Despite the effectiveness of anthracycline chemotherapy, cardiotoxicity remains a significant long-term secondary effect for children undergoing this therapy; cardiac toxicity is the most common treatment related cause of death in survivors. How anthracyclines lead to cardiotoxicity is the subject of debate.

One important pathway may be the generation of free radicals and the presence of redox-related damage resulting in the accumulation of iron. Anthracycline-generated free radicals induce lipid peroxidation that ultimately produces membrane damage and promotes apoptosis. In addition, mitochondrial damage, increased Ca²⁺ current along with inhibition of sarcoplasmic reticulum function, and decreased activity of Na⁺, K-ATPase, have all been implicated in doxorubicin-induced cardiotoxicity. Myocardial muscle contraction depends on release of Ca²⁺ from the sarcoplasmic reticulum (SR) and reuptake by the Ca²⁺ transport ATPase (SERCA). Recently, the Bassel-Duby and Olson team at UT Southwestern discovered a putative muscle-specific long noncoding RNA that encodes a peptide of 34 amino acids and named dwarf open reading frame (DWORF). DWORF localizes to the SR membrane, where it enhances the SERCA activity by displacing the SERCA inhibitors, phospholamban, sarcolipin, and myoregulin.

In recently published studies the group showed that DWORF mRNA is diminished in the failing heart. The group has made gain and loss of function DWORF mouse models that are being characterized by PET imaging. We hypothesize that doxorubicin-induced cardiotoxicity can be regulated by DWORF expression, opening the opportunity for new therapeutic approaches for chemotherapy induced cardiotoxicity.

Approximately 300 new children are diagnosed with and treated for cancer each year in our program, and many receive anthracyclines. Because of marked individual variation in cardiotoxicity, there is a need for a reliable and noninvasive method of early detection and serial monitoring for cardiotoxicity. This may then guide early management and therapy in attempts to avoid or delay the onset of clinical cardiotoxicity.

Early SPECT studies suggested alteration of perfusion and mitochondrial dysfunction may be an early marker of anthracycline toxicity. However, more recent studies suggest that perfusion is unaltered. PET offers the possibility to go beyond perfusion and evaluate metabolic or other changes that may occur with anthracycline toxicity.

This has been borne out in recent studies demonstrating increased glucose metabolism post anthracycline chemotherapy using [¹⁸F]FDG-PET.

Once the CRCFPO services became available, this User Group and CRCFPO started carrying out preclinical and clinical studies by pilot experiments. PET with corresponding radiotracers can identify cell death, defects in mitochondrial membrane function, glycolysis, and oxidative phosphorylation. Specifically, markers for cell apoptosis, reactive oxygen species, fatty acid metabolism, and glucose metabolism have been shown to be accurate in patients. MRI has also been used to monitor myocardial iron accumulation. We hypothesize that metabolic derangements are apparent prior to anatomic changes. The desired radiotracers allow early detection of myocyte injury and elucidate the mechanism of anthracycline-mediated cardiotoxicity. This will inform the design of future therapeutic trials. Therefore, an iterative translational research program has been proposed to investigate biomarkers and mechanisms in pre-clinical models and move them forward to clinical practice.

One preclinical model of anthracycline cardiotoxicity has been implemented. Three groups of animals are proposed: non-treated controls, anthracycline treated mice, anthracycline treated mice that have received a cardiac fibroblast reprogramming therapeutic regime or altered DWORF expression developed by the group of Dr. Eric Olson.

The mice are being imaged with:

1. [¹⁸F]FDG-PET to show glucose metabolism
2. [¹⁸F]FDM to assess the existence of reactive oxygen species and when it occurs
3. [¹¹C]acetate-PET to identify the defect in fatty acid metabolism
4. [¹⁸F]annexin for noninvasive assessment of apoptosis (other relevant radiotracers include ¹⁸F- or ⁶⁸Ga-labeled duramycin and [¹⁸F]ML-10)

These studies allow us to probe underlying pathologic mechanisms as well as changes in energy substrate preference (glucose vs. usual fatty acid) that commonly accompanies cardiac toxicity. The Olson group is assisting in setting up this model.

Secondly, a clinical observational study is being conducted with these radiotracers to evaluate for metabolic changes that might be associated with anthracycline-induced cardiotoxicity in children with leukemia, sarcoma or neuroblastoma.

It is estimated that 20-30 children with these cancers who are treated in the Division of Hematology/Oncology at UT Southwestern/Children’s Medical Center each year will be long-term survivors after high dose anthracycline therapy (about 300 mg/m²). This group is being followed yearly using cardiac MRI in the Division of Pediatric Cardiology at UT Southwestern/Children’s Medical Center for the duration of the study to correlate functional changes. They have been undergoing [¹⁸F]FDG-PET or [¹¹C]acetate-PET in a first of its kind longitudinal observational study.

Dynamic acquisition permits calculation of perfusion using first pass analysis. Left ventricular uptake is being quantified semi-quantitatively (SUV) and absolutely (kBq/mL). Activity is also being compared to reference tissue (muscle). Cumulative anthracycline dose is being plotted against tracer activity. Normal substrate preference in the heart is fatty acids but mitochondrial damage, if present, could lead to preferential glucose metabolism.

We are also enrolling children undergoing current anthracycline treatment and requiring [¹⁸F]FDG-PET/CT imaging for clinical surveillance. We estimate this will be a group of approximately 100 patients receiving a lower dose of anthracycline (50-80 mg/m²). Baseline and intermittent [¹⁸F]FDG-PET scans are being performed over the funding period and correlated with standard of care echocardiography or MRI. Analysis of the PET scans is as described above.

Note that [¹¹C]acetate PET or PET/CT scanning at UT Southwestern was initiated January 2017 under the U.S.-FDA IND and IRB approvals. Feedback from preclinical models are helping to identify potential novel radiotracers to use as biomarkers for acute injury in pediatric patients treated with anthracycline. Moreover, the results of clinical work can inform further radiotracer or imaging protocol development in preclinical models to refine imaging strategy, which will in turn help refine clinical protocols for imaging.

This combined clinical and preclinical strategy is enabling a strong feedback and feedforward iterative program. The goals are to demonstrate in preclinical models the utility of radiotracers targeting underlying pathophysiology and use that knowledge to optimize the design of therapeutic trials using the identified imaging biomarkers. Furthermore, if effective therapies become available for treating the underlying processes, e.g., free radical generation or apoptosis, then immediately we have imaging biomarkers that can impact individual patient care.

If successful, we will partner with other pediatric cancer programs in Texas or nationwide to disseminate the research protocols and infrastructure requirements in order to broadly benefit pediatric cancer patients with the cutting-edge imaging technologies developed by this core facility funding mechanism.