malignant disease. However, the architecture of autophagy-deficient, tumor-free pancreata was effaced, normal acinar tissue was largely replaced with low-grade pancreatic intraepithelial neoplasias (PanINs) and insulin expressing islet β-cells were reduced. Our data add further complexity to the interplay between Atg7 inhibition and Trp53 status in tumorigenesis.
Ribosomal biogenesis and protein synthesis are deregulated in most cancers, suggesting that interfering with translation machinery may hold significant therapeutic potential. Here, we show that loss of the tumor suppressor adenomatous polyposis coli (APC), which constitutes the initiating event in the adenoma carcinoma sequence for colorectal cancer (CRC), induces the expression of RNA polymerase I (RNAPOL1) transcription machinery, and subsequently upregulates ribosomal DNA (rDNA) transcription. Targeting RNAPOL1 with a specific inhibitor, CX5461, disrupts nucleolar integrity, and induces a disbalance of ribosomal proteins. Surprisingly, CX5461-induced growth arrest is irreversible and exhibits features of senescence and terminal differentiation. Mechanistically, CX5461 promotes differentiation in an MYC-interacting zinc-finger protein 1 (MIZ1)- and retinoblastoma protein (Rb)-dependent manner. In addition, the inhibition of RNAPOL1 renders CRC cells vulnerable towards senolytic agents. We validated this therapeutic effect of CX5461 in murine- and patient-derived organoids, and in a xenograft mouse model. These results show that targeting ribosomal biogenesis together with targeting the consecutive, senescent phenotype using approved drugs is a new therapeutic approach, which can rapidly be transferred from bench to bedside.
Background: Treatment options for NAFLD are still limited. Bariatric surgery, such as Roux-en-Y gastric bypass (RYGB), has been shown to improve metabolic and histologic markers of NAFLD. Glucagon-like-peptide-1 (GLP-1) analogues lead to improvements in phase 2 clinical trials. We directly compared the effects of RYGB with a treatment using liraglutide and/or peptide tyrosine tyrosine 3-36 (PYY\(_{3-36}\)) in a rat model for early NAFLD. Methods: Obese male Wistar rats (high-fat diet (HFD)-induced) were randomized into the following treatment groups: RYGB, sham-operation (sham), liraglutide (0.4 mg/kg/day), PYY\(_{3-36}\) (0.1 mg/kg/day), liraglutide+PYY\(_{3-36}\), and saline. After an observation period of 4 weeks, liver samples were histologically evaluated, ELISAs and RNA sequencing + RT-qPCRs were performed. Results: RYGB and liraglutide+PYY\(_{3-36}\) induced a similar body weight loss and, compared to sham/saline, marked histological improvements with significantly less steatosis. However, only RYGB induced significant metabolic improvements (e.g., adiponectin/leptin ratio 18.8 ± 11.8 vs. 2.4 ± 1.2 in liraglutide+PYY\(_{3-36}\)- or 1.4 ± 0.9 in sham-treated rats). Furthermore, RNA sequencing revealed a high number of differentially regulated genes in RYGB treated animals only. Conclusions: The combination therapy of liraglutide+PYY\(_{3-36}\) partly mimics the positive effects of RYGB on weight reduction and on hepatic steatosis, while its effects on metabolic function lack behind RYGB.
Neurodegeneration by α-synuclein-specific T cells in AAV-A53T-α-synuclein Parkinson’s disease mice
(2022)
Background
Antigen-specific neuroinflammation and neurodegeneration are characteristic for neuroimmunological diseases. In Parkinson’s disease (PD) pathogenesis, α-synuclein is a known culprit. Evidence for α-synuclein-specific T cell responses was recently obtained in PD. Still, a causative link between these α-synuclein responses and dopaminergic neurodegeneration had been lacking. We thus addressed the functional relevance of α-synuclein-specific immune responses in PD in a mouse model.
Methods
We utilized a mouse model of PD in which an Adeno-associated Vector 1/2 serotype (AAV1/2) expressing human mutated A53T-α-Synuclein was stereotactically injected into the substantia nigra (SN) of either wildtype C57BL/6 or Recombination-activating gene 1 (RAG1)\(^{-/-}\) mice. Brain, spleen, and lymph node tissues from different time points following injection were then analyzed via FACS, cytokine bead assay, immunohistochemistry and RNA-sequencing to determine the role of T cells and inflammation in this model. Bone marrow transfer from either CD4\(^{+}\)/CD8\(^{-}\), CD4\(^{-}\)/CD8\(^{+}\), or CD4\(^{+}\)/CD8\(^{+}\) (JHD\(^{-/-}\)) mice into the RAG-1\(^{-/-}\) mice was also employed. In addition to the in vivo studies, a newly developed A53T-α-synuclein-expressing neuronal cell culture/immune cell assay was utilized.
Results
AAV-based overexpression of pathogenic human A53T-α-synuclein in dopaminergic neurons of the SN stimulated T cell infiltration. RNA-sequencing of immune cells from PD mouse brains confirmed a pro-inflammatory gene profile. T cell responses were directed against A53T-α-synuclein-peptides in the vicinity of position 53 (68–78) and surrounding the pathogenically relevant S129 (120–134). T cells were required for α-synuclein-induced neurodegeneration in vivo and in vitro, while B cell deficiency did not protect from dopaminergic neurodegeneration.
Conclusions
Using T cell and/or B cell deficient mice and a newly developed A53T-α-synuclein-expressing neuronal cell culture/immune cell assay, we confirmed in vivo and in vitro that pathogenic α-synuclein peptide-specific T cell responses can cause dopaminergic neurodegeneration and thereby contribute to PD-like pathology.
Purpose
Patients suffering from aggressive systemic peripheral lymphoma with primary central nervous system involvement (PCL) are a rare and sparsely investigated population. Recommended treatment regimens include a combination of intrathecal and systemic chemotherapy as well as whole brain radiotherapy while offering relatively poor survival.
Methods
We conducted a single-center retrospective study that analyzed safety and outcome of 4 + 4 cycles Rituximab (R)-CHOP and R-high-dose Methotrexate (HD-MTX) for newly diagnosed, transplant-eligible patients ("Ping-Pong"), followed by Cytarabine (AraC)/Thiotepa (TT), BCNU/TT, and autologous hematologic stem cell transplantation (aHSCT). We retrospectively analyzed a set of 16 patients with high-intermediate or high-risk IPI status.
Results
Overall response rate to Ping-Pong was 100% measured by CT/MRI, including 93.75% complete remissions after BCNU/TT followed by PBSCT. One patient failed to qualify for high-dose chemotherapy due to progression when receiving Cytarabine/TT. All patients experienced grade III adverse events, 3 of them a grade IV adverse event. Estimated progression-free survival is 93.75% after a 4.8-year follow-up currently.
Conclusion
Our study suggests high effectivity of R-CHOP with mid-cycle MTX with aHSCT consolidation towards acceptable OS results in this challenging patient population.
Personalized oncology is a rapidly evolving area and offers cancer patients therapy options that are more specific than ever. However, there is still a lack of understanding regarding transcriptomic similarities or differences of metastases and corresponding primary sites. Applying two unsupervised dimension reduction methods (t-Distributed Stochastic Neighbor Embedding (t-SNE) and Uniform Manifold Approximation and Projection (UMAP)) on three datasets of metastases (n = 682 samples) with three different data transformations (unprocessed, log10 as well as log10 + 1 transformed values), we visualized potential underlying clusters. Additionally, we analyzed two datasets (n = 616 samples) containing metastases and primary tumors of one entity, to point out potential familiarities. Using these methods, no tight link between the site of resection and cluster formation outcome could be demonstrated, or for datasets consisting of solely metastasis or mixed datasets. Instead, dimension reduction methods and data transformation significantly impacted visual clustering results. Our findings strongly suggest data transformation to be considered as another key element in the interpretation of visual clustering approaches along with initialization and different parameters. Furthermore, the results highlight the need for a more thorough examination of parameters used in the analysis of clusters.
Animal models are important tools to investigate the pathogenesis and develop treatment strategies for breast cancer in humans. In this study, we developed a new three-dimensional in vivo arteriovenous loop model of human breast cancer with the aid of biodegradable materials, including fibrin, alginate, and polycaprolactone. We examined the in vivo effects of various matrices on the growth of breast cancer cells by imaging and immunohistochemistry evaluation. Our findings clearly demonstrate that vascularized breast cancer microtissues could be engineered and recapitulate the in vivo situation and tumor-stromal interaction within an isolated environment in an in vivo organism. Alginate–fibrin hybrid matrices were considered as a highly powerful material for breast tumor engineering based on its stability and biocompatibility. We propose that the novel tumor model may not only serve as an invaluable platform for analyzing and understanding the molecular mechanisms and pattern of oncologic diseases, but also be tailored for individual therapy via transplantation of breast cancer patient-derived tumors.
The transcription factors of the nuclear factor of activated T cell (NFAT) family play a crucial role in multiple aspects of T cell function. It has recently been reported that NFATs play an important role in the suppressive function of CD4\(^+\)CD25\(^+\)Foxp3\(^+\) regulatory T (T\(_{reg}\)) cells. In this study, we have investigated the role of NFATs in the thymic development of T\(_{reg}\) cells in mice. We show that NFAT factors are dispensable for the development of Foxp3\(^+\) T\(_{reg}\) cells in the thymus but are critical for the maintenance of both the phenotype and survival of T\(_{reg}\) cells in the thymus as well as in peripheral lymphoid organs. Specifically, the homeostasis of CD4\(^+\)CD25\(^+\)Foxp3\(^+\) but not the CD4\(^+\)CD25\(^-\)Foxp3\(^+\) fraction is severely perturbed when NFAT signaling is blocked, leading to a strongly reduced T\(_{reg}\) population. We underscored this intriguing effect of NFAT on CD4\(^+\)CD25\(^+\)Foxp3\(^+\) T\(_{reg}\) cells to the disruption of survival signals provided by interleukin 2 (IL-2). Accordingly, blocking T\(_{reg}\) cell death by abolishing the activity of pro-apoptotic Bcl-2 family member Bim, compensated for the survival defects induced due to a lack of NFAT-IL-2-IL-2R signaling. Inhibition of NFAT activity led to a strong reduction in the number of Foxp3\(^+\) T\(_{reg}\) cells; however, it did not influence the level of Foxp3 expression on an individual cell basis. In addition, we show a differential effect of IL-2 and IL-7 signaling on Foxp3\(^+\) T\(_{reg}\) versus CD4\(^+\)CD25\(^-\) T cell development, again underlining the dispensability of NFAT signaling in the development, but not in the maintenance of Foxp3\(^+\) T\(_{reg}\) cells.
Background
Despite advances in treatment of patients with non-small cell lung cancer, carriers of certain genetic alterations are prone to failure. One such factor frequently mutated, is the tumor suppressor PTEN. These tumors are supposed to be more resistant to radiation, chemo- and immunotherapy.
Results
We demonstrate that loss of PTEN led to altered expression of transcriptional programs which directly regulate therapy resistance, resulting in establishment of radiation resistance. While PTEN-deficient tumor cells were not dependent on DNA-PK for IR resistance nor activated ATR during IR, they showed a significant dependence for the DNA damage kinase ATM. Pharmacologic inhibition of ATM, via KU-60019 and AZD1390 at non-toxic doses, restored and even synergized with IR in PTEN-deficient human and murine NSCLC cells as well in a multicellular organotypic ex vivo tumor model.
Conclusion
PTEN tumors are addicted to ATM to detect and repair radiation induced DNA damage. This creates an exploitable bottleneck. At least in cellulo and ex vivo we show that low concentration of ATM inhibitor is able to synergise with IR to treat PTEN-deficient tumors in genetically well-defined IR resistant lung cancer models.
Differences in stem cell marker and osteopontin expression in primary and recurrent glioblastoma
(2022)
Background
Despite of a multimodal approach, recurrences can hardly be prevented in glioblastoma. This may be in part due to so called glioma stem cells. However, there is no established marker to identify these stem cells.
Methods
Paired samples from glioma patients were analyzed by immunohistochemistry for expression of the following stem cell markers: CD133, Musashi, Nanog, Nestin, octamer-binding transcription factor 4 (Oct4), and sex determining region Y-box 2 (Sox2). In addition, the expression of osteopontin (OPN) was investigated. The relative number of positively stained cells was determined. By means of Kaplan–Meier analysis, a possible association with overall survival by marker expression was investigated.
Results
Sixty tissue samples from 30 patients (17 male, 13 female) were available for analysis. For Nestin, Musashi and OPN a significant increase was seen. There was also an increase (not significant) for CD133 and Oct4. Patients with mutated Isocitrate Dehydrogenase-1/2 (IDH-1/2) status had a reduced expression for CD133 and Nestin in their recurrent tumors. Significant correlations were seen for CD133 and Nanog between OPN in the primary and recurrent tumor and between CD133 and Nestin in recurrent tumors. By confocal imaging we could demonstrate a co-expression of CD133 and Nestin within recurrent glioma cells. Patients with high CD133 expression had a worse prognosis (22.6 vs 41.1 months, p = 0.013). A similar trend was seen for elevated Nestin levels (24.9 vs 41.1 months, p = 0.08).
Conclusions
Most of the evaluated markers showed an increased expression in their recurrent tumor. CD133 and Nestin were associated with survival and are candidate markers for further clinical investigation.
Oncogenic transformation of lung epithelial cells is a multistep process, frequently starting with the inactivation of tumour suppressors and subsequent development of activating mutations in proto-oncogenes, such as members of the PI3K or MAPK families. Cells undergoing transformation have to adjust to changes, including altered metabolic requirements. This is achieved, in part, by modulating the protein abundance of transcription factors. Here, we report that the ubiquitin carboxyl-terminal hydrolase 28 (USP28) enables oncogenic reprogramming by regulating the protein abundance of proto-oncogenes such as c-JUN, c-MYC, NOTCH and ∆NP63 at early stages of malignant transformation. USP28 levels are increased in cancer compared with in normal cells due to a feed-forward loop, driven by increased amounts of oncogenic transcription factors such as c-MYC and c-JUN. Irrespective of oncogenic driver, interference with USP28 abundance or activity suppresses growth and survival of transformed lung cells. Furthermore, inhibition of USP28 via a small-molecule inhibitor resets the proteome of transformed cells towards a ‘premalignant’ state, and its inhibition synergizes with clinically established compounds used to target EGFR\(^{L858R}\)-, BRAF\(^{V600E}\)- or PI3K\(^{H1047R}\)-driven tumour cells. Targeting USP28 protein abundance at an early stage via inhibition of its activity is therefore a feasible strategy for the treatment of early-stage lung tumours, and the observed synergism with current standard-of-care inhibitors holds the potential for improved targeting of established tumours.
Altered features of tumor cells acquired across therapy can result in the survival of treatment-resistant clones that may cause minimal residual disease (MRD). Despite the efficacy of ibrutinib in treating relapsed/refractory mantle cell lymphoma, the obstacle of residual cells contributes to relapses of this mature B-cell neoplasm, and the disease remains incurable. RNA-seq analysis of an ibrutinib-sensitive mantle cell lymphoma cell line following ibrutinib incubation of up to 4 d, corroborated our previously postulated resistance mechanism of a metabolic switch to reliance on oxidative phosphorylation (OXPHOS) in surviving cells. Besides, we had shown that treatment-persisting cells were characterized by increased CD52 expression. Therefore, we hypothesized that combining ibrutinib with another agent targeting these potential escape mechanisms could minimize the risk of survival of ibrutinib-resistant cells. Concomitant use of ibrutinib with OXPHOS-inhibitor IACS-010759 increased toxicity compared to ibrutinib alone. Targeting CD52 was even more efficient, as addition of CD52 mAb in combination with human serum following ibrutinib pretreatment led to rapid complement-dependent-cytotoxicity in an ibrutinib-sensitive cell line. In primary mantle cell lymphoma cells, a higher toxic effect with CD52 mAb was obtained, when cells were pretreated with ibrutinib, but only in an ibrutinib-sensitive cohort. Given the challenge of treating multi-resistant mantle cell lymphoma patients, this work highlights the potential use of anti-CD52 therapy as consolidation after ibrutinib treatment in patients who responded to the BTK inhibitor to achieve MRD negativity and prolong progression-free survival.
Diabetes Mellitus Is a Possible Risk Factor for Nodo-paranodopathy With Antiparanodal Autoantibodies
(2022)
Background and Objectives
Nodo-paranodopathies are peripheral neuropathies with dysfunction of the node of Ranvier. Affected patients who are seropositive for antibodies against adhesion molecules like contactin-1 and neurofascin show distinct clinical features and a disruption of the paranodal complex. An axoglial dysjunction is also a characteristic finding of diabetic neuropathy. Here, we aim to investigate a possible association of antibody-mediated nodo-paranodopathy and diabetes mellitus (DM).
Methods
We retrospectively analyzed clinical data of 227 patients with chronic inflammatory demyelinating polyradiculoneuropathy and Guillain-Barré syndrome from multiple centers in Germany who had undergone diagnostic testing for antiparanodal antibodies targeting neurofascin-155, pan-neurofascin, contactin-1–associated protein 1, and contactin-1. To study possible direct pathogenic effects of antiparanodal antibodies, we performed immunofluorescence binding assays on human pancreatic tissue sections.
Results The frequency of DM was 33.3% in seropositive patients and thus higher compared with seronegative patients (14.1%, OR = 3.04, 95% CI = 1.31–6.80). The relative risk of DM in seropositive patients was 3.4-fold higher compared with the general German population. Seropositive patients with DM most frequently harbored anti–contactin-1 antibodies and had higher antibody titers than seropositive patients without DM. The diagnosis of DM preceded the onset of neuropathy in seropositive patients. No immunoreactivity of antiparanodal antibodies against pancreatic tissue was detected.
Discussion
We report an association of nodo-paranodopathy and DM. Our results suggest that DM may be a potential risk factor for predisposing to developing nodo-paranodopathy and argue against DM being induced by the autoantibodies. Our findings set the basis for further research investigating underlying immunopathogenetic connections.
Epithelial-to-mesenchymal transition (EMT) is discussed to be centrally involved in invasion, stemness, and drug resistance. Experimental models to evaluate this process in its biological complexity are limited. To shed light on EMT impact and test drug response more reliably, we use a lung tumor test system based on a decellularized intestinal matrix showing more in vivo-like proliferation levels and enhanced expression of clinical markers and carcinogenesis-related genes. In our models, we found evidence for a correlation of EMT with drug resistance in primary and secondary resistant cells harboring KRAS\(^{G12C}\) or EGFR mutations, which was simulated in silico based on an optimized signaling network topology. Notably, drug resistance did not correlate with EMT status in KRAS-mutated patient-derived xenograft (PDX) cell lines, and drug efficacy was not affected by EMT induction via TGF-β. To investigate further determinants of drug response, we tested several drugs in combination with a KRAS\(^{G12C}\) inhibitor in KRAS\(^{G12C}\) mutant HCC44 models, which, besides EMT, display mutations in P53, LKB1, KEAP1, and high c-MYC expression. We identified an aurora-kinase A (AURKA) inhibitor as the most promising candidate. In our network, AURKA is a centrally linked hub to EMT, proliferation, apoptosis, LKB1, and c-MYC. This exemplifies our systemic analysis approach for clinical translation of biomarker signatures.
Hematopoietic stem and progenitor cell (HSPC) maintenance and the differentiation of various lineages is a highly complex but precisely regulated process. Multiple signaling pathways and an array of transcription factors influence HSPC maintenance and the differentiation of individual lineages to constitute a functional hematopoietic system. Nuclear factor of activated T cell (NFAT) family transcription factors have been studied in the context of development and function of multiple mature hematopoietic lineage cells. However, until now their contribution in HSPC physiology and HSPC differentiation to multiple hematopoietic lineages has remained poorly understood. Here, we show that NFAT proteins, specifically NFATc1, play an indispensable role in the maintenance of HSPCs. In the absence of NFATc1, very few HSPCs develop in the bone marrow, which are functionally defective. In addition to HSPC maintenance, NFATc1 also critically regulates differentiation of lymphoid, myeloid, and erythroid lineage cells from HSPCs. Deficiency of NFATc1 strongly impaired, while enhanced NFATc1 activity augmented, the differentiation of these lineages, which further attested to the vital involvement of NFATc1 in regulating hematopoiesis. Hematopoietic defects due to lack of NFATc1 activity can lead to severe pathologies such as lymphopenia, myelopenia, and a drastically reduced lifespan underlining the critical role NFATc1 plays in HSPC maintenance and in the differentaion of various lineages. Our findings suggest that NFATc1 is a critical component of the myriad signaling and transcriptional regulators that are essential to maintain normal hematopoiesis.
High programmed cell death 1 ligand 1 (PD-L1) protein expression and copy number alterations (CNAs) of the corresponding genomic locus 9p24.1 in Hodgkin- and Reed–Sternberg cells (HRSC) have been shown to be associated with favourable response to anti-PD-1 checkpoint inhibition in relapsed/refractory (r/r) classical Hodgkin lymphoma (cHL). In the present study, we investigated baseline 9p24.1 status as well as PD-L1 and major histocompatibility complex (MHC) class I and II protein expression in 82 biopsies from patients with early stage unfavourable cHL treated with anti-PD-1-based first-line treatment in the German Hodgkin Study Group (GHSG) NIVAHL trial (ClinicalTrials.gov Identifier: NCT03004833). All evaluated specimens showed 9p24.1 CNA in HRSC to some extent, but with high intratumoral heterogeneity and an overall smaller range of alterations than reported in advanced-stage or r/r cHL. All but two cases (97%) showed PD-L1 expression by the tumour cells in variable amounts. While MHC-I was rarely expressed in >50% of HRSC, MHC-II expression in >50% of HRSC was found more frequently. No obvious impact of 9p24.1 CNA or PD-L1 and MHC-I/II expression on early response to the highly effective anti-PD-1-based NIVAHL first-line treatment was observed. Further studies evaluating an expanded panel of potential biomarkers are needed to optimally stratify anti-PD-1 first-line cHL treatment.
Active vitamin D (1,25(OH)2D3) is known to exert direct anti-cancer actions on various malignant tissues through binding to the vitamin D receptor (VDR). These effects have been demonstrated in breast, prostate, renal and thyroid cancers, which all have a high propensity to metastasise to bone. In addition, there is evidence that vitamin D catabolism via 24-hydroxylase (CYP24A1) is altered in tumour cells, thus, reducing local active vitamin D levels in cancer cells. The aim of this study was to assess VDR and CYP24A1 expression in various types of bone metastases by using immunohistochemistry. Overall, a high total VDR protein expression was detected in 59% of cases (39/66). There was a non-significant trend of high-grade tumours towards the low nuclear VDR expression (p = 0.07). Notably, patients with further distant metastases had a reduced nuclear VDR expression (p = 0.03). Furthermore, a high CYP24A1 expression was detected in 59% (39/66) of bone metastases. There was a significant positive correlation between nuclear VDR and CYP24A1 expression (p = 0.001). Collectively, the VDR and CYP24A1 were widely expressed in a multitude of bone metastases, pointing to a potential role of vitamin D signalling in cancer progression. This is of high clinical relevance, as vitamin D deficiency is frequent in patients with bone metastases.
Protocadherins (PCDHs) belong to the cadherin superfamily and represent the largest subgroup of calcium-dependent adhesion molecules. In the genome, most PCDHs are arranged in three clusters, α, β, and γ on chromosome 5q31. PCDHs are highly expressed in the central nervous system (CNS). Several PCDHs have tumor suppressor functions, but their individual role in primary brain tumors has not yet been elucidated. Here, we examined the mRNA expression of PCDHGC3, a member of the PCDHγ cluster, in non-cancerous brain tissue and in gliomas of different World Health Organization (WHO) grades and correlated it with the clinical data of the patients. We generated a PCDHGC3 knockout U343 cell line and examined its growth rate and migration in a wound healing assay. We showed that PCDHGC3 mRNA and protein were significantly overexpressed in glioma tissue compared to a non-cancerous brain specimen. This could be confirmed in glioma cell lines. High PCDHGC3 mRNA expression correlated with longer progression-free survival (PFS) in glioma patients. PCDHGC3 knockout in U343 resulted in a slower growth rate but a significantly faster migration rate in the wound healing assay and decreased the expression of several genes involved in WNT signaling. PCDHGC3 expression should therefore be further investigated as a PFS-marker in gliomas. However, more studies are needed to elucidate the molecular mechanisms underlying the PCDHGC3 effects.
Simple Summary
In glioblastoma, tumor recurrence is inevitable and the prognosis of patients is poor, despite multidisciplinary treatment approaches involving surgical resection, radiotherapy and chemotherapy. Recently, Tumor Treating Fields (TTFields) have been added to the therapeutic set-up. These alternating electric fields are applied to glioblastoma at 200 kHz frequency via arrays placed on the shaved scalp of patients. Patients show varying response to this therapy. Molecular effects of TTFields have been investigated largely in cell cultures and animal models, but not in patient tissue samples. Acquisition of matched treatment-naïve and recurrent patient tissues is a challenge. Therefore, we suggest three reliable patient-derived three-dimensional ex vivo models (primary cells grown as microtumors on murine organotypic hippocampal slices, organoids and tumor slice cultures) which may facilitate prediction of patients’ treatment responses and provide important insights into clinically relevant cellular and molecular alterations under TTFields.
Abstract
Glioblastoma (GBM) displays a wide range of inter- and intra-tumoral heterogeneity contributing to therapeutic resistance and relapse. Although Tumor Treating Fields (TTFields) are effective for the treatment of GBM, there is a lack of ex vivo models to evaluate effects on patients’ tumor biology or to screen patients for treatment efficacy. Thus, we adapted patient-derived three-dimensional tissue culture models to be compatible with TTFields application to tissue culture. Patient-derived primary cells (PDPC) were seeded onto murine organotypic hippocampal slice cultures (OHSC), and microtumor development with and without TTFields at 200 kHz was observed. In addition, organoids were generated from acute material cultured on OHSC and treated with TTFields. Lastly, the effect of TTFields on expression of the Ki67 proliferation marker was evaluated on cultured GBM slices. Microtumors exhibited increased sensitivity towards TTFields compared to monolayer cell cultures. TTFields affected tumor growth and viability, as the size of microtumors and the percentage of Ki67-positive cells decreased after treatment. Nevertheless, variability in the extent of the response was preserved between different patient samples. Therefore, these pre-clinical GBM models could provide snapshots of the tumor to simulate patient treatment response and to investigate molecular mechanisms of response and resistance.
Background: Primary cutaneous follicular B-cell lymphoma (PCFBCL) represents an indolent subtype of Non-Hodgkin’s lymphomas, being clinically characterized by slowly growing tumors of the skin and common cutaneous relapses, while only exhibiting a low propensity for systemic dissemination or fatal outcome. Up to now, only few studies have investigated underlying molecular alterations of PCFBCL with respect to somatic mutations. Objectives: Our aim was to gain deeper insight into the pathogenesis of PCFBCL and to delineate discriminatory molecular features of this lymphoma subtype. Methods: We performed hybridization-based panel sequencing of 40 lymphoma-associated genes of 10 cases of well-characterized PCFBCL. In addition, we included two further ambiguous cases of atypical B-cell-rich lymphoid infiltrate/B-cell lymphoma of the skin for which definite subtype attribution had not been possible by routine investigations. Results: In 10 out of 12 analyzed cases, we identified genetic alterations within 15 of the selected 40 target genes. The most frequently detected alterations in PCFBCL affected the TNFRSF14, CREBBP, STAT6 and TP53 genes. Our analysis unrevealed novel mutations of the BCL2 gene in PCFBCL. All patients exhibited an indolent clinical course. Both the included arbitrary cases of atypical B-cell-rich cutaneous infiltrates showed somatic mutations within the FAS gene. As these mutations have previously been designated as subtype-specific recurrent alterations in primary cutaneous marginal zone lymphoma (PCMZL), we finally favored the diagnosis of PCMZL in these two cases based on these molecular findings. Conclusions: To conclude, our molecular data support that PCFBCL shows distinct somatic mutations which may aid to differentiate PCFBCL from pseudo-lymphoma as well as from other indolent and aggressive cutaneous B-cell lymphomas. While the detected genetic alterations of PCFBCL did not turn out to harbor any prognostic value in our cohort, our molecular data may add adjunctive discriminatory features for diagnostic purposes on a molecular level.
(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.
Hyper-IgM syndrome type 2 (HIGM2) is a B cell intrinsic primary immunodeficiency caused by mutations in AICDA encoding activation-induced cytidine deaminase (AID) which impair immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM). Whereas autosomal-recessive AID-deficiency (AR-AID) affects both CSR and SHM, the autosomal-dominant form (AD-AID) due to C-terminal heterozygous variants completely abolishes CSR but only partially affects SHM. AR-AID patients display enhanced germinal center (GC) reactions and autoimmune manifestations, which are not present in AD-AID, suggesting that SHM but not CSR regulates GC reactions and peripheral B cell tolerance. Herein, we describe two siblings with HIGM2 due to a novel homozygous AICDA mutation (c.428-1G > T) which disrupts the splice acceptor site of exon 4 and results in the sole expression of a truncated AID variant that lacks 10 highly conserved amino acids encoded by exon 4 (AID-ΔE4a). AID-ΔE4a patients suffered from defective CSR and enhanced GC reactions and were therefore indistinguishable from other AR-AID patients. However, the AID-ΔE4a variant only partially affected SHM as observed in AD-AID patients. In addition, AID-ΔE4a but not AD-AID patients revealed impaired targeting of mutational hotspot motives and distorted mutational patterns. Hence, qualitative defects in AID function and altered SHM rather than global decreased SHM activity may account for the disease phenotype in these patients.
Routine coronal paraffin-sections through the dorsal frontal and parieto-occipital cortex of a total of sixty cases with divergent causes of death were immunohistochemically (IHC) stained with an antibody against TMEM119. Samples of cerebrospinal fluid (CSF) of the same cases were collected by suboccipital needle-puncture, subjected to centrifugation and processed as cytospin preparations stained with TMEM119. Both, cytospin preparations and sections were subjected to computer-assisted density measurements. The density of microglial TMEM119-positive cortical profiles correlated with that of cytospin results and with the density of TMEM119-positive microglial profiles in the medullary layer. There was no statistically significant correlation between the density of medullary TMEM119-positive profiles and the cytospin data. Cortical microglial cells were primarily encountered in supragranular layers I, II, and IIIa and in infragranular layers V and VI, the region of U-fibers and in circumscribed foci or spread in a diffuse manner and high density over the white matter. We have evidence that cortical microglia directly migrate into CSF without using the glympathic pathway. Microglia in the medullary layer shows a strong affinity to the adventitia of deep vessels in the myelin layer. Selected rapidly fatal cases including myocardial infarcts and drowning let us conclude that microglia in cortex and myelin layer can react rapidly and its reaction and migration is subject to pre-existing external and internal factors. Cytospin preparations proved to be a simple tool to analyze and assess complex changes in the CNS after rapid fatal damage. There is no statistically significant correlation between cytospin and postmortem interval. Therefore, the quantitative analyses of postmortem cytospins obviously reflect the neuropathology of the complete central nervous system. Cytospins provide forensic pathologists a rather simple and easy to perform method for the global assessment of CNS affliction.
The subclassification of diffuse large B-cell lymphoma (DLBCL) into germinal center B-cell-like (GCB) and activated B-cell-like (ABC) subtypes has become mandatory in the 2017 update of the WHO classification of lymphoid neoplasms and will continue to be used in the WHO 5\(^{th}\) edition. The RNA-based Lymph2Cx assay has been validated as a reliable surrogate of high-throughput gene expression profiling assays for distinguishing between GCB and ABC DLBCL and provides reliable results from formalin-fixed, paraffin-embedded (FFPE) material. This test has been previously used in clinical trials, but experience from real-world routine application is rare. We routinely applied the Lymph2Cx assay to day-to-day diagnostics on a series of 147 aggressive B-cell lymphoma cases and correlated our results with the immunohistochemical subclassification using the Hans algorithm and fluorescence in situ hybridization findings using break-apart probes for MYC, BCL2, and BCL6. The routine use of the Lymph2Cx assay had a high technical success rate (94.6%) with a low rate of failure due to poor material and/or RNA quality. The Lymph2Cx assay was discordant with the Hans algorithm in 18% (23 of 128 cases). Discordant cases were mainly classified as GCB by the Hans algorithm and as ABC by Lymph2Cx (n = 11, 8.6%). Only 5 cases (3.9%) were classified as non-GCB by the Hans algorithm and as GCB by Lymph2Cx. Additionally, 5.5% of cases (n = 7) were left unclassified by Lymph2Cx, whereas they were defined as GCB (n = 4) or non-GCB (n = 3) by the Hans algorithm. Our data support the routine applicability of the Lymph2Cx assay.
Idiopathic Parkinson’s disease (PD) is characterized by a progredient degeneration of the brain, starting at deep subcortical areas such as the dorsal motor nucleus of the glossopharyngeal and vagal nerves (DM) (stage 1), followed by the coeruleus–subcoeruleus complex; (stage 2), the substantia nigra (SN) (stage 3), the anteromedial temporal mesocortex (MC) (stage 4), high-order sensory association areas and prefrontal fields (HC) (stage 5) and finally first-order sensory association areas, premotor areas, as well as primary sensory and motor field (FC) (stage 6). Autoimmunity might play a role in PD pathogenesis. Here we analyzed whether anti-brain autoantibodies differentially recognize different human brain areas and identified autoantigens that correlate with the above-described dissemination of PD pathology in the brain. Brain tissue was obtained from deceased individuals with no history of neurological or psychiatric disease and no neuropathological abnormalities. Tissue homogenates from different brain regions (DM, SN, MC, HC, FC) were subjected to SDS-PAGE and Western blot. Blots were incubated with plasma samples from 30 PD patients and 30 control subjects and stained with anti-IgG antibodies to detect anti-brain autoantibodies. Signals were quantified. Prominent autoantigens were identified by 2D-gel-coupled mass spectrometry sequencing. Anti-brain autoantibodies are frequent and occur both in healthy controls and individuals with PD. Glial fibrillary acidic protein (GFAP) was identified as a prominent autoantigen recognized in all plasma samples. GFAP immunoreactivity was highest in DM areas and lowest in FC areas with no significant differences in anti-GFAP autoantibody titers between healthy controls and individuals with PD. The anti-GFAP autoimmunoreactivity of different brain areas correlates with the dissemination of histopathological neurodegeneration in PD. We hypothesize that GFAP autoantibodies are physiological but might be involved as a cofactor in PD pathogenesis secondary to a leakage of the blood–brain barrier.