Sirolimus Treatment in Pediatric Patients With Ataxia-Telangiectasia

After baseline evaluation, oral sirolimus was initiated at a dose of 1 mg/m²/day divided into two daily doses. Treatment duration was 6 months, safety was assessed throughout the trial. Laboratory studies and adverse events were assessed every 2 months.

Neurological function was assessed using the Scale for the Assessment and Rating of Ataxia (SARA). Total scores range from 0 to 40 points, with higher scores indicating greater severity of ataxia. Scores were assessed at baseline and after 6 months of sirolimus treatment.

Baseline and follow-up immunological assessments were performed every 2 months and included serum immunoglobulin levels and lymphocyte subpopulation analysis. Lymphocyte proliferation was assessed using CFSE labeling (C34554; molecular probes). In brief, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque density gradient centrifugation and labeled with carboxifluorescein succinimidyl ester (CFSE). Cells were then stimulated with anti-CD3 (clone OKT3) plus anti CD28 (clone CD28.2) monoclonal antibodies (1µg/ml each). In parallel, PBMCs were stimulated with phytohemagglutinin (PHA) plus interleukin 2 (IL-2). Lymphocyte proliferation was quantified by flow cytometric analysis of CFSE dilution. Expression of the ATM protein was assessed in peripheral blood mononuclear cells by Western blot analysis.

Conjunctival telangiectasia was assessed using standardized digital photographs. It was quantified as the percentage of segmented vascular area relative to the total conjunctival region of interest. Measurements were obtained at baseline and after 6 months of sirolimus treatment.

Conjunctival photographs were analyzed using Fiji (ImageJ version 2.16.0/1.54p). The conjunctival region of interest (ROI) was manually delineated, excluding eyelids, eyelashes, and surrounding background, and the total ROI area was measured in pixels. Color segmentation was performed using the Color Threshold function in the HSB color space with fixed parameters: Hue, 0-25; Saturation, 110-255; and Brightness, 90-255. Images were converted into binary masks, and the segmented vascular area within the ROI was measured in pixels. Conjunctival telangiectasia was expressed as the percentage of segmented vascular area relative to the total conjunctival ROI area. All images were analyzed by the same investigator using an identical workflow and fixed thresholding parameters.

Cognitive performance was assessed using the Cerebellar Cognitive Affective Syndrome Scale (CCAS). The total raw score ranges from 0 to 120 points, with higher scores indicating better cognitive performance. Assessments were performed at baseline and after 6 months of sirolimus treatment.

Everyday executive functioning was assessed using the Behavior Rating Inventory of Executive Function, Second Edition (BRIEF-2). The Global Executive Composite was analyzed using T-scores, with higher scores indicating greater executive dysfunction. Assessments were performed at baseline and after 6 months of sirolimus treatment.

Emotional and behavioral functioning was assessed using the Child Behavior Checklist (CBCL). The Total Problems score was analyzed using T-scores, with higher scores indicating greater emotional and behavioral problems. Assessments were performed at baseline and after 6 months of sirolimus treatment.