Abstract Background Cancer therapy-related cardiac dysfunction (CTRCD) is a major side effect of anthracycline chemotherapy (AC). Apart from cardiac effects, early vascular toxicity has been described and increased vascular stiffness and endothelial dysfunction are associated with future cardiac events. Whether vascular dysfunction has a role as an early, potential modifiable biomarker for CTRCD, is not clear. Purpose To assess the dynamics of endothelial function and vascular stiffness in response to AC. Methods In this single-centre cohort study, female breast cancer patients scheduled for AC were prospectively enrolled (01/2020-05/2023). Cardiac evaluation (echocardiography, hs-troponin I (hs-TnI) and NT-proBNP) and vascular function measurements were performed at 5 timepoints: before chemotherapy (T0), directly after taxane chemotherapy (T1), at the end of AC (T2), and at three (T3) and twelve months after chemotherapy (T4). CTRCD was defined according to the ESC guidelines. Endothelial function was assessed non-invasively, with flow mediated dilatation (FMD) and peripheral arterial tonometry (PAT), vascular stiffness was measured by Pulse Wave Velocity (PWV). Results Baseline and treatment characteristics of the 33 included patients are displayed in Figure 1. Cardiac function declined overall after exposure to AC, as evidenced by a reduction in LVEF (p=0.009), worsening of GLS (p=0.014) and a significant rise in hs-TnI (p<0.001). There was a trend for higher NT-proBNP levels (p=0.054). Overall, 20 patients developed mild CTRCD (60.6%), while five (15.2%) developed moderate CTRCD. After AC, blood pressure decreased and heart rate increased significantly (p<0.001). Endothelial function measured by FMD did not show a significant change during follow-up (p=0.096) but measurement by PAT showed a significant decline after AC (corrected reactive hyperaemia index fRHI p=0.043). Interestingly, PWV, corrected for diastolic blood pressure (DBP), declined after chemotherapy (p=0.014). A decline in endothelial function became evident after taxane chemotherapy, worsened following AC, and normalized during longer follow-up. In contrast, the decrease in vascular stiffness remained apparent up to one year after AC. The observed changes in fRHI and PWV were independent of the development of CTRCD (repeated measure ANOVA with CTRCD as factor: p=0.606 and 0.549 respectively). Conclusion There is an overall decline in systolic cardiac function and a high incidence of (asymptomatic) CTRCD in patients with breast cancer treated with taxane and AC. We observed an early and transient endothelial dysfunction. Surprisingly vascular stiffness shows a lasting decrease in the first year after AC, which is at least partly driven by a sustained decline in arterial blood pressure. These observed changes in vascular function were not related to the occurrence of CTRCD. Therefore, further work is needed to elucidate the longer-term effects of AC on vascular function.Figure 1For image description, please refer to the figure legend and surrounding text. Figure 2For image description, please refer to the figure legend and surrounding text.

Truncating variants in TTN (TTNtv) are present in around 7% of patients with anthracycline-induced cancer therapy-related cardiac dysfunction (CTRCD). In dilated cardiomyopathy, proximal I-band TTNtv (TTNtvI) harbour less pathogenic potential than distant A-band TTNtv (TTNtvA). To assess if a location-dependent effect of TTNtv is at play in an anthracycline-induced CTRCD, in order to refine risk stratification in patients. Distinct isogenic TTNtv hiPSC lines were created using CRISPR/Cas9 starting from an in-house reprogrammed hiPSC line of a healthy female control individual. Exon 48 (E48), located in the I-band, and exon 357 (E357), located in the A-band were targeted, based on variants identified in our CTRCD population. Differentiation of iPSCs to cardiomyocytes was performed using an in-house established protocol, each of the lines was differentiated at least two times. Expression of cardiac markers and TTN was evaluated with immunocytochemistry. For assessment of contractility, bright-field videos of contracting monolayers were obtained at day 27-44 and analysed using Musclemotion software. Next, doxorubicin (DOX) was added for 24 hours (0, 0.1, 1 or 10 µM) and contractility imaging was repeated. After DOX-treatment, apoptosis (as Caspase 3 activity) and calpain activity were assessed using luciferase assays (Caspase-Glo 3/7 Assay and Calpain-GloTM Protease Assay respectively). We created two isogenic hiPSC lines with a heterozygous TTNtvI (E48): E48_C1: c.13836_13853del and E48_C2: c.13836_13838delTTTinsGATACATACAA, and two with a heterozygous TTNtvA (E357): E357_C1: c. 100373delT and E357_C2: c. 100372_100373insT. In all cell lines, 24 hours treatment with DOX resulted in increased nuclear staining for distal titin. Overall, cells became less elongated and shorter segments of striated titin were observed at higher DOX doses, indicating an impact on cell architecture. Apoptosis significantly increased with increasing DOX dose and according to cell line, with lowest values in the isogenic control and highest values in TTNtvA. Similarly, Calpain activity increased with higher DOX dose, and this was most outspoken in TTNtvA, present in TTNtvI, but absent in the control cell line. The impact on contractility however was less clear, as no linear DOX dose related effects were observed. However, DOX-dose and targeted exons still showed a significant interaction (p<0.0001) indicating a different sensibility to DOX according to which exon was targeted. For each DOX dose, TTNtvA and TTNtvI hiPSC-CMs had significantly lower contractility than the control cell line. DOX influenced cell architecture and nuclear presence of distal titin in all cell lines. Heterozygous TTNtvI and TTNtvA hiPSC-CMs show increased apoptosis and calpain activity in response to DOX treatment, suggesting an increased predilection for cardiotoxicity. Effects were more prominent in TTNtvA than in TTNtvI hiPSC-CMs.Figure 1 Figure 2

In dilated cardiomyopathy, truncating variants in titin (TTNtv) show position-dependent effects: proximal I-band (TTNtvI) harbour less pathogenic potential than distal A-band TTNtv (TTNtvA). TTNtv are present in 7-10% of patients with anthracycline-induced cancer therapy-related cardiac dysfunction (CTRCD). To assess if a location-dependent effect of TTNtv is at play in anthracycline-induced CTRCD, in order to refine risk stratification in patients. Distinct isogenic TTNtv human induced pluripotent stem cell (hiPSC) lines were created using CRISPR/Cas9 starting from an in-house reprogrammed hiPSC line of a healthy female. Exon 48 (E48), located in the I-band, and exon 357 (E357), located in the A-band were targeted, based on TTNtv identified in our CTRCD population. The engineered TTNtvI and TTNtvA lines were validated. Differentiation of each iPSCs line to cardiomyocytes was performed at least two times. Expression of cardiac markers was evaluated with immunocytochemistry. For assessment of contractility, bright-field videos of contracting monolayers were obtained at day 27-44 (VOX microscope) and analysed using Musclemotion software. Next, doxorubicin (DOX) was added for 24 hours (0, 0.1, 1 or 10 µM) and contractility imaging was repeated. After DOX-treatment, apoptosis (Caspase 3 activity) and calpain activity were assessed using luciferase assays (Caspase-Glo 3/7 Assay, Calpain-GloTM Protease Assay), as DOX induces calpain-dependent titin proteolysis. We created isogenic hiPSC lines with a heterozygous TTNtv located in the I-band: E48_C1: c.13836_13853del and E48_C2: c.13836_13838delTTTinsGATACATACAA, or with a heterozygous TTNtv located in the A-band: E357_C1: c. 100373delT and E357_C2: c. 100372_100373insT. In all cell lines, DOX resulted in increased nuclear staining for distal titin, with a dose-response effect observed. Overall, cells became less elongated. Apoptosis significantly increased with increasing DOX dose and according to cell line, with lowest values in the isogenic control and highest in TTNtvA (Fig 1). Similarly, Calpain activity increased with higher DOX dose, and this was most outspoken in TTNtvA, present in TTNtvI, but absent in the control line. For contractility, DOX-dose and targeted exons showed a significant interaction (p<0.0001) indicating a different sensibility to DOX according to which exon was targeted. However, for control and TTNtvI lines, no linear DOX dose related effects were seen, while TTNtvA hiPSC-CMs showed worst contractility when treated with the highest. For each DOX dose, TTNtvA and TTNtvI hiPSC-CMs had significantly lower contractility than the control cell line (Fig 2). DOX influenced cell architecture in all cell lines. Heterozygous TTNtvI and TTNtvA hiPSC-CMs show increased apoptosis and calpain activity in response to DOX treatment compared to an isogenic control cell line. Effects were more prominent in TTNtvA than in TTNtvI hiPSC-CMs.Figure 1 Figure 2

Abstract Background Close monitoring of development of cardiovascular toxicity during anthracycline (AC) chemotherapy is crucial for early diagnosis and guidance of therapy. SerpinA3, a serine protease inhibitor, was recently described as prognostic marker for heart failure and elevated plasma levels are associated with increased mortality. The effect of AC on SERPINA3 levels in a cancer population has not been studied yet Purpose To investigate whether SERPINA3 plasma levels could serve as a marker for cancer therapy-related cardiac dysfunction and whether its levels are influenced by AC chemotherapy Methods Adult cancer patients requiring AC chemotherapy were enrolled between January 2020 and June 2022. Patients with a history of heart failure or prior cardiotoxicity were excluded. Measurement of cTnI and NT-proBNP and echocardiography (LVEF and GLS) were performed at four timepoints: before the start of AC (V1), directly after completion of chemotherapy (V2), 3 months thereafter (V3) and 1 year after V2 (V4). CTRCD was diagnosed according to the recent ESC guidelines. Results Fifty-one patients fulfilled inclusion criteria and agreed to participate in the study. Patients were on average 54 years old and the majority were female (80.4%). After anthracyclines, 38 patients developed asymptomatic CTRCD, graded as mild in 29 patients and moderate (LVEF 40-49%) in patients. SerpinA3 plasma levels at V3 were significantly higher in patients with moderate CTRCD (247.7; [196.4-428.9]) compared to patients with no CTRCD (142.7; [126.5-170.2]; p=0.004). In patients developing moderate CTRCD, a significant increase in SerpinA3 from baseline to V2 ( 209.0 [162.025-2.7]; 313.3 [212.6- 387.1, p=0.028) and remained elevated till V3 (247.7 [196.4-428.9], p=0.038). In patients with no CTRCD SerpinA3 showed an opposite trend with no significant difference at V2 compared to V1 (190.1 [165.2-230.5] vs 196.3 [170.8-297.1]; p= 0.972), but a subsequent significant decline from V2 to V3 (142.7 [126.5-170.2]; p=0.003). In patients with mild CTRCD a similar, but non-significant trend as in no CTRCD patients was seen(211.4 [167.8-302.6] vs 218.3 [171.9-277.7]; p=0.346 vs 167.5 [140.5-286.8]; p=0.122). These findings were confirmed by the absolute change in SerpinA3 from baseline to V4 which differed significantly in moderate CTRCD (+51.3; [7.1-176.4]) compared to both patients with no CTRCD (-47.5;[ -78.0- -35.4]; p=0.019) and patients with mild CTRCD (-52.0;[ -78.6- 32.8]; p=0.043). In patients with moderate CTRCD SerpinA3 values at V4 remained significantly elevated in patients without full cardiac recovery 1 year after the end of chemotherapy (4/5 patients; p=0.050). Conclusion SerpinA3 is increased after anthracycline chemotherapy in patients with moderate CTRCD. A lasting increase of serpinA3 was observed in in patients who did not show a complete recovery of cardiac function, potentially indicative of a worse cardiac prognosis.Figure 1Figure 2

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): 1.Flanders Research Foundation- FWO (doctoral research grant) 2.UZA Foundation-Wolvenbos Grant 2019 Background Anthracycline chemotherapy is a cornerstone in the treatment of several malignancies. However, it causes substantial toxicity, of which cardiotoxicity is the most frequent and most severe (1). Ideally, patients at risk for cardiotoxicity are identified prior to treatment, however, traditional patient- and treatment-related factors fail to fully predict the individual risk at the moment. Improved risk stratification for ANT-induced cardiotoxicity could possibly be reached by taking genetic risk factors into account. Purpose We assessed the prevalence of variants in genes encoding for cardiac proteins in the development of anthracycline-induced cardiotoxicity. Methods Patients presenting with early (< 1y) or late (> 1y) anthracycline-induced cardiotoxicity between 1995 and 2020 were included. Cardiotoxicity was defined as a decline in left ventricular ejection fraction (LVEF) to <50% and a ≥10% reduction from baseline by echocardiography. Genetic analysis was offered using a haloplex gene panel composed of 59 known cardiomyopathy-related genes. Variants were classified according to ACMG guidelines(2). Both variants of unknown significance (VUS, class 3) and likely pathogenic/pathogenic variants (class 4 and 5) were taken into account. Frequency of genetic variants was compared to a matched positive control cohort of patients with dilated cardiomyopathy (DCM). Results Forty-two patients that had developed cardiotoxicity (mean age at time of diagnosis 54yrs) fulfilled the inclusion criteria and agreed to genetic testing. The majority of patients had been treated for lymphoma (52.4%) or breast cancer (38.1%). Total equivalent dose of anthracyclines averaged 285.5 mg/m² doxorubicin. Patients showed an average reduction in LVEF of 27.1% (+- 10.1). In 18 patients (42.9 %), a variant could be identified; 15 patients carried a variant of unknown significance (VUS) and 3 carried a likely pathogenic variant. In total 29 variants different variants were identified in 18 distinct individuals. Genetic yield was independent of anthracycline-dose and the presence of other cardiovascular risk factors. The genetic yield in cardiotoxicity patients did not differ significantly from this in DCM-controls (18/42 VUS, 5/42 (likely) pathogenic variants, p = 0.19). After initiation of standard heart failure therapy, 6 (33.3%) of variant carriers showed incomplete recovery of cardiac function, compared to only 3 (12.5%) of patients who did not carry a variant (p=0.103). Conclusion Prevalence of genetic variants in anthracycline-induced cardiotoxicity is similar to that in isolated dilated cardiomyopathy. This finding supports a second-hit mechanism in which anthracycline chemotherapy, uncovers a genetically determined cardiomyopathy. As such, genetic variants in cardiomyopathy genes could improve individualized risk stratification prior to anthracycline chemotherapy and can assist in personalized prevention of this feared side effect of chemotherapy.

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Fund for Scientific Research (FWO) Flanders INSPIRE project (H2020-MSCA-ITN program) Background The chemotherapeutic doxorubicin (DOX) is frequently used to treat a wide variety of cancers, but the cardiotoxic side effects limits its clinical use in some patients. Additionally, DOX contributes to vascular toxicity, which may be an early manifestation of DOX-associated toxicity. Therefore, there is an interest to evaluate vascular function in patients receiving DOX-based treatment regimens. Purpose We aimed to assess arterial stiffness and endothelial dysfunction as potential markers of vascular toxicity in both DOX-treated cancer patients and a murine model. Methods Female breast cancer patients with a need for adjuvant or neoadjuvant DOX-based treatment were prospectively included. Chemotherapeutic regimen consisted of 12 weekly cycles of taxane treatment, followed by 4 cycles of DOX (and cyclophosphamide) treatment. Vascular function (endothelial function (FMD and RHI), arterial stiffness (Aix and cfPWV) and cardiac function (LVEF, hsTnI, NT-proBNP) were performed at baseline (T1), after completion of taxane treatment (T2) and after completion of DOX treatment (T3). In addition to the clinical study, male C57Bl6/J mice were intraperitoneally injected with 2 mg /kg (low dose) or 4 mg/kg DOX (high dose) once per week for 6 weeks. Arterial stiffness was assessed in vivo by measuring abdominal aorta pulse wave velocity (aaPWV) by high-frequency ultrasound imaging combined with ex vivo vascular function evaluation. Results Twenty patients (mean age 51.2 +/- 10.1 yrs) treated with DOX were included. After DOX treatment (T3 vs T2), left ventricular ejection fraction (LVEF) was decreased, while hsTnI and NT-proBNP levels were increased, indicating cardiotoxicity (Table 1). Likewise, LVEF was reduced in DOX-treated mice after 3 weeks, which persisted until the end of the treatment (6 weeks). In patients, RHI was impaired at T2 and T3 compared to T1, which indicates progressive endothelial dysfunction during treatment (Table 1). Consistent with these findings, DOX treatment in mice resulted in endothelial dysfunction, as evidenced by a lower basal nitric oxide (NO) index and reduced acetylcholine-induced endothelium-dependent vasorelaxation (Figure 1B & 1C). Finally, cfPWV was decreased at T2 and T3 compared to T1, even after correction for BP, suggesting rather an improvement of arterial stiffness (Table 1). Although DOX treatment in mice resulted in a similar trend towards lower aaPWV at the end of treatment (6 weeks), we observed an initial increase in aaPWV after 2 weeks (Figure 1A). Conclusion Collectively, these findings suggest that, apart from cardiotoxicity, DOX treatment results in early and consistent endothelial dysfunction, while evaluation of arterial stiffness may be time-sensitive. Hence, endothelial dysfunction may be a more reliable and sensitive marker than arterial stiffness to evaluate DOX-induced vascular toxicity in patients.