Purpose: Lymphopenia is a common adverse event following radiotherapy, but its prognostic relevance following comprehensive involved-site radiation (ISRT) for oligometastatic disease is unknown. We evaluated lymphocyte kinetics after ISRT for oligometastases and tested whether treatment-related lymphopenia was associated with overall survival (OS) and modified progression-free survival (mPFS). Patients and Methods: We performed a single-institution registry study of consecutive patients with 1 to 5 distant metastases treated with comprehensive ISRT by a single radiation oncologist from 2014 to 2023. Systemic therapy was administered at clinician discretion. Absolute lymphocyte count (ALC) was collected at baseline, during radiation and at 1, 3 and 12 months after radiotherapy. Lymphopenia was graded using CTCAE v5.0 (grade 1, ALC < 1000 cells/µL; grade ≥ 3, ALC < 500 cells/µL). OS and mPFS (defined as death or metastatic progression not salvageable with further local therapy) were estimated by the Kaplan–Meier method. Associations were evaluated using univariable and multivariable Cox proportional hazards models. Results: Among 177 patients, the 5-year OS was 39.6% and the median OS was 42.8 months. Median ALC declined from 1400 cells/µL at baseline to 800 cells/µL during radiotherapy (p < 0.01) and 700 cells/µL at a median of 0.7 months after radiation (p < 0.01). Partial recovery was observed at 1000 cells/µL at 3 months (p < 0.001) and 1000 cells/µL at 1 year (p < 0.01). Baseline ALC <1000 cells/µL was associated with worse OS on univariable analysis (p = 0.04) but not on multivariable analysis. Although 27% developed grade ≥ 3 lymphopenia within 3 months of radiation, the 5-year overall survival was 41.4% without lymphopenia versus 31.9% with lymphopenia (p = 0.60). On multivariable analysis, ECOG performance status (HR 1.9, p < 0.01), age (HR 1.04, p < 0.01), albumin (HR 0.6, p = 0.03), and pre-radiation chemotherapy (HR 3.1, p < 0.01) independently predicted overall survival. Conclusions: Comprehensive ISRT for oligometastatic disease was associated with a sustained decrease in median ALC. Treatment-related lymphopenia was not independently associated with OS in this heterogeneous cohort. The disease control benefit of metastasis-directed therapy may outweigh potential detrimental immunologic effects of radiation-induced lymphopenia.

Ventricular remodeling occurring in heart failure (HF) involves structural disarray of the sarcolemma T-tubule (TT)–sarcoplasmic reticulum (SR) dyad junctions, thereby disrupting the close apposition of L-type Ca²⁺ channels (Ca_V1.2) with ryanodine receptors (RyR2) that trigger SR Ca²⁺ release and myofilament contraction. In a rat ischemic heart failure model expressing low thyroid hormone (TH) function, we used 3D stochastic optical reconstruction microscopy (STORM) to image RyR2 clusters with Ca_V1.2 channels, and the associated protein junctophilin-2 (Jph2). We tested whether treatment with T3, the biologically active form of TH, throughout progression of the disease would preserve T-tubule structure and dyadic ion channel organization. Confocal microscopy of isolated cardiomyocytes (CMs) stained with ANEPPS membrane dye showed significantly decreased TT density in diseased CMs while T3 treatment attenuated TT disorganization. 3D STORM images of dyadic ion channels labeled with fluorescent-tagged antibodies to RyR-Dylight550, Jph-CF647 and Ca_V1.2/IgG-Dylight488 were captured. A density-based algorithm defined RyR2 clusters, and a 400 nm spherical 3D volume of interest around each RyR2 cluster’s centroid determined the number of Ca_V1.2 and Jph2 localizations associated with each RyR2 cluster. Analysis revealed significant reduction in RyR2 cluster size and number with reduced co-localized Jph2 in failing CMs. T3 treatment increased RyR2 cluster numbers and cluster volumes albeit non-significantly, with increased co-clustering of Jph2. The number of Ca_V1.2 co-localized with RyR2 clusters trended lower in the failing CMs. These results support maintaining TH homeostasis in optimizing the nanoscale organization of Ca²⁺ ion channels in triggering Ca²⁺ release and myofibrillar contraction in patients with heart disease.