Increased Telomere Length Is a Negative a Predicting Factor for IST in Children with Severe Aplastic Anemia?
Authors: Pawelec Katarzyna , Janiak Marek , Matysiak Michał , Wlodarski Pawel Krzysztof 
Published: October 2019

Abstract
Abstract 4381
Shortening of telomeres is observed in 1/3 of peripheral blond samples, collected from patients with severe aplastic anemia (SAA). Probably, this phenomenon is associated with poor response to immunosuppressive therapy –IST (antithymocyte globulin -ATG and cyklosporin-CSA). The aim of this study was to assess the length of telomeres in peripheral blood of children with SAA treated with antithymocyte globulin (ATG) and cyklosporin (CSA)

Materials and Methods:
Peripheral blood samples were collected from 13 children with confirmed SAA. Patients (9 girls and 4 boys) aged 7–19 yrs have all received rabbit ATG in a dose of 3.75 mg/kg/day for 5 days and CSA in a dose of 5mg/kg/day for 12–14 months). Remission was assessed on 180th and 360th day of treatment. Control peripheral blood samples were obtained from 12 healthy children (3 girls and 9 boys, aged 3–17 yrs), 12 healthy adultand from 4 SAA children parents. Informed consent was obtained from all adult donors and from parents of participating children.
DNA extraction and qPCR Genomic DNA was extracted from whole blood using AxyPrep Blood Genomic DNA Miniprep Kit (Axygene). Telomere length was determined using the quantitive PCR (qPCR) method described by Elisa Pavesi et al (1). Briefly, two qPCR reactions were run for each DNA sample: amplification of telomere product and amplification of a single copy gene (SEP15). Each qPCR reaction contained: Power SYBR1 Green PCR Master Mix (Applied Biosystems), template DNA and primers (270nM Tel1 and 900nM Tel2 or 500nM of each SEP15 primers). Telomere/single copy gene (T/S) ratios for samples were calculated using Human reference DNA (Applied Biosystems) standard curve. Reactions were preformed in Real Time PCR CFX96 system (Biorad). Statistica software version 9.1 (StatSoft) was used for statistical analysis

Results:
Among 13 SAA patients telomere shortening was found in 9 children while 4 patients had significantly elongated telomeres. All of the latter ones responded poorly to IST treatment: 3 of them did not respond (NR) to IST (neither on day 180 nor on 360) and one patient had a partial remission (PR). 2 patients in the NR group were randomized to unrelated bone marrow transplantation and one had another course of IST (no unrelated donor), after which he reached a PR. All patients with elongated telomeres had normal serum adriostendion level. Telomere elongation was also found in parents compared to the control group of adults (n=12, age range 31–57). Among 9 patients with shortened telomeres, 4 achieved complete remission (CR), 1 - PR and 4 did not respond (NR) to treatment on check times points.

Conclusions:
Our observations indicate that SAA patients with shortened telomeres respond to immunosuppressive therapy better then those with elongated telomeres. Telomere length may be considered therefore as a predicting factor in this patients. Studies on a larger group of patients should be performed to confirm our observations.
Short telomere definition –samples, which T/S value is less than the first Quartile of controls. Long telomere - samples, which T/S value is in III quartile of controls. Very long telomere - samples, which T/S value is higher than the IV Quartile of controls.

Disclosures:
No relevant conflicts of interest to declare.