Molecular Determinants of Sensitivity to Polatuzumab Vedotin in Diffuse Large B Cell Lymphoma.

Polatuzumab Vedotin (Pola-V) is an antibody-drug conjugate directed to the CD79B subunit of the B cell receptor (BCR). When combined with conventional immunochemotherapy, Pola-V improves outcomes in DLBCL. To identify determinants of Pola-V sensitivity, we used CRISPR-Cas9 screening for genes that modulated Pola-V toxicity for lymphomas or the surface expression of its target, CD79B. Our results reveal the striking impact of CD79B glycosylation on Pola-V epitope availability on the lymphoma cell surface and on Pola-V toxicity. Genetic, pharmacological, and enzymatic approaches that remove sialic acid from N-linked glycans enhanced lymphoma killing by Pola-V. Pola-V toxicity was also modulated by KLHL6, an E3 ubiquitin ligase that is recurrently inactivated in germinal center derived lymphomas. We reveal how KLHL6 targets CD79B for degradation in normal and malignant germinal center B cells, thereby determining expression of the surface BCR complex. Our findings suggest precision medicine strategies to optimize Pola-V as a lymphoma therapeutic.

The ß-Secretase BACE1 Drives Fibroblast Activation in Systemic Sclerosis through the APP/ß-Catenin/Notch Signaling Axis.

BACE1 is well-known for its role in the development of Alzheimer's disease. Recent publications, including our own, have demonstrated a role for this enzyme in other chronic diseases. The aim of this study was to investigate the role of BACE1 in the autoimmune disease systemic sclerosis (SSc). BACE1 protein levels were elevated in the skin of patients with SSc. Inhibition of BACE1 with small-molecule inhibitors or small interfering RNA blocked SSc and fibrotic stimuli-mediated fibroblast activation. Furthermore, we show that BACE1 regulation of dermal fibroblast activation is dependent on ß-catenin and Notch signaling. The neurotropic factor brain-derived neurotrophic factor negatively regulates BACE1 expression and activity in dermal fibroblasts. Finally, sera from patients with SSc show higher ß-amyloid and lower brain-derived neurotrophic factor levels than healthy controls. The ability of BACE1 to regulate SSc fibroblast activation reveals a therapeutic target in SSc. Several BACE1 inhibitors have been shown to be safe in clinical trials for Alzheimer's disease and could be repurposed to ameliorate fibrosis progression.

Behaviour support in dentistry: A Delphi study to agree terminology in behaviour management.

OBJECTIVES: Dental behaviour support (DBS) describes all specific techniques practiced to support patients in their experience of professional oral healthcare. DBS is roughly synonymous with behaviour management, which is an outdated concept. There is no agreed terminology to specify the techniques used to support patients who receive dental care. This lack of specificity may lead to imprecision in describing, understanding, teaching, evaluating and implementing behaviour support techniques in dentistry. Therefore, this e-Delphi study aimed to develop a list of agreed labels and descriptions of DBS techniques used in dentistry and sort them according to underlying principles of behaviour. METHODS: Following a registered protocol, a modified e-Delphi study was applied over two rounds with a final consensus meeting. The threshold of consensus was set a priori at 75%. Agreed techniques were then categorized by four coders, according to behavioural learning theory, to sort techniques according to their mechanism of action. RESULTS: The panel (n = 35) agreed on 42 DBS techniques from a total of 63 candidate labels and descriptions. Complete agreement was achieved regarding all labels and descriptions, while agreement was not achieved regarding distinctiveness for 17 techniques. In exploring underlying principles of learning, it became clear that multiple and differing principles may apply depending on the specific context and procedure in which the technique may be applied. DISCUSSION: Experts agreed on what each DBS technique is, what label to use, and their description, but were less likely to agree on what distinguishes one technique from another. All techniques were describable but not comprehensively categorizable according to principles of learning. While objective consistency was not attained, greater clarity and consistency now exists. The resulting list of agreed terminology marks a significant foundation for future efforts towards understanding DBS techniques in research, education and clinical care.

The androgen receptor interacts with GATA3 to transcriptionally regulate a luminal epithelial cell phenotype in breast cancer.

BACKGROUND: The androgen receptor (AR) is a tumor suppressor in estrogen receptor (ER) positive breast cancer, a role sustained in some ER negative breast cancers. Key factors dictating AR genomic activity in a breast context are largely unknown. Herein, we employ an unbiased chromatin immunoprecipitation-based proteomic technique to identify endogenous AR interacting co-regulatory proteins in ER positive and negative models of breast cancer to gain new insight into mechanisms of AR signaling in this disease. RESULTS: The DNA-binding factor GATA3 is identified and validated as a novel AR interacting protein in breast cancer cells irrespective of ER status. AR activation by the natural ligand 5α-dihydrotestosterone (DHT) increases nuclear AR-GATA3 interactions, resulting in AR-dependent enrichment of GATA3 chromatin binding at a sub-set of genomic loci. Silencing GATA3 reduces but does not prevent AR DNA binding and transactivation of genes associated with AR/GATA3 co-occupied loci, indicating a co-regulatory role for GATA3 in AR signaling. DHT-induced AR/GATA3 binding coincides with upregulation of luminal differentiation genes, including EHF and KDM4B, established master regulators of a breast epithelial cell lineage. These findings are validated in a patient-derived xenograft model of breast cancer. Interaction between AR and GATA3 is also associated with AR-mediated growth inhibition in ER positive and ER negative breast cancer. CONCLUSIONS: AR and GATA3 interact to transcriptionally regulate luminal epithelial cell differentiation in breast cancer regardless of ER status. This interaction facilitates the tumor suppressor function of AR and mechanistically explains why AR expression is associated with less proliferative, more differentiated breast tumors and better overall survival in breast cancer.

Diabetes and infection: review of the epidemiology, mechanisms and principles of treatment.

An association between diabetes and infection has been recognised for many years, with infection being an important cause of death and morbidity in people with diabetes. The COVID-19 pandemic has re-kindled an interest in the complex relationship between diabetes and infection. Some infections occur almost exclusively in people with diabetes, often with high mortality rates without early diagnosis and treatment. However, more commonly, diabetes is a complicating factor in many infections. A reciprocal relationship occurs whereby certain infections and their treatments may also increase the risk of diabetes. People with diabetes have a 1.5- to 4-fold increased risk of infection. The risks are the most pronounced for kidney infection, osteomyelitis and foot infection, but are also increased for pneumonia, influenza, tuberculosis, skin infection and general sepsis. Outcomes from infection are worse in people with diabetes, with the most notable example being a twofold higher rate of death from COVID-19. Hyperglycaemia has deleterious effects on the immune response. Vascular insufficiency and neuropathy, together with altered skin, mucosal and gut microbial colonisation, contribute to the increased risk of infection. Vaccination is important in people with diabetes although the efficacy of certain immunisations may be compromised, particularly in the presence of hyperglycaemia. The principles of treatment largely follow those of the general population with certain notable exceptions.

Response-adapted, time-limited venetoclax, umbralisib, and ublituximab for relapsed/refractory chronic lymphocytic leukemia.

ABSTRACT: Many patients with chronic lymphocytic leukemia (CLL) will develop treatment resistance to Bruton tyrosine kinase (BTK) inhibitors. Phosphatidylinositol-3-kinase (PI3K) inhibitors, including umbralisib, have significant clinical activity in relapsed/refractory CLL, but prolonged exposure is associated with potential toxicities. Owing to the synergistic antitumor effects of combined PI3K and BCL-2 inhibition, we sought to explore the feasibility of response-adapted, time-limited therapy to optimize disease control while mitigating the risks of prolonged treatment. We conducted a phase 1/2 clinical trial to determine the safety and efficacy of venetoclax in combination with umbralisib and the anti-CD20 monoclonal antibody, ublituximab, (U2-VeN) in patients with relapsed/refractory CLL (N = 46) and Richter transformation (N = 5). After 12 cycles, treatment was stopped for patients with CLL who achieved undetectable minimal residual disease (uMRD). Adverse events of special interest included diarrhea in 50% of patients (11% grade 3/4), and aspartate aminotransferase and/or alanine aminotransferase elevation in 15 patients (33%), with 3 (7%) grade 3/4. There were no cases of tumor lysis syndrome related to venetoclax, with outpatient initiation in 96% of patients. The intent-to-treat overall response rate for CLL was 98% with best response of 100% in evaluable patients (42% complete responses). The end-of-treatment rate of uMRD at 10-4 in bone marrow was 77% (30/39), including a 71% uMRD rate among 14 patients refractory to prior BTK inhibitor. Time-limited venetoclax and U2 is safe and highly effective combination therapy for patients with relapsed/refractory CLL including those who have been previously treated with covalent BTK inhibitors. This trial was registered on www.clinicaltrials.gov as #NCT03379051.

Protocol for quantitative proteomic analysis of heterogeneous adipose tissue-residing progenitor subpopulations in mice.

Recent studies have revealed cellular heterogeneity of mesenchymal stromal cells and immune cells in adipose tissue and emphasized the need for quantitative analysis of small numbers of functionally distinct cells using state-of-the-art "omics" technologies. Here, we present an optimized protocol for precise protein quantification from minute amounts of samples. We describe steps for isolation of mouse adipose progenitor cells, proteomics sample preparation, mass spectrometry measurement, and computational analysis. This protocol can be adapted to other samples with limited amounts. For complete details on the use and execution of this protocol, please refer to Shan et al. (2022).1.

Going Asexual: A Survey of Mites of the Genus Thyreophagus (Acari: Acaridae) Revealing a Large Number of New Parthenogenetic Species in the Holarctic Region.

Mites of the genus Thyreophagus (Acari: Acaridae) are distributed worldwide; they inhabit concealed habitats and include several beneficial and economically important species. However, species identification is difficult because many species are poorly described or delimited and their phoretic stages are unknown or uncorrelated. Furthermore, Thyreophagus is interesting because it includes entirely asexual (parthenogenetic) species. However, among the 34 described species of Thyreophagus, the asexual status is confirmed through laboratory rearing for only two species. Here, we provide detailed descriptions of five new species from North America (four) and Europe (one) based on adults and phoretic heteromorphic deutonymphs. Four of these species were asexual, while one was sexual. For most of these mites, the asexual status was confirmed and phoretic deutonymphs were obtained through rearing in the lab. We show that asexual mites retain seemingly functional copulatory and sperm storage systems, indicating that these lineages have relatively short evolutionary lifespans. One North American species, Thyreophagus ojibwe, was found in association with the native American chestnut Castanea dentata, suggesting a possibility that this mite can be used to control chestnut blight in North America. We also provide a diagnostic key to females, males, and heteromorphic deutonymphs of the Thyreophagus species in the world.

Visualisation of drug distribution in skin using correlative optical spectroscopy and mass spectrometry imaging.

A correlative methodology for label-free chemical imaging of soft tissue has been developed, combining non-linear optical spectroscopies and mass spectrometry to achieve sub-micron spatial resolution and critically improved drug detection sensitivity. The approach was applied to visualise the kinetics of drug reservoir formation within human skin following in vitro topical treatment with a commercial diclofenac gel. Non-destructive optical spectroscopic techniques, namely stimulated Raman scattering, second harmonic generation and two photon fluorescence microscopies, were used to provide chemical and structural contrast. The same tissue sections were subsequently analysed by secondary ion mass spectrometry, which offered higher sensitivity for diclofenac detection throughout the epidermis and dermis. A method was developed to combine the optical and mass spectrometric datasets using image registration techniques. The label-free, high-resolution visualisation of tissue structure coupled with sensitive chemical detection offers a powerful method for drug biodistribution studies in the skin that impact directly on topical pharmaceutical product development.

Healthcare Utilization Disparities of Adolescent and Young Adults Compared to the Older Lymphoma Population.

BACKGROUND: Adolescent and Young Adults (AYAs) are an underserved, high-risk population. Identifying health care utilization patterns, and particularly acute care visits, is important as these are high-intensity, expensive services. We investigated whether differences exist in health care utilization between the AYA lymphoma population compared to their older adult counterparts. MATERIALS AND METHODS: Two correlated outcomes were used to measure health care utilization: 4 or more acute visits (emergency department or urgent care) and number of nonacute visits (office or telephone visits). We studied 442 patients with aggressive lymphoma patients 15 years or older at time of diagnosis managed at our cancer center within 2 years of their diagnosis. A multivariate generalized linear mixed model simultaneously estimated the effect of baseline predictors on 4 or more acute care visit with robust Poisson regression and nonacute visit counts with negative binomial regression allowing for a within-subject random effect. RESULTS: AYAs had increased risk of having ≥4 acute visits (RR = 1.96; P = .047) compared to their older counterparts. Obesity (RR = 2.04, P = .015) and living less than 50 miles from the cancer center (RR = 3.48, P = .015) were independently associated with higher risk of acute care usage. Acute care visits for psychiatric or substance use related reasons were significantly higher (P = .0001) among AYA (10/114, 8.8%) vs. non-AYA (3/328, 0.9%). CONCLUSION: Disease-targeted interventions to address high acute health care utilization is needed amongst AYAs. Additionally, early multidisciplinary involvement after cancer diagnosis particularly with psychiatric expertise amongst AYAs and palliative care involvement in both groups is needed.

The Novel DNA Binding Mechanism of Ridinilazole, a Precision Clostridiodes difficile Antibiotic.

Clostridioides difficile infection (CDI) causes substantial morbidity and mortality worldwide with limited antibiotic treatment options. Ridinilazole is a precision bisbenzimidazole antibiotic being developed to treat CDI and reduce unacceptably high rates of infection recurrence in patients. Although in late clinical development, the precise mechanism of action by which ridinilazole elicits its bactericidal activity has remained elusive. Here, we present conclusive biochemical and structural data to demonstrate that ridinilazole has a primary DNA binding mechanism, with a co-complex structure confirming binding to the DNA minor groove. Additional RNA-seq data indicated early pleiotropic changes to transcription, with broad effects on multiple C. difficile compartments and significant effects on energy generation pathways particularly. DNA binding and genomic localization was confirmed through confocal microscopy utilizing the intrinsic fluorescence of ridinilazole upon DNA binding. As such, ridinilazole has the potential to be the first antibiotic approved with a DNA minor groove binding mechanism of action.

Rituximab induced cytokine release with high serum IP-10 (CXCL10) concentrations is associated with infusion reactions.

Monoclonal antibody induced infusion reactions (IRs) can be serious and even fatal. We used clinical data and blood samples from 37 treatment naïve patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) initiating therapy for progressive disease with a single 50 mg dose of intravenous (IV) rituximab at 25 mg/h. Twenty-four (65 %) patients had IRs at a median of 78 min (range 35-128) and rituximab dose of 32 mg (range 15-50). IR risk did not correlate with patient or CLL characteristics, CLL counts or CD20 levels, or serum rituximab or complement concentrations. Thirty-five (95 %) patients had cytokine release response with a ≥ 4-fold increase in serum concentration of ≥ 1 inflammatory cytokine. IRs were associated with significantly higher post-infusion serum concentrations of gamma interferon induced cytokines IP-10, IL-6 and IL-8. IP-10 concentrations increased ≥ 4-fold in all patients with an IR and were above the upper limit of detection (40,000 pg/ml) in 17 (71 %). In contrast, to only three (23 %) patients without an IR had an ≥ 4-fold increase in serum concentrations of IP-10 (highest 22,013 pg/ml). Our data suggest that cytokine release could be initiated by activation of effector cells responsible for clearance of circulating CLL cells with IRs occurring in those with higher levels of gamma interferon induced cytokines. These novel insights could inform future research to better understand and manage IRs and understand the role of cytokines in the control of cytotoxic immune responses to mAb.

The role of splenectomy in management of splenic B-cell lymphomas.

INTRODUCTION: Splenic B-cell lymphomas are rare and understudied entities. Splenectomy is frequently required for specific pathological diagnosis in patients with splenic B-cell lymphomas other than classical hairy cell leukemia (cHCL), and can be effective and durable therapy. Our study investigated the diagnostic and therapeutic role of splenectomy for non-cHCL indolent splenic B-cell lymphomas. METHODS: Observational study of patients with non-cHCL splenic B-cell lymphoma undergoing splenectomy between 1 August 2011 and 1 August 2021 at the University of Rochester Medical Center. The comparison cohort was patients categorized as having non-cHCL splenic B-cell lymphoma who did not undergo splenectomy. RESULTS: Forty-nine patients (median age 68 years) had splenectomy (SMZL n = 33, HCLv n = 9, SDRPL n = 7) with median follow up of 3.9 years post splenectomy. One patient had fatal post-operative complications. Post-operative hospitalization was ≤ 4 days for 61% and ≤ 10 days for 94% of patients. Splenectomy was initial therapy for 30 patients. Of the 19 patients who had previous medical therapy, splenectomy changed their lymphoma diagnosis in 5 (26%). Twenty-one patients without splenectomy were clinically categorized as having non-cHCL splenic B-cell lymphoma. Nine required medical treatment for progressive lymphoma and of these 3 (33%) required re-treatment for lymphoma progression compared to 16% of patients following first line splenectomy. CONCLUSION: Splenectomy is useful for the diagnosis of non-cHCL splenic B-cell lymphomas with comparable risk/benefit profile and remission duration to medical therapy. Patients with suspected non-cHCL splenic lymphomas should be considered for referral to a high-volume center with experience in performing splenectomies for definitive diagnosis and treatment.

Arrhythmogenic Cardiotoxicity Associated With Contemporary Treatments of Lymphoproliferative Disorders.

Background There are limited data on risk of arrhythmias among patients with lymphoproliferative disorders. We designed this study to determine the risk of atrial and ventricular arrhythmia during treatment of lymphoma in a real-world setting. Methods and Results The study population comprised 2064 patients included in the University of Rochester Medical Center Lymphoma Database from January 2013 to August 2019. Cardiac arrhythmias-atrial fibrillation/flutter, supraventricular tachycardia, ventricular arrhythmia, and bradyarrhythmia-were identified using International Classification of Diseases, Tenth Revision (ICD-10) codes. Multivariate Cox regression analysis was used to assess the risk of arrhythmic events with treatments categorized as Bruton tyrosine kinase inhibitor (BTKi), mainly ibrutinib/non-BTKi treatment versus no treatment. Median age was 64 (54-72) years, and 42% were women. The overall rate of any arrhythmia at 5 years following the initiation of BTKi was (61%) compared with (18%) without treatment. Atrial fibrillation/flutter was the most common type of arrhythmia accounting for 41%. Multivariate analysis showed that BTKi treatment was associated with a 4.3-fold (P<0.001) increased risk for arrhythmic event (P<0.001) compared with no treatment, whereas non-BTKi treatment was associated with a 2-fold (P<0.001) risk increase. Among subgroups, patients without a history of prior arrhythmia exhibited a pronounced increase in the risk for the development of arrhythmogenic cardiotoxicity (3.2-fold; P<0.001). Conclusions Our study identifies a high burden of arrhythmic events after initiation of treatment, which is most pronounced among patients treated with the BTKi ibrutinib. Patients undergoing treatments for lymphoma may benefit from prospective focused cardiovascular monitoring prior, during, and after treatment regardless of arrhythmia history.

Multi-omics analyses demonstrate a critical role for EHMT1 methyltransferase in transcriptional repression during oogenesis.

EHMT1 (also known as GLP) is a multifunctional protein, best known for its role as an H3K9me1 and H3K9me2 methyltransferase through its reportedly obligatory dimerization with EHMT2 (also known as G9A). Here, we investigated the role of EHMT1 in the oocyte in comparison to EHMT2 using oocyte-specific conditional knockout mouse models (Ehmt2 cKO, Ehmt1 cKO, Ehmt1/2 cDKO), with ablation from the early phase of oocyte growth. Loss of EHMT1 in Ehmt1 cKO and Ehmt1/2 cDKO oocytes recapitulated meiotic defects observed in the Ehmt2 cKO; however, there was a significant impairment in oocyte maturation and developmental competence in Ehmt1 cKO and Ehmt1/2 cDKO oocytes beyond that observed in the Ehmt2 cKO. Consequently, loss of EHMT1 in oogenesis results, upon fertilization, in mid-gestation embryonic lethality. To identify H3K9 methylation and other meaningful biological changes in each mutant to explore the molecular functions of EHMT1 and EHMT2, we performed immunofluorescence imaging, multi-omics sequencing, and mass spectrometry (MS)-based proteome analyses in cKO oocytes. Although H3K9me1 was depleted only upon loss of EHMT1, H3K9me2 was decreased, and H3K9me2-enriched domains were eliminated equally upon loss of EHMT1 or EHMT2. Furthermore, there were more significant changes in the transcriptome, DNA methylome, and proteome in Ehmt1/2 cDKO than Ehmt2 cKO oocytes, with transcriptional derepression leading to increased protein abundance and local changes in genic DNA methylation in Ehmt1/2 cDKO oocytes. Together, our findings suggest that EHMT1 contributes to local transcriptional repression in the oocyte, partially independent of EHMT2, and is critical for oogenesis and oocyte developmental competence.