Hypomethylating agents are associated with high rates of hematologic toxicity in patients with secondary myeloid neoplasms developing after acquired aplastic anemia.

Not available.

Mild Hypoxia Accelerates Cerebral Cavernous Malformation Disease Through CX3CR1-CX3CL1 Signaling.

BACKGROUND: Heterogeneity in the severity of cerebral cavernous malformations (CCMs) disease, including brain bleedings and thrombosis that cause neurological disabilities in patients, suggests that environmental, genetic, or biological factors act as disease modifiers. Still, the underlying mechanisms are not entirely understood. Here, we report that mild hypoxia accelerates CCM disease by promoting angiogenesis, neuroinflammation, and vascular thrombosis in the brains of CCM mouse models. METHODS: We used genetic studies, RNA sequencing, spatial transcriptome, micro-computed tomography, fluorescence-activated cell sorting, multiplex immunofluorescence, coculture studies, and imaging techniques to reveal that sustained mild hypoxia via the CX3CR1-CX3CL1 signaling pathway influences cell-specific neuroinflammatory interactions, contributing to heterogeneity in CCM severity. RESULTS: Histological and expression profiles of CCM neurovascular lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) in male and female mice found that sustained mild hypoxia (12% O2, 7 days) accelerates CCM disease. Our findings indicate that a small reduction in oxygen levels can significantly increase angiogenesis, neuroinflammation, and thrombosis in CCM disease by enhancing the interactions between endothelium, astrocytes, and immune cells. Our study indicates that the interactions between CX3CR1 and CX3CL1 are crucial in the maturation of CCM lesions and propensity to CCM immunothrombosis. In particular, this pathway regulates the recruitment and activation of microglia and other immune cells in CCM lesions, which leads to lesion growth and thrombosis. We found that human CX3CR1 variants are linked to lower lesion burden in familial CCMs, proving it is a genetic modifier in human disease and a potential marker for aggressiveness. Moreover, monoclonal blocking antibody against CX3CL1 or reducing 1 copy of the Cx3cr1 gene significantly reduces hypoxia-induced CCM immunothrombosis. CONCLUSIONS: Our study reveals that interactions between CX3CR1 and CX3CL1 can modify CCM neuropathology when lesions are accelerated by environmental hypoxia. Moreover, a hypoxic environment or hypoxia signaling caused by CCM disease influences the balance between neuroinflammation and neuroprotection mediated by CX3CR1-CX3CL1 signaling. These results establish CX3CR1 as a genetic marker for patient stratification and a potential predictor of CCM aggressiveness.

CPX-351 Pharmacokinetics and Safety in Adults with Hematologic Malignancies and Renal Function Impairment: Phase 1 Trial.

This open-label phase 1 study (clinicaltrials.gov, NCT03555955) assessed CPX-351 pharmacokinetics (PK) and safety in patients with hematologic malignancies with normal or impaired renal function. Patients were enrolled into three cohorts based on their creatinine clearance (CrCl): ≥90 mL/min (Cohort 1, normal renal function, n = 7), 30 to <59 mL/min (Cohort 2, moderate renal impairment, n = 8), or <30 mL/min (Cohort 3, severe renal impairment, n = 6). Patients received intravenous CPX-351 for initial induction; blood and urine samples were collected for PK analysis. The primary objective was to assess the PK parameters for cytarabine, daunorubicin, and their respective metabolites, arabinosyluracil (Ara-U) and daunorubicinol. Renal impairment did not significantly impact the cytarabine, daunorubicin, or daunorubicinol exposure, but it caused a slight increase in the Ara-U exposure. The CPX-351 side effect profile was similar in patients with impaired renal function compared to those with normal renal function. All the patients reported ≥1 treatment-emergent adverse event (TEAE), most commonly febrile neutropenia and nausea (57% each) and hyperglycemia (43%); no patients discontinued treatment due to TEAEs. These data suggest that CPX-351 dose adjustment is not required for patients with hematologic malignancies with moderate or severe renal impairment.

Heartbreaker: Detection and prevention of cardiotoxicity in hematological malignancies.

Cancer survivors are at significant risk of cardiovascular (CV) morbidity and mortality; patients with hematologic malignancies have a higher rate of death due to heart failure compared to all other cancer subtypes. The majority of conventional hematologic cancer treatments is associated with increased risk of acute and long-term CV toxicity. The incidence of cancer therapy induced CV toxicity depends on the combination of patient characteristics and on the type, dose, and duration of the therapy. Early diagnosis of CV toxicity, appropriate referral, more specific cardiac monitoring follow-up and timely interventions in target patients can decrease the risk of CV adverse events, the interruption of oncological therapy, and improve the patient's prognosis. Herein, we summarize the CV effects of conventional treatments used in hematologic malignancies with a focus on definitions and incidence of the most common CV toxicities, guideline recommended early detection approaches, and preventive strategies before and during cancer treatments.

Influence of Income, Education, and Medicaid Expansion on Palliative Care in Acute Myeloid Leukemia Using the National Cancer Database.

Palliative care is integral to symptom management, and we examined its relationship with income, education, and Medicaid expansion in acute myeloid leukemia. This was a retrospective cross-sectional study using the National Cancer Database that included patients with acute myeloid and monocytic leukemias > 18 years of age treated at Commission on Cancer facilities from 2004 to 2016. Univariate and multivariate models were adjusted for demographic variables and facility characteristics. There were 124,988 patients, but only 106,495 had palliative care data, and of this 4111 (3%) received palliative care. The most educated had the highest odds of receiving palliative care (odds ratio, OR 1.23, 95% CI 1.08-1.41; P = 0.002), but the highest income bracket (≥ $63,333) had the lowest odds (OR 0.82, 95% CI 0.72-0.93; P = 0.003). Residence in states with Medicaid expansion (January 2014 onward) had greater palliative care utilization. Palliative care use was associated with higher education but underutilized with higher incomes. Increased access with Medicaid expansion suggests the importance of public insurance.

Donor-Derived Malignancy and Transplantation Morbidity: Risks of Patient and Donor Genetics in Allogeneic Hematopoietic Stem Cell Transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a key treatment option for hematologic malignancies (HMs), although it carries significant risks. Up to 30% of patients relapse after allo-HSCT, of which up to 2% to 5% are donor-derived malignancies (DDMs). DDMs can arise from a germline genetic predisposition allele or clonal hematopoiesis (CH) in the donor. Increasingly, genetic testing reveals that patient and donor genetic factors contribute to the development of DDM and other allo-HSCT complications. Deleterious germline variants in CEBPA, DDX41, GATA2, and RUNX1 predispose to inferior allo-HSCT outcomes. DDM has been linked to donor-acquired somatic CH variants in DNMT3A, ASXL1, JAK2, and IDH2, often with additional new variants. We do not yet have evidence to standardize donor genetic sequencing prior to allo-HSCT. The presence of hereditary HM disorders should be considered in patients with myeloid malignancies and their related donors, and screening of unrelated donors should include family and personal history of cytopenia and HMs. Excellent multidisciplinary care is critical to ensure efficient timelines for screening and necessary discussions among medical oncologists, genetic counselors, recipients, and potential donors. After allo-HSCT, HM relapse monitoring with genetic testing effectively results in genetic sequencing of the donor, as the transplanted hematopoietic system is donor-derived, which presents ethical challenges for disclosure to patients and donors. We encourage consideration of the recent National Marrow Donor Program policy that allows donors to opt-in for notification about detection of their genetic variants after allo-HSCT, with appropriate genetic counseling when feasible. We look forward to prospective investigation of the impact of germline and acquired somatic genetic variants on hematopoietic stem cell mobilization/engraftment, graft-versus-host disease, and DDM to facilitate improved outcomes through knowledge of genetic risk.

Infection and Vaccine Induced Spike Antibody Responses Against SARS-CoV-2 Variants of Concern in COVID-19-Naïve Children and Adults.

Although a more efficient adaptive humoral immune response has been proposed to underlie the usually favorable outcome of pediatric COVID-19, the breadth of viral and vaccine cross-reactivity toward the ever-mutating Spike protein among variants of concern (VOCs) has not yet been compared between children and adults. We assessed antibodies to conformational Spike in COVID-19-naïve children and adults vaccinated by BNT162b2 and ChAdOx1, and naturally infected with SARS-CoV-2 Early Clade, Delta, and Omicron. Sera were analyzed against Spike including naturally occurring VOCs Alpha, Beta, Gamma, Delta, and Omicron BA.1, BA.2, BA.5, BQ.1.1, BA2.75.2, and XBB.1, and variants of interest Epsilon, Kappa, Eta, D.2, and artificial mutant Spikes. There was no notable difference between breadth and longevity of antibody against VOCs in children and adults. Vaccinated individuals displayed similar immunoreactivity profiles across variants compared with naturally infected individuals. Delta-infected patients had an enhanced cross-reactivity toward Delta and earlier VOCs compared to patients infected by Early Clade SARS-CoV-2. Although Omicron BA.1, BA.2, BA.5, BQ.1.1, BA2.75.2, and XBB.1 antibody titers were generated after Omicron infection, cross-reactive binding against Omicron subvariants was reduced across all infection, immunization, and age groups. Some mutations, such as 498R and 501Y, epistatically combined to enhance cross-reactive binding, but could not fully compensate for antibody-evasive mutations within the Omicron subvariants tested. Our results reveal important molecular features central to the generation of high antibody titers and broad immunoreactivity that should be considered in future vaccine design and global serosurveillance in the context of limited vaccine boosters available to the pediatric population.

Real-world effectiveness of intensive chemotherapy with 7&3 versus venetoclax and hypomethylating agent in acute myeloid leukemia.

Intensive chemotherapy with cytarabine and anthracycline (7&3) remains the standard therapy for patients medically fit for induction, but the assessment of fitness remains controversial. Venetoclax and hypomethylating agent (ven/HMA) combination therapy has improved outcomes in unfit patients but no prospective study has assessed ven/HMA versus 7&3 as initial therapy in older, fit patients. Given no studies and expectation of ven/HMA use in patients outside of trial criteria, we evaluated retrospective outcomes among newly diagnosed patients. A nationwide electronic health record (EHR)-derived database and the University of Pennsylvania EHR identified 312 patients receiving 7&3 and 488 receiving ven/HMA who were 60-75 years old without history of organ failure. Ven/HMA patients were older and more likely to have secondary AML, adverse cytogenetics, and adverse mutations. Median overall survival (OS) for patients receiving intensive chemotherapy was 22 versus 10 months for ven/HMA (HR 0.53, 95% CI 0.40-0.60). Controlling for measured baseline characteristic imbalances reduced survival advantage by half (HR 0.71, 95% CI 0.53-0.94). A sub-group of patients with equipoise, likelihood at least 30%-70% of receiving either treatment, had similar OS outcomes (HR 1.10, 95% CI 0.75-1.6). Regarding safety outcomes, 60-day mortality was higher for ven/HMA (15% vs. 6% at 60 days) despite higher documented infections and febrile neutropenia for 7&3. In this multicenter real-word dataset, patients selected for intensive chemotherapy had superior OS but a large group had similar outcomes with ven/HMA. Prospective randomized studies, controlling for both measured and unmeasured confounders, must confirm this outcome.

Older Adults With Newly Diagnosed AML: Hot Topics for the Practicing Clinician.

Over the past decade, our understanding of AML pathogenesis and pathophysiology has improved significantly with mutational profiling. This has led to translational advances in therapeutic options, as there have been 10 new US Food and Drug Administration (FDA) approvals for AML therapies since 2017, half of which target specific driver mutations in FLT3, IDH1, or IDH2. These new agents have expanded the therapeutic armamentarium for AML, particularly for patients who are considered ineligible for intensive chemotherapy with anthracycline- and cytarabine-containing regimens. These new treatment options are relevant because the median age at diagnosis is 68 years, and outcomes for patients older than 60 years have historically been dismal. However, the optimal approach to incorporating novel agents into frontline regimens remains a clinical challenge, particularly with regard to sequencing of therapies, considering the role of allogeneic hematopoietic stem cell transplantation and managing toxicities.

Obesity in adult acute myeloid leukemia is not associated with inferior response or survival even when dose capping anthracyclines: An ECOG-ACRIN analysis.

BACKGROUND: Obesity (body mass index [BMI] ≥30 kg/m2 ) is an important epidemiological risk factor for developing acute myeloid leukemia (AML). Therefore, the authors studied the association of obesity with clinical and genetic phenotype and its impact on outcome in adults with AML. METHODS: The authors analyzed BMI in 1088 adults who were receiving intensive remission induction and consolidation therapy in two prospective, randomized therapeutic clinical trials of the Eastern Cooperative Oncology Group-American College of Radiology Imaging Network: E1900 (ClinicalTrials.gov identifier NCT00049517; patients younger than 60 years) and E3999 (ClinicalTrials.gov identifier NCT00046930; patients aged 60 years or older). RESULTS: Obesity was prevalent at diagnosis (33%) and, compared with nonobesity, was associated with intermediate-risk cytogenetics group (p = .008), poorer performance status (p = .01), and a trend toward older age (p = .06). Obesity was not associated with somatic mutations among a selected 18-gene panel that was tested in a subset of younger patients. Obesity was not associated with clinical outcome (including complete remission, early death, or overall survival), and the authors did not identify any patient subgroup that had inferior outcomes based on BMI. Obese patients were significantly more likely to receive <90% of the intended daunorubicin dose despite protocol specification, particularly in the E1900 high-dose (90 mg/m2 ) daunorubicin arm (p = .002); however, this did not correlate with inferior overall survival on multivariate analysis (hazard ratio, 1.39; 95% confidence interval, 0.90-2.13; p = .14). CONCLUSIONS: Obesity is associated with unique clinical and disease-related phenotypic features in AML and may influence physician treatment decisions regarding daunorubicin dosing. However, the current study demonstrates that obesity is not a factor in survival, and strict adherence to body surface area-based dosing is not necessary because dose adjustments do not affect outcomes.

Systematic review and meta-analysis: Prognostic impact of time from diagnosis to treatment in patients with acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) has been considered an oncologic emergency that requires initiation of chemotherapy immediately after diagnosis. With the introduction of novel targeted therapies, there is a potential benefit associated with delaying definitive treatment for identification of actionable therapeutic targets. Unfortunately, cytogenetic/molecular testing can take >7 days to return, and there is not a consensus regarding the prognostic impact of time from diagnosis to treatment (TDT) in AML. METHODS: A literature review and meta-analysis of studies done to date that evaluate TDT was conducted. Studies that reported baseline characteristics, TDT, and outcomes for patients with AML were selected. Outcomes included overall survival (OS), complete remission (CR), and mortality. Studies that measured CR rates within each TDT range and data to calculate odds ratios were included in the meta-analysis. The remaining outcomes were synthesized descriptively for literature review. RESULTS: Thirteen studies were identified, which comprised a total of 14,946 patients. Median TDT values were between 1 and 8 days. Several studies found a significant association between prolonged TDT and older age and lower proliferation burden. Four of 11 studies did not detect a significant relationship between TDT and OS. No studies found a significant association between TDT and early death. Six of eight studies did not find a significant association between TDT and CR rate. The meta-analysis found a significant association between prolonged TDT and decreased achievement of CR (p < .05). CONCLUSIONS: Results were highly variable but suggest it may be feasible to pursue cytogenetic/molecular testing in patients who are clinically stable, particularly in those aged 60 years and older.

Hospital facility characteristics and socioeconomic factors on outcomes and treatment in patients with multiple myeloma: National Cancer Database analysis.

Previous studies have shown that socioeconomic factors play an important role in multiple myeloma (MM) health outcomes. We postulated that the type of treatment facilities and their volume of cases also affect overall survival, utilization of various therapies including palliative care services in newly diagnosed MM. Using the National Cancer Database (NCDB), we analyzed 174,551 newly diagnosed MM participants from across the country. We found that at high volume facility centers (over 90th percentile of new patient volume from 2004 to 2016), the median overall survival (OS) was 62.3 months versus 35.3 months at lower volume facilities (p <0.001). Similarly, high volume academic cancer centers had an improved median OS of 66.4 months (65.3-67.4 CI) versus 39.2 months (37.9-40.4 months CI) in lower volume academic centers (p <0.001). The odds of utilizing chemotherapy, immunotherapy, and autologous transplants were higher in academic cancer centers compared to community cancer centers, after adjusting for demographic and socioeconomic factors (OR 1.10, 1.23, and 2.06 respectively, all with p<0.001). There was significantly decreased odds of receiving palliative care (OR 0.89, 95% CI 0.85-0.93) in high volume facilities compared to low volume. Palliative care services were more frequently utilized at integrated network cancers and comprehensive community cancer centers compared to community cancer centers, with similar odds of receiving palliative care between community and academic facility types. Our results likely reflect increased provider experience and resources in higher volume and academic facilities. This highlights the need to integrate resources and improve access to community programs.

Recent advances in targeted therapies in acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. While survival for younger patients over the last several decades has improved nearly sixfold with the optimization of intensive induction chemotherapy and allogeneic stem cell transplantation (alloHSCT), this effect has been largely mitigated in older and less fit patients as well as those with adverse-risk disease characteristics. However, the last 10 years has been marked by major advances in the molecular profiling of AML characterized by a deeper understanding of disease pathobiology and therapeutic vulnerabilities. In this regard, the classification of AML subtypes has recently evolved from a morphologic to a molecular and genetic basis, reflected by recent updates from the World Health Organization and the new International Consensus Classification system. After years of stagnation in new drug approvals for AML, there has been a rapid expansion of the armamentarium against this disease since 2017. Low-intensity induction therapy with hypomethylating agents and venetoclax has substantially improved outcomes, including in those previously considered to have a poor prognosis. Furthermore, targeted oral therapies against driver mutations in AML have been added to the repertoire. But with an accelerated increase in treatment options, several questions arise such as how to best sequence therapy, how to combine therapies, and if there is a role for maintenance therapy in those who achieve remission and cannot undergo alloHSCT. Moreover, certain subtypes of AML, such as those with TP53 mutations, still have dismal outcomes despite these recent advances, underscoring an ongoing unmet need and opportunity for translational advances. In this review, we will discuss recent updates in the classification and risk stratification of AML, explore the literature regarding low-intensity and novel oral combination therapies, and briefly highlight investigative agents currently in early clinical development for high-risk disease subtypes.

TP53 in AML and MDS: The new (old) kid on the block.

MDS and AML are clonal hematopoietic stem cell disorders of increasing incidence, having a variable prognosis based, among others, on co-occurring molecular abnormalities. TP53 mutations are frequently detected in these myeloid neoplasms and portend a poor prognosis with known therapeutic resistance. This article provides a timely review of the complexity of TP53 alterations, providing updates in diagnosis and prognosis based on new 2022 International Consensus Classification (ICC) and World Health Organization (WHO) guidelines. The article addresses optimal testing strategies and reviews current and arising therapeutic approaches. While the treatment landscape for this molecular subgroup is under active development, further exploration is needed to optimize the care of this group of patients with unmet needs.

Tracking the clonal dynamics of SARS-CoV-2-specific T cells in children and adults with mild/asymptomatic COVID-19.

Children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop less severe coronavirus disease 2019 (COVID-19) than adults. The mechanisms for the age-specific differences and the implications for infection-induced immunity are beginning to be uncovered. We show by longitudinal multimodal analysis that SARS-CoV-2 leaves a small footprint in the circulating T cell compartment in children with mild/asymptomatic COVID-19 compared to adult household contacts with the same disease severity who had more evidence of systemic T cell interferon activation, cytotoxicity and exhaustion. Children harbored diverse polyclonal SARS-CoV-2-specific naïve T cells whereas adults harbored clonally expanded SARS-CoV-2-specific memory T cells. A novel population of naïve interferon-activated T cells is expanded in acute COVID-19 and is recruited into the memory compartment during convalescence in adults but not children. This was associated with the development of robust CD4+ memory T cell responses in adults but not children. These data suggest that rapid clearance of SARS-CoV-2 in children may compromise their cellular immunity and ability to resist reinfection.

Doxorubicin pharmacokinetics and toxicity in patients with aggressive lymphoma and hepatic impairment.

Aggressive lymphomas are curable with doxorubicin-based chemotherapy. In patients presenting with elevated serum bilirubin, doxorubicin is commonly dose reduced or delayed based on limited pharmacokinetic data. We evaluated plasma pharmacokinetics of doxorubicin and its metabolite doxorubicinol as well as toxicity in 59 patients with normal bilirubin levels and 10 patients with elevated bilirubin levels. Patients received full-dose EPOCH +/-rituximab. Median (range) age was 51 (18-75) years. Patients with elevated bilirubin levels had higher international prognostic index and poorer performance status. Although median doxorubicin clearance was lower and median plasma doxorubicin and doxorubicinol concentrations were higher in patients with elevated bilirubin levels, values were within the concentration range observed in patients with normal levels. Rates of febrile neutropenia were similar between groups, but there was greater grade 4 neutropenia and thrombocytopenia during the first but not subsequent treatment cycles in patients with elevated bilirubin. More grade 3/4 gastrointestinal and neurotoxicity occurred in patients with elevated bilirubin during the first but not subsequent cycles. Although toxicity was greater on cycle 1, the adverse effects were managed safely. These results show that empiric dose reductions of continuous infusion doxorubicin may not be necessary in patients with elevated bilirubin levels. This trial was registered at www.clinicaltrials.gov as #NCT00001337, #NCT00069238, and #NCT00005780.

Diminished humoral and cellular responses to SARS-CoV-2 vaccines in patients with chronic lymphocytic leukemia.

Previous studies have demonstrated low rates of seroconversion to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) messenger RNA (mRNA) vaccines in patients with chronic lymphocytic leukemia (CLL). In this national collaboration of 11 cancer centers in the United States, we aimed to further characterize and understand vaccine-induced immune responses, including T-cell responses, and the impact of CLL therapeutics (#NCT04852822). Eligible patients were enrolled in 2 cohorts (1) at the time of initial vaccination and (2) at the time of booster vaccination. The serologic response rates (anti-S) from 210 patients in the initial vaccination cohort and 117 in the booster vaccination cohort were 56% (95% confidence interval [CI], 50-63) and 68% (95% CI, 60-77), respectively. Compared with patients not on therapy, those receiving B-cell-directed therapy were less likely to seroconvert (odds ratio [OR], 0.27; 95% CI, 0.15-0.49). Persistence of response was observed at 6 months; anti-S titers increased with the administration of booster vaccinations. In the initial vaccination cohort, positive correlations were observed between the quantitative serologic response and CD4 T-cell response for the Wuhan variant and, to a lesser degree, for the Omicron variant (Spearman P = 0.45 Wuhan; P = 0.25 Omicron). In the booster vaccination cohort, positive correlations were observed between serologic responses and CD4 T-cell responses for both variants (P = 0.58 Wuhan; P = 0.57 Omicron) and to a lesser degree for CD8 T-cell responses (P = 0.33 Wuhan; P = 0.22 Omicron). Although no deaths from coronavirus disease 2019 (COVID-19) have been reported after booster vaccinations, patients should use caution as newer variants emerge and escape vaccine-induced immunity. This trial was registered at www.clinicaltrials.gov as #NCT04852822.

Circulating Plasma miRNA Homologs in Mice and Humans Reflect Familial Cerebral Cavernous Malformation Disease.

Patients with familial cerebral cavernous malformation (CCM) inherit germline loss of function mutations and are susceptible to progressive development of brain lesions and neurological sequelae during their lifetime. To date, no homologous circulating molecules have been identified that can reflect the presence of germ line pathogenetic CCM mutations, either in animal models or patients. We hypothesize that homologous differentially expressed (DE) plasma miRNAs can reflect the CCM germline mutation in preclinical murine models and patients. Herein, homologous DE plasma miRNAs with mechanistic putative gene targets within the transcriptome of preclinical and human CCM lesions were identified. Several of these gene targets were additionally found to be associated with CCM-enriched pathways identified using the Kyoto Encyclopedia of Genes and Genomes. DE miRNAs were also identified in familial-CCM patients who developed new brain lesions within the year following blood sample collection. The miRNome results were then validated in an independent cohort of human subjects with real-time-qPCR quantification, a technique facilitating plasma assays. Finally, a Bayesian-informed machine learning approach showed that a combination of plasma levels of miRNAs and circulating proteins improves the association with familial-CCM disease in human subjects to 95% accuracy. These findings act as an important proof of concept for the future development of translatable circulating biomarkers to be tested in preclinical studies and human trials aimed at monitoring and restoring gene function in CCM and other diseases.

Neuroinflammation Plays a Critical Role in Cerebral Cavernous Malformation Disease.

BACKGROUND: Cerebral cavernous malformations (CCMs) are neurovascular lesions caused by loss of function mutations in 1 of 3 genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3). CCMs affect ≈1 out of 200 children and adults, and no pharmacologic therapy is available. CCM lesion count, size, and aggressiveness vary widely among patients of similar ages with the same mutation or even within members of the same family. However, what determines the transition from quiescent lesions into mature and active (aggressive) CCM lesions is unknown. METHODS: We use genetic, RNA-sequencing, histology, flow cytometry, and imaging techniques to report the interaction between CCM endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils (CCM endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils interaction) during the pathogenesis of CCMs in the brain tissue. RESULTS: Expression profile of astrocytes in adult mouse brains using translated mRNAs obtained from the purification of EGFP (enhanced green fluorescent protein)-tagged ribosomes (Aldh1l1-EGFP/Rpl10a) in the presence or absence of CCM lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) identifies a novel gene signature for neuroinflammatory astrocytes. CCM-induced reactive astrocytes have a neuroinflammatory capacity by expressing genes involved in angiogenesis, chemotaxis, hypoxia signaling, and inflammation. RNA-sequencing analysis on RNA isolated from brain endothelial cells in chronic Pdcd10BECKO mice (CCM endothelium), identified crucial genes involved in recruiting inflammatory cells and thrombus formation through chemotaxis and coagulation pathways. In addition, CCM endothelium was associated with increased expression of Nlrp3 and Il1b. Pharmacological inhibition of NLRP3 (NOD [nucleotide-binding oligomerization domain]-' LRR [leucine-rich repeat]- and pyrin domain-containing protein 3) significantly decreased inflammasome activity as assessed by quantification of a fluorescent indicator of caspase-1 activity (FAM-FLICA [carboxyfluorescein-fluorochrome-labeled inhibitors of caspases] caspase-1) in brain endothelial cells from Pdcd10BECKO in chronic stage. Importantly, our results support the hypothesis of the crosstalk between astrocytes and CCM endothelium that can trigger recruitment of inflammatory cells arising from brain parenchyma (microglia) and the peripheral immune system (leukocytes) into mature active CCM lesions that propagate lesion growth, immunothrombosis, and bleedings. Unexpectedly, partial or total loss of brain endothelial NF-κB (nuclear factor κB) activity (using Ikkbfl/fl mice) in chronic Pdcd10BECKO mice does not prevent lesion genesis or neuroinflammation. Instead, this resulted in a trend increase in the number of lesions and immunothrombosis, suggesting that therapeutic approaches designed to target inflammation through endothelial NF-κB inhibition may contribute to detrimental side effects. CONCLUSIONS: Our study reveals previously unknown links between neuroinflammatory astrocytes and inflamed CCM endothelium as contributors that trigger leukocyte recruitment and precipitate immunothrombosis in CCM lesions. However, therapeutic approaches targeting brain endothelial NF-κB activity may contribute to detrimental side effects.