Multifactorial White Matter Damage in the Acute Phase and Pre-Existing Conditions May Drive Cognitive Dysfunction after SARS-CoV-2 Infection: Neuropathology-Based Evidence.

BACKGROUND: There is an urgent need to better understand the mechanisms underlying acute and long-term neurological symptoms after COVID-19. Neuropathological studies can contribute to a better understanding of some of these mechanisms. METHODS: We conducted a detailed postmortem neuropathological analysis of 32 patients who died due to COVID-19 during 2020 and 2021 in Austria. RESULTS: All cases showed diffuse white matter damage with a diffuse microglial activation of a variable severity, including one case of hemorrhagic leukoencephalopathy. Some cases revealed mild inflammatory changes, including olfactory neuritis (25%), nodular brainstem encephalitis (31%), and cranial nerve neuritis (6%), which were similar to those observed in non-COVID-19 severely ill patients. One previously immunosuppressed patient developed acute herpes simplex encephalitis. Acute vascular pathologies (acute infarcts 22%, vascular thrombosis 12%, diffuse hypoxic-ischemic brain damage 40%) and pre-existing small vessel diseases (34%) were frequent findings. Moreover, silent neurodegenerative pathologies in elderly persons were common (AD neuropathologic changes 32%, age-related neuronal and glial tau pathologies 22%, Lewy bodies 9%, argyrophilic grain disease 12.5%, TDP43 pathology 6%). CONCLUSIONS: Our results support some previous neuropathological findings of apparently multifactorial and most likely indirect brain damage in the context of SARS-CoV-2 infection rather than virus-specific damage, and they are in line with the recent experimental data on SARS-CoV-2-related diffuse white matter damage, microglial activation, and cytokine release.

Computational drug repositioning of clopidogrel as a novel therapeutic option for focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a glomerular lesion often associated with nephrotic syndrome. It is also associated with a high risk of progression to end-stage kidney disease. Current treatment of FSGS is limited to systemic corticosteroids or calcineurin inhibition, along with inhibitors of the renin-angiotensin-aldosterone system. FSGS is heterogeneous in etiology, and novel therapies targeting specific, dysregulated molecular pathways represent a major unmet medical need. We have generated a network-based molecular model of FSGS pathophysiology using previously established systems biology workflows to allow computational evaluation of compounds for their predicted interference with molecular processes contributing to FSGS. We identified the anti-platelet drug clopidogrel as a therapeutic option to counterbalance dysregulated FSGS pathways. This prediction of our computational screen was validated by testing clopidogrel in the adriamycin FSGS mouse model. Clopidogrel improved key FSGS outcome parameters and significantly reduced urinary albumin to creatinine ratio (P < 0.01) and weight loss (P < 0.01), and ameliorated histopathological damage (P < 0.05). Clopidogrel is used to treat several cardiovascular diseases linked to chronic kidney disease. Clopidogrel's favorable safety profile and its efficacy in the adriamycin mouse FSGS model thus recommend it as an attractive drug repositioning candidate for clinical trial in FSGS.

Deep Morphology Learning Enhances Ex Vivo Drug Profiling-Based Precision Medicine.

Drug testing in patient biopsy-derived cells can identify potent treatments for patients suffering from relapsed or refractory hematologic cancers. Here we investigate the use of weakly supervised deep learning on cell morphologies (DML) to complement diagnostic marker-based identification of malignant and nonmalignant cells in drug testing. Across 390 biopsies from 289 patients with diverse blood cancers, DML-based drug responses show improved reproducibility and clustering of drugs with the same mode of action. DML does so by adapting to batch effects and by autonomously recognizing disease-associated cell morphologies. In a post hoc analysis of 66 patients, DML-recommended treatments led to improved progression-free survival compared with marker-based recommendations and physician's choice-based treatments. Treatments recommended by both immunofluorescence and DML doubled the fraction of patients achieving exceptional clinical responses. Thus, DML-enhanced ex vivo drug screening is a promising tool in the identification of effective personalized treatments. SIGNIFICANCE: We have recently demonstrated that image-based drug screening in patient samples identifies effective treatment options for patients with advanced blood cancers. Here we show that using deep learning to identify malignant and nonmalignant cells by morphology improves such screens. The presented workflow is robust, automatable, and compatible with clinical routine. This article is highlighted in the In This Issue feature, p. 476.

Functional Precision Medicine Provides Clinical Benefit in Advanced Aggressive Hematologic Cancers and Identifies Exceptional Responders.

Personalized medicine aims to match the right drug with the right patient by using specific features of the individual patient's tumor. However, current strategies of personalized therapy matching provide treatment opportunities for less than 10% of patients with cancer. A promising method may be drug profiling of patient biopsy specimens with single-cell resolution to directly quantify drug effects. We prospectively tested an image-based single-cell functional precision medicine (scFPM) approach to guide treatments in 143 patients with advanced aggressive hematologic cancers. Fifty-six patients (39%) were treated according to scFPM results. At a median follow-up of 23.9 months, 30 patients (54%) demonstrated a clinical benefit of more than 1.3-fold enhanced progression-free survival compared with their previous therapy. Twelve patients (40% of responders) experienced exceptional responses lasting three times longer than expected for their respective disease. We conclude that therapy matching by scFPM is clinically feasible and effective in advanced aggressive hematologic cancers. SIGNIFICANCE: This is the first precision medicine trial using a functional assay to instruct n-of-one therapies in oncology. It illustrates that for patients lacking standard therapies, high-content assay-based scFPM can have a significant value in clinical therapy guidance based on functional dependencies of each patient's cancer.See related commentary by Letai, p. 290.This article is highlighted in the In This Issue feature, p. 275.

Metabolic drug survey highlights cancer cell dependencies and vulnerabilities.

Interrogation of cellular metabolism with high-throughput screening approaches can unravel contextual biology and identify cancer-specific metabolic vulnerabilities. To systematically study the consequences of distinct metabolic perturbations, we assemble a comprehensive metabolic drug library (CeMM Library of Metabolic Drugs; CLIMET) covering 243 compounds. We, next, characterize it phenotypically in a diverse panel of myeloid leukemia cell lines and primary patient cells. Analysis of the drug response profiles reveals that 77 drugs affect cell viability, with the top effective compounds targeting nucleic acid synthesis, oxidative stress, and the PI3K/mTOR pathway. Clustering of individual drug response profiles stratifies the cell lines into five functional groups, which link to specific molecular and metabolic features. Mechanistic characterization of selective responses to the PI3K inhibitor pictilisib, the fatty acid synthase inhibitor GSK2194069, and the SLC16A1 inhibitor AZD3965, bring forth biomarkers of drug response. Phenotypic screening using CLIMET represents a valuable tool to probe cellular metabolism and identify metabolic dependencies at large.

Secondary basophilic leukemia in Ph-negative myeloid neoplasms: A distinct subset with poor prognosis.

During progression of myeloid neoplasms, the basophil compartment may expand substantially and in some of these patients, a basophilic leukemia is diagnosed. In patients with Ph-chromosome+ chronic myeloid leukemia, acceleration of disease is typically accompanied by marked basophilia. In other myeloid neoplasms, secondary leukemic expansion of basophils is rarely seen. We report on 5 patients who suffered from a myelodysplastic syndrome, myeloproliferative neoplasm, or acute leukemia and developed a massive expansion of basophils during disease progression. In 4 of 5 patients, peripheral blood basophil counts reached 40%, and the diagnosis "secondary basophilic leukemia" was established. As assessed by flow cytometry, neoplastic basophils expressed CD9, CD18, CD25, CD33, CD63, PD-L1, CD123, and CLL-1. In addition, basophils were found to display BB1 (basogranulin), 2D7, tryptase and KIT. In 4 of 5 patients the disease progressed quickly and treatment with azacitidine was started. However, azacitidine did not induce major clinical responses, and all patients died from progressive disease within 3 Y. In in vitro experiments, the patients´ cells and the basophilic leukemia cell line KU812 showed variable responses to targeted drugs, including azacitidine, venetoclax, hydroxyurea, and cytarabine. A combination of venetoclax and azacitidine induced cooperative antineoplastic effects in these cells. Together, secondary basophilic leukemia has a poor prognosis and monotherapy with azacitidine is not sufficient to keep the disease under control for longer time-periods. Whether drug combination, such as venetoclax+azacitidine, can induce better outcomes in these patients remains to be determined in future clinical studies.

Immune Checkpoint Inhibitor Therapy Induces Inflammatory Activity in the Large Arteries of Lymphoma Patients under 50 Years of Age.

Background: Immune checkpoint inhibitors (ICI) have transformed the management of various cancers. Serious and potentially fatal cardiovascular toxicity, as well as a progression of atherosclerosis, have been described, mainly in elderly and comorbid patients. Methods: We investigated 117 arterial segments of 12 young (under 50 years of age), otherwise healthy lymphoma patients pre/post-ICI treatment using 2-[18F]fluorodeoxyglucose (FDG) positron emission tomography (PET). Maximum FDG standardized uptake values (SUVmax) and target-to-background ratios (TBRs) were calculated along arterial segments. Additionally, metabolic activities (SUVmax) of the bone marrow, spleen, and liver were analyzed. The levels of high-sensitivity C-reactive protein (hsCRP) were assessed. Results: ICI therapy induced arterial inflammatory activity, detected by increased TBR in arterial segments without pre-existing inflammation (TBRneg_pre = 1.20 ± 0.22 vs. TBRneg_post = 1.71 ± 0.45, p < 0.001), whereas already-inflamed lesions remained unchanged. Dormant calcified segments (Hounsfield Units-HU ≥ 130) showed a significant increase in TBR values after ICI treatment (TBRcalc_pre = 1.36 ± 0.38 vs. TBRcalc_post = 1.76 ± 0.42, p < 0.001). FDG uptake measured in other organs and hsCRP levels remained unchanged after ICI therapy. Conclusions: Although the effects of ICI therapy on arterial inflammation are still incompletely understood, cancer immunotherapy might be a critical moderator of atherosclerosis with a subsequently increased risk of future cerebro- and/or cardiovascular events in young oncological patients.

Influence of TP53 Mutation on Survival of Diffuse Large B-Cell Lymphoma in the CAR T-Cell Era.

Refractory/relapsed diffuse large B-cell lymphoma (DLBCL) is associated with poor outcome. The clinical behavior and genetic landscape of DLBCL is heterogeneous and still not fully understood. TP53 mutations in DLBCL have been identified as markers of poor prognosis and are often associated with therapeutic resistance. Chimeric antigen receptor T-cell therapy is an innovative therapeutic concept and represents a game-changing therapeutic option by supporting the patient's own immune system to kill the tumor cells. We investigated the impact of TP53 mutations on the overall survival of refractory/relapsed DLBCL patients treated with comparable numbers of therapy lines. The minimum number of therapy lines was 2 (median 4), including either anti-CD19 CAR T-cell therapy or conventional salvage therapy. A total of 170 patients with DLBCL and high-grade B-cell lymphoma with MYC, BCL2, and/or BCL6 rearrangements (DHL/THL), diagnosed and treated in our hospital between 2000 and 2021, were included. Twenty-nine of them received CAR T-cell therapy. TP53 mutations were found in 10/29 (35%) and 31/141 (22%) of patients in the CAR T-cell and conventional groups, respectively. Among the 141 patients not treated with CAR T cells, TP53 mutation was an independent prognostic factor for overall survival (OS) (median 12 months with TP53 vs. not reached without TP53 mutation, p < 0.005), but in the CAR T cell treated group, this significance could not be shown (median OS 30 vs. 120 months, p = 0.263). The findings from this monocentric retrospective study indicate that TP53 mutation status does not seem to affect outcomes in DLBCL patients treated with CAR T-cell therapy. Detailed evaluation in large cohorts is warranted.

Tissue distribution of epirubicin after severe extravasation in humans.

PURPOSE: As critical parameter after extravasation of cytotoxic vesicants, anthracyclines were determined in removed tissue from patients requiring surgical intervention due to tissue necrosis. We monitored their distribution within the affected lesion to establish a possible dose-toxicity relation. METHODS: From six patients scheduled for surgery, removed tissue flaps were systematically analysed by HPLC (epirubicin: 5 subjects; doxorubicin: 1 subject). RESULTS: After extravasation, tissue concentrations were highly variable with an individual anthracycline distribution pattern ranging from a few nanograms up to 17 µg per 100 mg tissue, which indicated a substantial difference in tissue sensitivity among patients. The resection borders coincided with the extension of the erythema and guided the surgical intervention after demarcation of the lesion, which occurred usually 2 or 3 weeks after extravasation. At that time, drug was hardly detected at the resection borders. Wound drains were negative for the extravasated drugs while showing a time profile of vascular growth factors and inflammatory cytokines, which was highly similar to routine surgery. In all six patients, surgical debridement with immediate wound closure led to healing within approximately 2 weeks, when therapy was resumed in all patients with reasonable time delay. CONCLUSION: Surgical intervention after demarcation of the extravasation lesion allows for almost uninterrupted continuation of treatment independent of the amount of extravasated anthracycline. As even minor amounts of the vesicants may trigger tissue necrosis, preventive measures merit the highest priority.

BH3 profiling identifies ruxolitinib as a promising partner for venetoclax to treat T-cell prolymphocytic leukemia.

Conventional therapies for patients with T-cell prolymphocytic leukemia (T-PLL), such as cytotoxic chemotherapy and alemtuzumab, have limited efficacy and considerable toxicity. Several novel agent classes have demonstrated preclinical activity in T-PLL, including inhibitors of the JAK/STAT and T-cell receptor pathways, as well as histone deacetylase (HDAC) inhibitors. Recently, the BCL-2 inhibitor venetoclax also showed some clinical activity in T-PLL. We sought to characterize functional apoptotic dependencies in T-PLL to identify a novel combination therapy in this disease. Twenty-four samples from patients with primary T-PLL were studied by using BH3 profiling, a functional assay to assess the propensity of a cell to undergo apoptosis (priming) and the relative dependence of a cell on different antiapoptotic proteins. Primary T-PLL cells had a relatively low level of priming for apoptosis and predominantly depended on BCL-2 and MCL-1 proteins for survival. Selective pharmacologic inhibition of BCL-2 or MCL-1 induced cell death in primary T-PLL cells. Targeting the JAK/STAT pathway with the JAK1/2 inhibitor ruxolitinib or HDAC with belinostat both independently increased dependence on BCL-2 but not MCL-1, thereby sensitizing T-PLL cells to venetoclax. Based on these results, we treated 2 patients with refractory T-PLL with a combination of venetoclax and ruxolitinib. We observed a deep response in JAK3-mutated T-PLL and a stabilization of the nonmutated disease. Our functional, precision-medicine-based approach identified inhibitors of HDAC and the JAK/STAT pathway as promising combination partners for venetoclax, warranting a clinical exploration of such combinations in T-PLL.

Correction: Dependency on the TYK2/STAT1/MCL1 axis in anaplastic large cell lymphoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

STAT5 is Expressed in CD34+/CD38- Stem Cells and Serves as a Potential Molecular Target in Ph-Negative Myeloproliferative Neoplasms.

Janus kinase 2 (JAK2) and signal transducer and activator of transcription-5 (STAT5) play a key role in the pathogenesis of myeloproliferative neoplasms (MPN). In most patients, JAK2 V617F or CALR mutations are found and lead to activation of various downstream signaling cascades and molecules, including STAT5. We examined the presence and distribution of phosphorylated (p) STAT5 in neoplastic cells in patients with MPN, including polycythemia vera (PV, n = 10), essential thrombocythemia (ET, n = 15) and primary myelofibrosis (PMF, n = 9), and in the JAK2 V617F-positive cell lines HEL and SET-2. As assessed by immunohistochemistry, MPN cells displayed pSTAT5 in all patients examined. Phosphorylated STAT5 was also detected in putative CD34+/CD38- MPN stem cells (MPN-SC) by flow cytometry. Immunostaining experiments and Western blotting demonstrated pSTAT5 expression in both the cytoplasmic and nuclear compartment of MPN cells. Confirming previous studies, we also found that JAK2-targeting drugs counteract the expression of pSTAT5 and growth in HEL and SET-2 cells. Growth-inhibition of MPN cells was also induced by the STAT5-targeting drugs piceatannol, pimozide, AC-3-019 and AC-4-130. Together, we show that CD34+/CD38- MPN-SC express pSTAT5 and that pSTAT5 is expressed in the nuclear and cytoplasmic compartment of MPN cells. Whether direct targeting of pSTAT5 in MPN-SC is efficacious in MPN patients remains unknown.

Podocyte RNA sequencing reveals Wnt- and ECM-associated genes as central in FSGS.

Loss of podocyte differentiation can cause nephrotic-range proteinuria and Focal and Segmental Glomerulosclerosis (FSGS). As specific therapy is still lacking, FSGS frequently progresses to end-stage renal disease. The exact molecular mechanisms of FSGS and gene expression changes in podocytes are complex and widely unknown as marker changes have mostly been assessed on the glomerular level. To gain a better insight, we isolated podocytes of miR-193a overexpressing mice, which suffer from FSGS due to suppression of the podocyte master regulator Wt1. We characterised the podocytic gene expression changes by RNAseq and identified many novel candidate genes not linked to FSGS so far. This included strong upregulation of the receptor tyrosine kinase EphA6 and a massive dysregulation of circadian genes including the loss of the transcriptional activator Arntl. By comparison with podocyte-specific changes in other FSGS models we found a shared dysregulation of genes associated with the Wnt signaling cascade, while classical podocyte-specific genes appeared widely unaltered. An overlap with gene expression screens from human FSGS patients revealed a strong enrichment in genes associated with extra-cellular matrix (ECM) and metabolism. Our data suggest that FSGS progression might frequently depend on pathways that are often overlooked when considering podocyte homeostasis.

ECM Characterization Reveals a Massive Activation of Acute Phase Response during FSGS.

The glomerular basement membrane (GBM) and extra-cellular matrix (ECM) are essential to maintain a functional interaction between the glomerular podocytes and the fenestrated endothelial cells in the formation of the slit diaphragm for the filtration of blood. Dysregulation of ECM homeostasis can cause Focal segmental glomerulosclerosis (FSGS). Despite this central role, alterations in ECM composition during FSGS have not been analyzed in detail yet. Here, we characterized the ECM proteome changes in miR-193a-overexpressing mice, which suffer from FSGS due to suppression of Wilms' tumor 1 (WT1). By mass spectrometry we identified a massive activation of the acute phase response, especially the complement and fibrinogen pathways. Several protease inhibitors (ITIH1, SERPINA1, SERPINA3) were also strongly increased. Complementary analysis of RNA expression data from both miR-193a mice and human FSGS patients identified additional candidate genes also mainly involved in the acute phase response. In total, we identified more than 60 dysregulated, ECM-associated genes with potential relevance for FSGS progression. Our comprehensive analysis of a murine FSGS model and translational comparison with human data offers novel targets for FSGS therapy.

High activation of STAT5A drives peripheral T-cell lymphoma and leukemia.

Recurrent gain-of-function mutations in the transcription factors STAT5A and much more in STAT5B were found in hematopoietic malignancies with the highest proportion in mature T- and natural killer-cell neoplasms (peripheral T-cell lymphoma, PTCL). No targeted therapy exists for these heterogeneous and often aggressive diseases. Given the shortage of models for PTCL, we mimicked graded STAT5A or STAT5B activity by expressing hyperactive Stat5a or STAT5B variants at low or high levels in the hematopoietic system of transgenic mice. Only mice with high activity levels developed a lethal disease resembling human PTCL. Neoplasia displayed massive expansion of CD8+ T cells and destructive organ infiltration. T cells were cytokine-hypersensitive with activated memory CD8+ T-lymphocyte characteristics. Histopathology and mRNA expression profiles revealed close correlation with distinct subtypes of PTCL. Pronounced STAT5 expression and activity in samples from patients with different subsets underline the relevance of JAK/STAT as a therapeutic target. JAK inhibitors or a selective STAT5 SH2 domain inhibitor induced cell death and ruxolitinib blocked T-cell neoplasia in vivo We conclude that enhanced STAT5A or STAT5B action both drive PTCL development, defining both STAT5 molecules as targets for therapeutic intervention.

RECIL versus Lugano for Treatment Response Assessment in FDG-Avid Non-Hodgkin Lymphomas: A Head-to-Head Comparison in 54 Patients.

The response evaluation criteria in lymphoma (RECIL) classification for lymphoma treatment response assessment was introduced in 2017, but it has not yet been compared to the established Lugano classification. Also, the value of the provisional "minor response" (MiR) category of RECIL is unclear. In 54 patients with FDG-avid non-Hodgkin lymphomas (41 diffuse large B-cell lymphomas (DLBCL) and 13 follicular lymphomas), [18F]FDG-PET/CT-based response according to RECIL and Lugano was determined at interim and end-of-treatment (EOT) restaging. Rates of agreement and Cohen's kappa (κ) coefficients were calculated. The relationship between RECIL and Lugano responses and 2-year complete remission (CR) status of DLBCL patients was determined. At interim restaging, MiR was observed in 14.8%, and at EOT, in 5.6% of patients. When MiR was recoded as partial remission, agreement between RECIL and Lugano was 83.3% at interim restaging (κ = 0.69), and 90.7% at EOT (κ = 0.79). 85.4%, of DLBCL patients with responding disease at interim restaging according to both RECIL and Lugano achieved 2-year CR status; whereas, at EOT, 82.9% of patients with responding disease according to Lugano, and 85.4% of patients with responding disease according to RECIL, achieved 2-year CR status. Thus, RECIL and Lugano classifications show comparable performance for treatment response assessment, and a similar association with 2-year CR status in FDG-avid lymphomas.

Diagnostic and Prognostic Value of Soluble Urokinase-type Plasminogen Activator Receptor (suPAR) in Focal Segmental Glomerulosclerosis and Impact of Detection Method.

The plasma soluble urokinase-type plasminogen activator receptor (suPAR) is a biomarker for focal segmental glomerulosclerosis (FSGS), but its value is under discussion because of ambiguous results arising from different ELISA methods in previous studies. The aim of this study was to compare diagnostic performance of two leading suPAR ELISA kits and examine four objectives in 146 subjects: (1) plasma suPAR levels according to glomerular disease (primary, secondary and recurrent FSGS after kidney transplantation, other glomerulonephritis) and in healthy controls; (2) suPAR levels based on glomerular filtration rate; (3) sensitivity and specificity of suPAR for FSGS diagnosis and determination of optimal cut-offs; (4) suPAR as prognostic tool. Patients with FSGS showed significant higher suPAR values than patients with other glomerulonephritis and healthy individuals. This applied to subjects with and without chronic kidney disease. Although both suPARnostic™ assay and Quantikine Human uPAR ELISA Kit exerted high sensitivity and specificity for FSGS diagnosis, their cut-off values of 4.644 ng/mL and 2.789 ng/mL were significantly different. Higher suPAR was furthermore predictive for progression to end-stage renal disease. In summary, suPAR values must be interpreted in the context of population and test methods used. Knowing test specific cut-offs makes suPAR a valuable biomarker for FSGS.

Transformed mucosa-associated lymphoid tissue lymphomas: A single institution retrospective study including polymerase chain reaction-based clonality analysis.

Given the lack of consistent data regarding the clinico-pathological features and clonal lymphomagenesis of patients with mucosa-associated lymphoid tissue (MALT) lymphoma and histological transformation (HT), we have systematically analysed 379 patients (32% gastric, 68% extra-gastric; median follow-up 52 months) diagnosed with HT at the Medical University Vienna 1999-2017, and reassessed tissues of identified patients by polymerase chain reaction (PCR)-based clonality analysis. HT was documented in 12/379 patients (3·2%) and occurred at a median time of 22 months (range; 6-202 months) after diagnosis of MALT lymphoma. By PCR-based clonality analysis, we detected a clear-cut clonal relationship of MALT lymphoma and diffuse large B-cell lymphoma (DLBCL) in 8 of 11 analysed cases proving that the large majority of DLBCL following MALT lymphoma are clonally-related and constitute a real transformation. Interestingly, HT occurred within the first 2·5 years after diagnosis in patients with clonal relationship, whereas time to aggressive lymphoma was longer in patients identified as clonally-unrelated (most likely secondary) lymphoma (82-202 months), suggesting that HT is an early event in this disease. Survival of patients with HT was poor with 6/12 dying at 1·5-33 months after HT, however, patients with localized gastric transformation had a superior outcome with only 1/6 dying due to progression of lymphoma.