Statins Do Not Significantly Affect Oxidative Nitrosative Stress Biomarkers in the PREVENT Randomized Clinical Trial.

PURPOSE: Preventing Anthracycline Cardiovascular Toxicity with Statins (PREVENT) (NCT01988571) randomized breast cancer or lymphoma patients receiving anthracyclines to atorvastatin 40 mg daily or placebo. We evaluated the effects of atorvastatin on oxidative and nitrosative stress biomarkers, and explored whether these biomarkers could explain the lack of effect of atorvastatin on LVEF in PREVENT. PATIENTS AND METHODS: Blood samples were collected and cardiac magnetic resonance imaging was performed prior to doxorubicin initiation and at 6 and 24 months. Thirteen biomarkers (arginine-nitric oxide metabolites, paraoxonase-1 [PON-1] activity, and myeloperoxidase) were measured. Dimensionality reduction using principal component analysis was used to define biomarker clusters. Linear mixed-effects models determined the changes in biomarkers over time according to treatment group. Mediation analysis determined if biomarker clusters explained the lack of effect of atorvastatin on LVEF. RESULTS: Among 202 participants with available biomarkers, median age was 53 years; 86.6% had breast cancer; median LVEF was 62%. Cluster 1 levels, reflecting arginine methylation metabolites, were lower over time with atorvastatin, although this was not statistically significant (p=0.081); cluster 2 levels, reflecting PON-1 activity, were significantly lower with atorvastatin (p=0.024). There were no significant changes in other biomarker clusters (p>0.05). Biomarker clusters did not mediate an effect of atorvastatin on LVEF (p>0.05) Conclusions: Atorvastatin demonstrated very modest effects on oxidative/nitrosative stress biomarkers in this low cardiovascular risk population. Our findings provide potential mechanistic insight into the lack of effect of atorvastatin on LVEF in the PREVENT trial.

Rapid and accurate remethylation of DNA in Dnmt3a-deficient hematopoietic cells with restoration of DNMT3A activity.

Here, we characterize the DNA methylation phenotypes of bone marrow cells from mice with hematopoietic deficiency of Dnmt3a or Dnmt3b (or both enzymes) or expressing the dominant-negative Dnmt3aR878H mutation [R882H in humans; the most common DNMT3A mutation found in acute myeloid leukemia (AML)]. Using these cells as substrates, we defined DNA remethylation after overexpressing wild-type (WT) DNMT3A1, DNMT3B1, DNMT3B3 (an inactive splice isoform of DNMT3B), or DNMT3L (a catalytically inactive "chaperone" for DNMT3A and DNMT3B in early embryogenesis). Overexpression of DNMT3A for 2 weeks reverses the hypomethylation phenotype of Dnmt3a-deficient cells or cells expressing the R878H mutation. Overexpression of DNMT3L (which is minimally expressed in AML cells) also corrects the hypomethylation phenotype of Dnmt3aR878H/+ marrow, probably by augmenting the activity of WT DNMT3A encoded by the residual WT allele. DNMT3L reactivation may represent a previously unidentified approach for restoring DNMT3A activity in hematopoietic cells with reduced DNMT3A function.

Impact of Atrial Fibrillation on Heart Failure in Patients Treated With Anthracycline Chemotherapy.

Atrial fibrillation (AF) and anthracyclines are known risk factors for heart failure (HF). The magnitude of the effect of preexisting AF (preanthracycline AF) and newly developed AF (postanthracycline AF) in patients treated with anthracyclines on the occurrence of HF is unknown. The aim of our study was to characterize the impact of preanthracycline and postanthracycline AF on the subsequent occurrence of HF in patients treated with anthracyclines. In 5,598 patients treated with new anthracycline therapy at a tertiary center between 2008 and 2021, propensity score matching was used to match 204 pairs with or without preanthracycline AF and 135 pairs with or without postanthracycline AF. The primary outcome was new-onset symptomatic HF defined by the American Heart Association/American College of Cardiology guidelines. Patients with and without preanthracycline and postanthracycline AF were well matched for age, gender, medications, and cardiovascular risk factors. A total of 45 patients with preanthracycline AF and 23 matched patients developed HF (5-year cumulative incidence: 29% in the preanthracycline AF group and 13% in the matched group, p = 0.003; hazard ratio 2.1, 95% confidence interval 1.3 to 3.4, p = 0.004). A total of 161 patients (2.9%) developed postanthracycline AF. A total of 39 patients (5-year cumulative incidence: 40%) with postanthracycline AF and 9 matched patients (5-year cumulative incidence: 7%) developed HF (hazard ratio 6.1, 95% confidence interval 3.0 to 12.4, p <0.001). Preanthracycline AF and postanthracycline AF are associated with a high incidence of subsequent HF in patients treated with anthracyclines. Prospective studies of therapies are required to decrease HF in these high-risk patients.

Rationale and design of the Children's Oncology Group study AAML1831 integrated cardiac substudies in pediatric acute myeloid leukemia therapy.

Background: Pediatric acute myeloid leukemia (AML) therapy is associated with substantial short- and long-term treatment-related cardiotoxicity mainly due to high-dose anthracycline exposure. Early left ventricular systolic dysfunction (LVSD) compromises anthracycline delivery and is associated with inferior event-free and overall survival in de novo pediatric AML. Thus, effective cardioprotective strategies and cardiotoxicity risk predictors are critical to optimize cancer therapy delivery and enable early interventions to prevent progressive LVSD. While dexrazoxane-based cardioprotection reduces short-term cardiotoxicity without compromising cancer survival, liposomal anthracycline formulations have the potential to mitigate cardiotoxicity while improving antitumor efficacy. This overview summarizes the rationale and methodology of cardiac substudies within AAML1831, a randomized Children's Oncology Group Phase 3 study of CPX-351, a liposomal formulation of daunorubicin and cytarabine, in comparison with standard daunorubicin/cytarabine with dexrazoxane in the treatment of de novo pediatric AML. Methods/design: Children (age <22 years) with newly diagnosed AML were enrolled and randomized to CPX-351-containing induction 1 and 2 (Arm A) or standard daunorubicin and dexrazoxane-containing induction (Arm B). Embedded cardiac correlative studies aim to compare the efficacy of this liposomal anthracycline formulation to dexrazoxane for primary prevention of cardiotoxicity by detailed core lab analysis of standardized echocardiograms and serial cardiac biomarkers throughout AML therapy and in follow-up. In addition, AAML1831 will assess the ability of early changes in sensitive echo indices (e.g., global longitudinal strain) and cardiac biomarkers (e.g., troponin and natriuretic peptides) to predict subsequent LVSD. Finally, AAML1831 establishes expert consensus-based strategies in cardiac monitoring and anthracycline dose modification to balance the potentially competing priorities of cardiotoxicity reduction with optimal leukemia therapy. Discussion: This study will inform diagnostic, prognostic, preventative, and treatment strategies regarding cardiotoxicity during pediatric AML therapy. Together, these measures have the potential to improve leukemia-free and overall survival and long-term cardiovascular health in children with AML. Clinical trial registration: https://clinicaltrials.gov/, identifier NCT04293562.

The Association of Metformin With Heart Failure in Patients With Diabetes Mellitus Receiving Anthracycline Chemotherapy.

Background: The prevention of heart failure (HF) is an important issue in patients treated with anthracyclines. Metformin, widely used to treat diabetes mellitus (DM), protects from anthracycline-induced cardiotoxicity in vitro and in animal models. Objectives: The aim of our study was to test the association of metformin with the occurrence of symptomatic HF in patients with DM receiving anthracyclines. Methods: A total of 561 patients with DM received new anthracycline therapy between 2008 and 2021 in a tertiary care center; propensity score matching was used to compare patients with or without metformin treatment. The primary outcome was new onset symptomatic HF occurring within 1 year of the initiation of anthracyclines. Results: A total of 315 patients (65 ± 11 years of age, 33.7% male) were included. Patients with and without metformin were well matched for age, sex, type of cancer, medications, and cardiovascular risk factors. Six patients treated with metformin and 17 matched patients developed HF within 1 year of anthracycline initiation. The incidence of HF in patients treated with metformin was lower than patients without metformin within 1 year after anthracyclines (cumulative incidence: 3.6% vs 10.5%; P = 0.022; HR: 0.35; 95% CI: 0.14-0.90; P = 0.029). The use of metformin (HR: 0.71; 95% CI: 0.50-1.00; P = 0.049), was also associated with lower mortality. Conclusions: The use of metformin was associated with a lower incidence of HF and overall mortality in patients with DM receiving anthracyclines. Our findings should be further confirmed by randomized control trials.

Disruption of FBN1 by an Alu element insertion: A novel genetic cause of Marfan syndrome.

Alu elements are retrotransposons with ubiquitous presence in the human genome that have contributed to human genomic diversity and health. These approximately 300-bp sequences can cause or mediate disease by disrupting coding/splicing regions in the germline, by insertional mutagenesis in somatic cells, and in promoting formation of copy-number variants. Alu elements may also disrupt epigenetic regulation by affecting non-coding regulatory regions. There are increasing reports of apparently sporadic and inherited genetic disorders caused by Alu-related gene disruption, but Marfan syndrome resulting from Alu element insertion has not been previously described. We report a family with classic features of Marfan syndrome whose previous FBN1 genetic testing was inconclusive. Using contemporary next-generation sequencing and bioinformatics analysis, a pathogenic/disruptive Alu insertion occurring in the coding region of the FBN1 gene was identified (c.6564_6565insAlu; p. Glu2189fs) and was confirmed and specified further with Sanger sequencing. This identified the molecular basis of disease in the family that was missed using previous genetic testing technologies and highlights a novel pathogenic mechanism for Marfan syndrome. This case adds to the growing literature of Mendelian diseases caused by Alu retrotransposition, and it also shows the growing capability of genomic technologies for detecting atypical mutation events.

Congenital heart defects caused by FOXJ1.

FOXJ1 is expressed in ciliated cells of the airways, testis, oviduct, central nervous system and the embryonic left-right organizer. Ablation or targeted mutation of Foxj1 in mice, zebrafish and frogs results in loss of ciliary motility and/or reduced length and number of motile cilia, affecting the establishment of the left-right axis. In humans, heterozygous pathogenic variants in FOXJ1 cause ciliopathy leading to situs inversus, obstructive hydrocephalus and chronic airway disease. Here, we report a novel truncating FOXJ1 variant (c.784_799dup; p.Glu267Glyfs*12) identified by clinical exome sequencing from a patient with isolated congenital heart defects (CHD) which included atrial and ventricular septal defects, double outlet right ventricle (DORV) and transposition of the great arteries. Functional experiments show that FOXJ1 c.784_799dup; p.Glu267Glyfs*12, unlike FOXJ1, fails to induce ectopic cilia in frog epidermis in vivo or to activate the ADGB promoter, a downstream target of FOXJ1 in cilia, in transactivation assays in vitro. Variant analysis of patients with heterotaxy or heterotaxy-related CHD indicates that pathogenic variants in FOXJ1 are an infrequent cause of heterotaxy. Finally, we characterize embryonic-stage CHD in Foxj1 loss-of-function mice, demonstrating randomized heart looping. Abnormal heart looping includes reversed looping (dextrocardia), ventral looping and no looping/single ventricle hearts. Complex CHDs revealed by histological analysis include atrioventricular septal defects, DORV, single ventricle defects as well as abnormal position of the great arteries. These results indicate that pathogenic variants in FOXJ1 can cause isolated CHD.

Incidence of MACE in Patients Treated With CAR-T Cell Therapy: A Prospective Study.

Background: Previous retrospective studies have shown that chimeric antigen receptor T (CAR-T) cell therapy may be associated with major adverse cardiovascular events (MACE), especially in the context of cytokine-release syndrome (CRS) events. Objectives: The aim of this prospective observational study was to define the occurrence of MACE in adults undergoing treatment with CAR-T cell therapy and identify associated risk factors. Methods: Vital signs, blood samples, and an echocardiogram were collected prior to and 2 days, 1 week, 1 month, and 6 months after CAR-T cell infusion, and charts were consulted at 12 months. In the event of CRS, echocardiography was repeated within 72 hours. MACE were defined as cardiovascular death, symptomatic heart failure, acute coronary syndrome, ischemic stroke, and de novo cardiac arrhythmia. Results: A total of 44 patients were enrolled (mean age 58 ± 11 years, 77% men). The median follow-up duration was 487 days (Q1-Q3: 258-622 days). There were 24 episodes of CRS in 23 patients (52%) (13 grade 1, 10 grade 2, and 1 grade 3), with a median time to CRS of 4 days. Two patients had MACE (heart failure with preserved ejection fraction and atrial fibrillation) within 1 year and 6 and 7 days after CAR-T cell infusion. There was no change in left ventricular ejection fraction, but a modest decrease in global longitudinal strain was noted. Conclusions: There were few cardiac effects associated with contemporary CAR-T cell therapy. As MACE occurred after CRS episodes, aggressive treatment and close follow-up during CRS events are essential.

Potential Drivers for the Re-Emergence of Canine Leptospirosis in the United States and Canada.

Canine leptospirosis is an important zoonotic disease in many countries. This review examines potential drivers for increased diagnoses of canine leptospirosis in the United States and Canada, using the epidemiologic triad of agent-environment-host as a template. Leptospira spp. are classified into more than 250 serovars, but in many laboratories only 6 are routinely tested for in serologic agglutination tests of canine sera. Leptospiral infections in dogs may potentially go undetected with unemployed or currently employed diagnostic methods. Disease transmission from infected reservoir hosts usually occurs via urine-contaminated environmental sources such as water. Direct contact between infected and susceptible individuals, environmental factors such as climate changes in temperature and/or rainfall, and increasing number and urbanization of reservoir hosts may greatly increase dog exposure risks. A dog's lifestyle may influence exposure risk to leptospirosis, but vaccination based on proper identification of circulating serogroups dramatically reduces post-exposure infections. Regrettably, resistance to vaccination by veterinarians and dog owners leaves a large number of dogs at risk for this zoonotic disease.

Canine leptospirosis in Canada, test-positive proportion and risk factors (2009 to 2018): A cross-sectional study.

Over the past decade, there has been an apparent increased frequency and widened distribution of canine leptospirosis in Canada, however, this has been minimally investigated. Availability and clinical uptake of Leptospira polymerase chain reaction (PCR)-based testing of dogs in Canada may provide important insight into the epidemiology of this canine and zoonotic infectious disease. Study objectives were to evaluate clinical canine Leptospira PCR test results from a large commercial laboratory to determine temporal and spatial distribution in Canada and identify dog, geographic and temporal risk factors for test-positive dogs. This cross-sectional study analyzed data obtained from IDEXX Laboratories, Inc. on 10,437 canine Leptospira PCR tests (blood and/or urine) submitted by Canada-based veterinarians (July 2009 to May 2018). Multivariable logistic regression was used to identify risk factors for test-positive dogs. Test-positive proportion varied widely annually (4.8-14.0%) and by location. Provinces with the highest test-positive proportion over the study period were Nova Scotia (18.5%) and Ontario (9.6%), with the prairie provinces (Manitoba and Alberta combined) having the lowest proportion (1.0%); the northern territories could not be evaluated due to limited testing. In the final model, dog age, sex, breed, month, and year test performed, and location (urban/rural, province) of the practice submitting the sample were significant predictors of a positive Leptospira PCR test. Dogs less than one year of age (OR = 2.1; 95% CI: 1.6-2.9), male sex (OR = 1.3; 1.1-1.5), toy breed (OR = 3.3; 2.5-4.4), and samples submitted from an urban practice (OR = 1.3; 1.0-1.8) had the greatest odds of a positive Leptospira PCR test as compared to referent groups. Significant two-way interactions between province-month and year-month highlight the complex spatial and temporal influences on leptospirosis occurrence in this region. Our work suggests a high incidence of canine leptospirosis regionally within Canada. Identifiable dog and location factors may assist in future targeted prevention efforts.

Somatic Dnmt3a inactivation leads to slow, canonical DNA methylation loss in murine hematopoietic cells.

Mutations in the gene encoding DNA methyltransferase 3A (DNMT3A) are the most common cause of clonal hematopoiesis and are among the most common initiating events of acute myeloid leukemia (AML). Studies in germline and somatic Dnmt3a knockout mice have identified focal, canonical hypomethylation phenotypes in hematopoietic cells; however, the kinetics of methylation loss following acquired DNMT3A inactivation in hematopoietic cells is essentially unknown. Therefore, we evaluated a somatic, inducible model of hematopoietic Dnmt3a loss, and show that inactivation of Dnmt3a in murine hematopoietic cells results in a relatively slow loss of methylation at canonical sites throughout the genome; in contrast, remethylation of Dnmt3a deficient genomes in hematopoietic cells occurs much more quickly. This data suggests that slow methylation loss may contribute, at least in part, to the long latent period that characterizes clonal expansion and leukemia development in individuals with acquired DNMT3A mutations in hematopoietic stem cells.

BRCA1/2 Mutations and Cardiovascular Function in Breast Cancer Survivors.

OBJECTIVE: Animal models suggest that BRCA1/2 mutations increase doxorubicin-induced cardiotoxicity risk but data in humans are limited. We aimed to determine whether germline BRCA1/2 mutations are associated with cardiac dysfunction in breast cancer survivors. METHODS: In a single-center cross-sectional study, stage I-III breast cancer survivors were enrolled according to three groups: (1) BRCA1/2 mutation carriers treated with doxorubicin; (2) BRCA1/2 mutation non-carriers treated with doxorubicin; and (3) BRCA1/2 mutation carriers treated with non-doxorubicin cancer therapy. In age-adjusted analysis, core-lab quantitated measures of echocardiography-derived cardiac function and cardiopulmonary exercise testing (CPET) were compared across the groups. A complementary in vitro study was performed to assess the impact of BRCA1 loss of function on human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) survival following doxorubicin exposure. RESULTS: Sixty-seven women with mean (standard deviation) age of 50 (11) years were included. Age-adjusted left ventricular ejection fraction (LVEF) was lower in participants receiving doxorubicin regardless of BRCA1/2 mutation status (p = 0.03). In doxorubicin-treated BRCA1/2 mutation carriers and non-carriers, LVEF was lower by 5.4% (95% CI; -9.3, -1.5) and 4.8% (95% CI; -9.1, -0.5), respectively compared to carriers without doxorubicin exposure. No significant differences in VO2max were observed across the three groups (poverall = 0.07). Doxorubicin caused a dose-dependent reduction in viability of iPSC-CMs in vitro without differences between BRCA1 mutant and wild type controls (p > 0.05). CONCLUSIONS: BRCA1/2 mutation status was not associated with differences in measures of cardiovascular function or fitness. Our findings do not support a role for increased cardiotoxicity risk with BRCA1/2 mutations in women with breast cancer.

Genetic resiliency associated with dominant lethal TPM1 mutation causing atrial septal defect with high heritability.

Analysis of large-scale human genomic data has yielded unexplained mutations known to cause severe disease in healthy individuals. Here, we report the unexpected recovery of a rare dominant lethal mutation in TPM1, a sarcomeric actin-binding protein, in eight individuals with large atrial septal defect (ASD) in a five-generation pedigree. Mice with Tpm1 mutation exhibit early embryonic lethality with disrupted myofibril assembly and no heartbeat. However, patient-induced pluripotent-stem-cell-derived cardiomyocytes show normal beating with mild myofilament defect, indicating disease suppression. A variant in TLN2, another myofilament actin-binding protein, is identified as a candidate suppressor. Mouse CRISPR knock-in (KI) of both the TLN2 and TPM1 variants rescues heart beating, with near-term fetuses exhibiting large ASD. Thus, the role of TPM1 in ASD pathogenesis unfolds with suppression of its embryonic lethality by protective TLN2 variant. These findings provide evidence that genetic resiliency can arise with genetic suppression of a deleterious mutation.

Paraoxonase-1 Activity in Breast Cancer Patients Treated With Doxorubicin With or Without Trastuzumab.

The objective of this study was to determine associations of paraoxonase-1 (PON-1) with development of cancer therapy-related cardiac dysfunction (CTRCD). PON-1 is a cardioprotective enzyme associated with high-density lipoprotein that prevents oxidized low-density lipoprotein formation. Given the role of oxidative stress in doxorubicin-induced cardiotoxicity, PON-1 activity may have relevance for the prediction of CTRCD. In 225 patients with breast cancer receiving doxorubicin with or without trastuzumab, we quantified PON-1 activity through its paraoxonase (Pon) and arylesterase (Aryl) enzymatic activity at baseline, during, and after doxorubicin completion. Echocardiograms were performed at baseline, during therapy, and annually. CTRCD was defined as a decrease in left ventricular ejection fraction by ≥10% from baseline to <50%. Associations between baseline biomarkers and clinical variables were determined using multivariable linear regression. Associations between changes in biomarker activity and time to CTRCD were evaluated using Cox regression. Pon was directly associated with Black race and inversely associated with Stage 2 cancer. Aryl was inversely associated with body mass index. After doxorubicin completion, activity levels of Pon and Aryl were significantly decreased (median ratio compared with baseline for Pon: 0.95 [Q1-Q3: 0.81-1.07, P < 0.001]; for Aryl: 0.97 [Q1-Q3: 0.85-1.08, P = 0.010]). A total of 184 patients had an available quantitated echocardiogram at baseline and at least 1 follow-up visit. Increases from baseline in Pon at doxorubicin completion were independently associated with increased CTRCD risk (per 10% increase: hazard ratio [HR]: 1.21; 95% confidence interval [CI]: 1.05-1.39; P = 0.007). Associations between increases in Aryl and CTRCD tended in the same direction but were of borderline statistical significance (HR: 1.17; 95% CI: 0.99-1.38; P = 0.071). In patients with breast cancer treated with doxorubicin with or without trastuzumab, increases in the Pon enzymatic activity level of PON-1 were associated with increased CTRCD risk. PON-1 activity may be relevant to mechanistic risk prediction of cardiotoxicity with anthracyclines.

Feasibility of a tailored home-based exercise intervention during neoadjuvant chemotherapy in breast cancer patients.

PURPOSE: To evaluate the feasibility of a home-based moderate-to-vigorous intensity, phased (introduction, intermediate, maintenance), exercise prescription in breast cancer patients receiving cardiotoxic neoadjuvant chemotherapy. METHODS: Nineteen breast cancer patients were randomized to intervention or control for the duration of chemotherapy (16-24 weeks). The intervention was one aerobic exercise session at 80-90% VO2max for 25 min/week and 65%-75% VO2max for ≥ 50 min/week. Adherence to the tailored home-based program was assessed by heart rate monitors. Acceptability, tolerability, feasibility, efficacy, change in VO2max, and patient reported outcomes, safety, and clinical events were assessed. RESULTS: 25.7% of eligible women consented (acceptability). Adherence was 87.6%. Women were not able to maintain exercise intensity as chemotherapy progressed (23.7% of exercise minutes were completed at prescribed heart rate during maintenance). Efficacy of the intervention was demonstrated by maintenance of VO2max (-1.0 ± 13.2%) compared to (-27.5 ± 7.4%) the control group. Further, during and after therapy, patients in the intervention arm reported less fatigue (control-baseline: 14.4 ± 15.9; midpoint: 19.0 ± 11.4; follow-up: 29.4 ± 20.0; intervention-baseline: 29.2 ± 24.6; midpoint: 24.6 ± 14.4; follow-up: 23.6 ± 11.9), impairment in activities (control-baseline: 13.7 ± 16.0; midpoint: 32.8 ± 17.0; follow-up: 58.6 ± 27.9; intervention-baseline: 38.7 ± 31.8; midpoint: 47.1 ± 27.5; follow-up: 47.5 ± 31.0), and pain (control-baseline: 80.8 ± 17.1; midpoint: 73.9 ± 20.7; follow-up: 50.7 ± 25.7; intervention-baseline: 68.7 ± 28.4; midpoint: 61.4 ± 22.5; follow-up: 65.3 ± 22.4). There were no differences in adverse events, treatment delays, or pathological complete response. CONCLUSIONS: Neoadjuvant breast cancer patients maintained approximately one hour/week of moderate-intensity exercise over the course of their treatment. Further, this volume of exercise was sufficient to maintain fitness capacity and quality of life compared to the control group. TRIAL REGISTRY: ClinicalTrials.gov Identifier: NCT03280836, prospectively registered 9/13/2017, https://clinicaltrials.gov/ct2/show/NCT03280836 .