External validation of a deep learning-based algorithm for detection of tall cells in papillary thyroid carcinoma: A multicenter study.

The tall cell subtype (TC-PTC) is an aggressive subtype of papillary thyroid carcinoma (PTC). The TC-PTC is defined as a PTC comprising at least 30% epithelial cells that are three times as tall as they are wide. In practice, this definition is difficult to adhere to, resulting in high inter-observer variability. In this multicenter study, we validated a previously trained deep learning (DL)-based algorithm for detection of tall cells on 160 externally collected hematoxylin and eosin (HE)-stained PTC whole-slide images. In a test set of 360 manual annotations of regions of interest from 18 separate tissue sections in the external dataset, the DL-based algorithm detected TCs with a sensitivity of 90.6% and a specificity of 88.5%. The DL algorithm detected non-TC areas with a sensitivity of 81.6% and a specificity of 92.9%. In the validation datasets, 20% and 30% TC thresholds correlated with a significantly shorter relapse-free survival. In conclusion, the DL algorithm detected TCs in unseen, external scanned HE tissue slides with high sensitivity and specificity without any retraining.

Inhibition of glutaminase-1 in DLBCL potentiates venetoclax-induced antitumor activity by promoting oxidative stress.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma in adults, but first-line immunochemotherapy fails to produce a durable response in about one-third of the patients. Because tumor cells often reprogram their metabolism, we investigated the importance of glutaminolysis, a pathway converting glutamine to generate energy and various metabolites, for the growth of DLBCL cells. Glutaminase-1 (GLS1) expression was robustly detected in DLBCL biopsy samples and cell lines. Both pharmacological inhibition and genetic knockdown of GLS1 induced cell death in DLBCL cells regardless of their subtype classification, whereas primary B cells remained unaffected. Interestingly, GLS1 inhibition resulted not only in reduced levels of intermediates of the tricarboxylic acid cycle but also in a strong mitochondrial accumulation of reactive oxygen species. Supplementation of DLBCL cells with α-ketoglutarate or with the antioxidant α-tocopherol mitigated oxidative stress and abrogated cell death upon GLS1 inhibition, indicating an essential role of glutaminolysis in the protection from oxidative stress. Furthermore, the combination of the GLS1 inhibitor CB-839 with the therapeutic BCL2 inhibitor ABT-199 not only induced massive reactive oxygen species (ROS) production but also exhibited highly synergistic cytotoxicity, suggesting that simultaneous targeting of GLS1 and BCL2 could represent a novel therapeutic strategy for patients with DLBCL.

BRD4 inhibition sensitizes diffuse large B-cell lymphoma cells to ferroptosis.

Diffuse large B-cell lymphoma (DLBCL), the most common form of non-Hodgkin lymphoma, is characterized by an aggressive clinical course. In approximately one-third of patients with DLBCL, first-line multiagent immunochemotherapy fails to produce a durable response. Molecular heterogeneity and apoptosis resistance pose major therapeutic challenges in DLBCL treatment. To circumvent apoptosis resistance, the induction of ferroptosis might represent a promising strategy for lymphoma therapy. In this study, a compound library, targeting epigenetic modulators, was screened to identify ferroptosis-sensitizing drugs. Strikingly, bromodomain and extra-terminal domain (BET) inhibitors sensitized cells of the germinal center B-cell-like (GCB) subtype of DLBCL to ferroptosis induction and the combination of BET inhibitors with ferroptosis inducers, such as dimethyl fumarate or RSL3, synergized in the killing of DLBCL cells in vitro and in vivo. On the molecular level, the BET protein BRD4 was found to be an essential regulator of ferroptosis suppressor protein 1 expression and thus to protect GCB-DLBCL cells from ferroptosis. Collectively, we identified and characterized BRD4 as an important player in ferroptosis suppression in GCB-DLBCL and provide a rationale for the combination of BET inhibitors with ferroptosis-inducing agents as a novel therapeutic approach for DLBCL treatment.

The Paracaspase MALT1 in Cancer.

Almost twenty years ago, the importance of the paracaspase MALT1 in antigen receptor-induced NF-κB activation was first described. Since then, several other immune receptors, G-protein-coupled receptors, and receptor tyrosine kinases were identified as relying on MALT1 to induce NF-κB activation. In various hematological malignancies and solid tumors, MALT1 is constitutively activated and drives chronic NF-κB target gene expression. Deregulated MALT1 activity in cancer thus promotes tumor cell survival, proliferation, and metastasis. Since the molecular function of MALT1 partially requires its protease activity, pharmacological targeting of MALT1 may represent a promising anti-cancer strategy. Here, we review the molecular features of MALT1 activation and function as well as the therapeutic potential of MALT1 inhibition in hematological malignancies and solid tumors.

Dimethyl fumarate induces ferroptosis and impairs NF-κB/STAT3 signaling in DLBCL.

Despite the development of novel targeted drugs, the molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) still poses a substantial therapeutic challenge. DLBCL can be classified into at least 2 major subtypes (germinal center B cell [GCB]-like and activated B cell [ABC]-like DLBCL), each characterized by specific gene expression profiles and mutation patterns. Here we demonstrate a broad antitumor effect of dimethyl fumarate (DMF) on both DLBCL subtypes, which is mediated by the induction of ferroptosis, a form of cell death driven by the peroxidation of phospholipids. As a result of the high expression of arachidonate 5-lipoxygenase in concert with low glutathione and glutathione peroxidase 4 levels, DMF induces lipid peroxidation and thus ferroptosis, particularly in GCB DLBCL. In ABC DLBCL cells, which are addicted to NF-κB and STAT3 survival signaling, DMF treatment efficiently inhibits the activity of the IKK complex and Janus kinases. Interestingly, the BCL-2-specific BH3 mimetic ABT-199 and an inhibitor of ferroptosis suppressor protein 1 synergize with DMF in inducing cell death in DLBCL. Collectively, our findings identify the clinically approved drug DMF as a promising novel therapeutic option in the treatment of both GCB and ABC DLBCLs.

Ferroptotic pores induce Ca2+ fluxes and ESCRT-III activation to modulate cell death kinetics.

Ferroptosis is an iron-dependent form of regulated necrosis associated with lipid peroxidation. Despite its key role in the inflammatory outcome of ferroptosis, little is known about the molecular events leading to the disruption of the plasma membrane during this type of cell death. Here we show that a sustained increase in cytosolic Ca2+ is a hallmark of ferroptosis that precedes complete bursting of the cell. We report that plasma membrane damage leading to ferroptosis is associated with membrane nanopores of a few nanometers in radius and that ferroptosis, but not lipid peroxidation, can be delayed by osmoprotectants. Importantly, Ca2+ fluxes during ferroptosis induce the activation of the ESCRT-III-dependent membrane repair machinery, which counterbalances the kinetics of cell death and modulates the immunological signature of ferroptosis. Our findings with ferroptosis provide a unifying concept that sustained increase of cytosolic Ca2+ prior to plasma membrane rupture is a common feature of regulated types of necrosis and position ESCRT-III activation as a general protective mechanism in these lytic cell death pathways.

Threonine Phosphorylation of IκBζ Mediates Inhibition of Selective Proinflammatory Target Genes.

Transcription factors of the NF-κB family play a crucial role for immune responses by activating the expression of chemokines, cytokines, and antimicrobial peptides involved in pathogen clearance. IκBζ, an atypical nuclear IκB protein and selective coactivator of particular NF-κB target genes, has recently been identified as an essential regulator for skin immunity. This study discovered that IκBζ is strongly induced in keratinocytes that sense the fungal glucan zymosan A. Additionally, IκBζ is essential for the optimal expression of proinflammatory genes, such as IL6, CXCL5, IL1B, or S100A9. Moreover, this study found that IκBζ was not solely regulated on the transcriptional level but also by phosphorylation events. This study identified several IκBζ phosphorylation sites, including a conserved cluster of threonine residues located in the N-terminus of the protein, which can be phosphorylated by MAPKs. Surprisingly, IκBζ phosphorylation at this threonine cluster promoted the recruitment of histone deacetylase 1 to specific target gene promoters and, thus, negatively controlled transcription. Taken together, this study proposes a model of how an antifungal response translates to the expression of proinflammatory cytokines and highlights an additional layer of complexity in the regulation of the NF-κB responses in keratinocytes.

Ultrafast Toluidine Blue Staining for Rapid On-Site Evaluation of Cytological Smears.

Rapid on-site evaluation (ROSE) is one of cytopathology's "unique selling propositions." The quality, speed, and ease of handling of the staining used is a critical factor for the efficacy of the ROSE procedure. Here, we describe a modification of rapid toluidine blue staining that can be performed within 25 s, provides excellent nuclear morphology, and is compatible with subsequent Papanicolaou staining of the slides. Furthermore, exposure to hazardous chemicals is minimized, as no organic solvents other than the alcohol-based fixative and glycerin for temporary mounting and coverslipping are required. We have used this protocol successfully in our ROSE practice and have not observed any discrepancies between toluidine blue- and permanent Papanicolaou-stained slides.

Targeting chronic NFAT activation with calcineurin inhibitors in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) represents the most common adult lymphoma and can be divided into 2 major molecular subtypes: the germinal center B-cell-like and the aggressive activated B-cell-like (ABC) DLBCL. Previous studies suggested that chronic B-cell receptor signaling and increased NF-κB activation contribute to ABC DLBCL survival. Here we show that the activity of the transcription factor NFAT is chronically elevated in both DLBCL subtypes. Surprisingly, NFAT activation is independent of B-cell receptor signaling, but mediated by an increased calcium flux and calcineurin-mediated dephosphorylation of NFAT. Intriguingly, although NFAT is activated in both DLBCL subtypes, long-term calcineurin inhibition with cyclosporin A or FK506, both clinically approved drugs, triggers potent cytotoxicity specifically in ABC DLBCL cells. The antitumor effects of calcineurin inhibitors are associated with the reduced expression of c-Jun, interleukin-6, and interleukin-10, which were identified as NFAT target genes that are particularly important for the survival of ABC DLBCL. Furthermore, calcineurin blockade synergized with BCL-2 and MCL-1 inhibitors in killing ABC DLBCL cells. Collectively, these findings identify constitutive NFAT signaling as a crucial functional driver of ABC DLBCL and highlight calcineurin inhibition as a novel strategy for the treatment of this aggressive lymphoma subtype.

MiRNAs Are Involved in Tall Cell Morphology in Papillary Thyroid Carcinoma.

Five percent of papillary thyroid carcinomas (PTC) show an adverse clinical outcome (ACO). The tall cell variant of papillary thyroid carcinomas (TCV) is a good predictor of an ACO, however, the identification of tall-cells is subjective. Micro RNAs are short non-coding ribonucleic acids (miRNA). Their expression in PTC could be a powerful, more objective predictor of prognosis. METHODS: Forty-four PTC underwent miRNA profiling, twenty-four of them were TCV. The miRNA dataset was validated by analysis of expression of known target proteins (vascular endothelial growth factor (VEGF) and phosphatase and tensin homolog (PTEN)) in 125 patients including 48 TCV and 57 with an ACO. RESULTS: One hundred and forty-nine miRNAs were significantly associated with an ACO, seventy-one of them with TC-morphology. Twenty-two miRNAs were identified as targets for VEGF and thirty-two as targets for PTEN. In univariate and multivariable analysis, reduced expression of PTEN and an increased expression of VEGF were associated with shorter relapse free survival. A classifier, including TC-morphology, pT-stage, VEGF, and PTEN, predicted relapse with an 80% accuracy. CONCLUSIONS: Some miRNAs predict outcome in PTC and are involved in TC-morphology in PTC. These miRNAs may serve as more objective indicators of an ACO than tall cell morphology. PTEN and VEGF protein expression are prognostically relevant and are at least partially regulated by miRNAs.

Targeting CD47 in Anaplastic Thyroid Carcinoma Enhances Tumor Phagocytosis by Macrophages and Is a Promising Therapeutic Strategy.

Background: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human cancers, with a median survival of only three to six months. Standard treatment options and even targeted therapies have so far failed to improve long-term overall survival. Thus, novel treatment modalities for ATC, such as immunotherapy, are urgently needed. CD47 is a "don't eat me" signal, which prevents cancer cells from phagocytosis by binding to signal regulatory protein alpha on macrophages. So far, the role of macrophages and the CD47-signal regulatory protein alpha signaling axis in ATC is not well understood. Methods: This study analyzed 19 primary human ATCs for macrophage markers, CD47 expression, and immune checkpoints by immunohistochemistry. ATC cell lines and a fresh ATC sample were assessed by flow cytometry for CD47 expression and macrophage infiltration, respectively. CD47 was blocked in phagocytosis assays of co-cultured macrophages and ATC cell lines. Anti-CD47 antibody treatment was administered to ATC cell line xenotransplanted immunocompromised mice, as well as to tamoxifen-induced ATC double-transgenic mice. Results: Human ATC samples were heavily infiltrated by CD68- and CD163-expressing tumor-associated macrophages (TAMs), and expressed CD47 and calreticulin, the dominant pro-phagocytic molecule. In addition, ATC tissues expressed the immune checkpoint molecules programmed cell death 1 and programmed death ligand 1. Blocking CD47 promoted the phagocytosis of ATC cell lines by macrophages in vitro. Anti-CD47 antibody treatment of ATC xenotransplanted mice increased the frequency of TAMs, enhanced the expression of macrophage activation markers, augmented tumor cell phagocytosis, and suppressed tumor growth. In double-transgenic ATC mice, CD47 was expressed on tumor cells, and blocking CD47 increased TAM frequencies. Conclusions: Targeting CD47 or CD47 in combination with programmed cell death 1 may potentially improve the outcomes of ATC patients and may represent a valuable addition to the current standard of care.

Competitive Testing of the WHO 2010 versus the WHO 2017 Grading of Pancreatic Neuroendocrine Neoplasms: Data from a Large International Cohort Study.

BACKGROUND: The World Health Organization (WHO) and the American Joint Cancer Committee (AJCC) modified the grading of pancreatic neuroendocrine neoplasms from a three-tier (WHO-AJCC 2010) to a four-tier system by introducing the novel category of NET G3 (WHO-AJCC 2017). OBJECTIVES: This study aims at validating the WHO-AJCC 2017 and identifying the most effective grading system. METHOD: A total of 2,102 patients were enrolled; entry criteria were: (i) patient underwent surgery; (ii) at least 2 years of follow-up; (iii) observation time up to 2015. Data from 34 variables were collected; grading was assessed and compared for efficacy by statistical means including Kaplan-Meier method, Cox regression analysis, Harrell's C statistics, and Royston's explained variation in univariable and multivariable analyses. RESULTS: In descriptive analysis, the two grading systems demonstrated statistically significant differences for the major category sex but not for age groups. In Cox regression analysis, both grading systems showed statistically significant differences between grades for OS and EFS; however, no statistically significant difference was observed between the two G3 classes of WHO-AJCC 2017. In multivariable analysis for the two models fitted to compare efficacy, the two grading systems performed equally well with substantially similar optimal discrimination and well-explained variation for both OS and EFS. The WHO-AJCC 2017 grading system retained statistically significant difference between the two G3 classes for OS but not for EFS. CONCLUSIONS: The WHO-AJCC 2017 grading system is at least equally performing as the WHO-AJCC 2010 but allows the successful identification of the most aggressive PanNET subgroup. Grading is confirmed as probably the most powerful tool for predicting patient survival.

The IGF pathway is activated in insulinomas but downregulated in metastatic disease.

Clinical and molecular studies have implicated epidermal growth factor receptor (EGFR), insulin-like growth factor (IGF) and target of rapamycin (mTOR) signaling pathways in the regulation of pancreatic neuroendocrine tumor (PanNET) growth. Interpretation and comparison of these studies is complex due to clinical and molecular tumor heterogeneity. We therefore focused in this study on insulinomas, which we examined for mRNA and protein expression of EGFR, IGF and mTOR signaling pathway components by quantitative real-time PCR (n=48) and immunohistochemistry (n=86). Findings were compared with normal pancreatic islets and correlated with histopathological data and clinical outcome. Insulinomas showed low EGFR and high IGF2 expression. IGFBP2, IGFBP3 and IGFBP6 mRNA levels were 2-4 folds higher than in islets. High protein expression of IGF2, IGF1R and INSR (in 51-92% of the tumors) and low to moderate expression of mTORC1 pathway proteins p-PS6k and p-4EBP1 (7-28% of the tumors) were observed. Correlations were found between 1) ERK1 mRNA expression and that of numerous IGF pathway genes, 2) p-ERK and IGF1R protein expression and 3) decrease of IGF pathway components and both metastatic disease and shorter 10 years disease free survival. In conclusion, our observations suggest that high expression of IGF signaling pathway components is a hallmark of insulinomas, but does not necessarily lead to increased mTOR signaling. Reduced expression of IGF pathway components may be an adverse prognostic factor in insulinomas.

YB-1 Expression and Phosphorylation Regulate Tumorigenicity and Invasiveness in Melanoma by Influencing EMT.

Cutaneous melanoma represents one of the most aggressive human tumor entities possessing a high tendency to metastasize. Cancer cells frequently exploit a highly conserved developmental program, the epithelial-to-mesenchymal transition (EMT), to gain migratory and invasive properties promoting their metastatic spread. Cytoplasmic localization of the oncogenic transcription and translation factor Y-box binding protein 1 (YB-1) is a powerful inducer of EMT in breast carcinoma cells. Interestingly, EMT-like processes have also been observed in cutaneous melanoma despite its neural crest origin. Here, increased expression of YB-1 negatively affects patient survival in malignant melanoma and promotes melanoma cell tumorigenicity both in vitro and in vivo Intriguingly, this effect seems to be mainly mediated by cytoplasmic YB-1 that does not exhibit phosphorylation at serine-102 (S102). Moreover, S102 unphosphorylated YB-1 enhances the migratory and invasive potential of human melanoma cells in two-dimensional (2D) and three-dimensional (3D) culture systems and facilitates acquisition of a mesenchymal-like invasive phenotype in the chick embryo model. Collectively, these data demonstrate that the cytoplasmic activity of YB-1 stimulates tumorigenicity and metastatic potential of melanoma cells by promoting EMT-like properties.Implications: This study reveals for the first time that YB-1 efficiently drives tumorigenicity and invasiveness of melanoma cells in its S102 unphosphorylated cytoplasmic state and that YB-1 expression represents a negative prognostic factor in primary melanoma patients. Mol Cancer Res; 16(7); 1149-60. ©2018 AACR.

NF-κB Activation in Lymphoid Malignancies: Genetics, Signaling, and Targeted Therapy.

The NF-κB transcription factor family plays a crucial role in lymphocyte proliferation and survival. Consequently, aberrant NF-κB activation has been described in a variety of lymphoid malignancies, including diffuse large B-cell lymphoma, Hodgkin lymphoma, and adult T-cell leukemia. Several factors, such as persistent infections (e.g., with Helicobacter pylori), the pro-inflammatory microenvironment of the cancer, self-reactive immune receptors as well as genetic lesions altering the function of key signaling effectors, contribute to constitutive NF-κB activity in these malignancies. In this review, we will discuss the molecular consequences of recurrent genetic lesions affecting key regulators of NF-κB signaling. We will particularly focus on the oncogenic mechanisms by which these alterations drive deregulated NF-κB activity and thus promote the growth and survival of the malignant cells. As the concept of a targeted therapy based on the mutational status of the malignancy has been supported by several recent preclinical and clinical studies, further insight in the function of NF-κB modulators and in the molecular mechanisms governing aberrant NF-κB activation observed in lymphoid malignancies might lead to the development of additional treatment strategies and thus improve lymphoma therapy.

Implementation of a 'lean' cytopathology service: towards routine same-day reporting.

OBJECTIVES: To systematically assess the effects of a Lean management intervention in an academic cytopathology service. METHODS: We monitored outcomes including specimen turnaround times during stepwise implementation of a lean cytopathology workflow for gynaecological and non-gynaecological cytology. RESULTS: The intervention resulted in a major reduction of turnaround times for both gynaecological (3rd quartile 4.1 vs 2.3 working days) and non-gynaecological cytology (3rd quartile 1.9 vs. 1.2 working days). Introduction of fully electronic reporting had additional effect over continuous staining of slides alone. The rate of non-gynaecological specimens reported the same day increased from 4.5% to 56.5% of specimens received before noon. CONCLUSIONS: Lean management principles provide a useful framework for organization of a cytopathology workflow. Stepwise implementation beginning with a simplified gynaecological cytology workflow allowed involved staff to monitor the effects of individual changes and allowed for a smooth transition.

Human melanoma cells resistant to MAPK inhibitors can be effectively targeted by inhibition of the p90 ribosomal S6 kinase.

The clinical availability of small molecule inhibitors specifically targeting mutated BRAF marked a significant breakthrough in melanoma therapy. Despite a dramatic anti-tumour activity and improved patient survival, rapidly emerging resistance, however, greatly limits the clinical benefit. The majority of the already described resistance mechanisms involve a reactivation of the MAPK signalling pathway. The p90 ribosomal S6 kinase (RSK), a downstream effector of the MAPK signalling cascade, has been reported to enhance survival of melanoma cells in response to chemotherapy. Here, we can show that RSK activity is significantly increased in human melanoma cells with acquired resistance to the BRAFV600E/K inhibitor vemurafenib. Interestingly, inhibition of RSK signalling markedly impairs the viability of vemurafenib resistant melanoma cells and is effective both in two-dimensional and in three-dimensional culture systems, especially in a chronic, long-term application. The effect of RSK inhibition can be partly replicated by downregulation of the well-known RSK target, Y-box binding protein 1 (YB-1). Intriguingly, RSK inhibition also retains its efficacy in melanoma cells with combined resistance to vemurafenib and the MEK inhibitor trametinib. These data suggest that active RSK signalling might be an attractive novel therapeutic target in melanoma with acquired resistance to MAPK pathway inhibitors.