Acquired Aplastic anemia (AA) is a bone marrow failure syndrome characterized by pancytopenia and marrow hypoplasia, and is mediated by immune destruction of hematopoietic stem cells. Mutations in several genes including telomerase, a ribonucleoprotein enzyme complex, consisting of a reverse transcriptase enzyme (TERT), an RNA template (TERC), and several stabilizing proteins, and the associated shelterin complexes have been found in both congenital and idiopathic AA. In particular, several TERT and TERC mutations reduce telomerase activity in vitro and accelerate telomere attrition in vivo. Shortened telomeres have been observed in a third of idiopathic AA patients, but only 10% of these patients have mutations in genes of the telomerase complex. We have recently demonstrated that in addition to keeping telomeres from shortening, telomerase directly regulates transcriptional programs of developmentally relevant genes (Ghosh et al, Nat Cell Biol, 2012, 14, 1270). We postulate that changes in expression of telomerase associated genes, specifically TERT, contribute to the etiology of aplastic anemia. In an effort to better understand the molecular and clinical correlates of this disease, 24 idiopathic AA patient samples were collected at a tertiary medical center in Bangalore, India. Following informed consent, we performed RT-PCR analysis on harvested RNA from each patient and measured levels of TERT expression compared to that of normal controls (n=6). An 8 fold reduction in TERT expression was observed in 17/24 patients, while 7/24 patients maintained normal TERT expression. In general, TERT-low patients were younger in age (mean age 29y) compared with the TERT-normal patients (mean age 40y). TERT-low patients were more likely to have severe aplastic anemia (SAA) leading to higher mortality and poorer response to therapy, with 6/17 patients dying and 4/17 not responding to ATG therapy. Targeted panel sequencing of the 24 samples on an Illumina platform revealed that while TERT-normal patients had no mutations in genes associated with the telomerase/shelterin complex, TERT-low patients carried predicted pathogenic variants in TERT, TEP1, TINF2, NBN, TPP1, HSP90A and POT1 genes, all associated with the telomerase complex. Somatic gene variants were also identified in other AA associated genes, PRF1 and CDAN1, in the TERT-low cohort. In addition, novel predicted pathogenic mutations associated with the shelterin complex were found in two TERT-low patients in the TNKS gene. We also detected mutations in TET2, BCORL1, FLT-3, MLP and BRAF genes in TERT-low patients. Mutations in these genes are associated with clonal evolution, disease progression and poor prognosis. Our observations were further illustrated in a single patient where normal TERT expression was noted at initial clinical presentation. ATG therapy led to CR, but the patient returned within a year and succumbed to E.coli related sepsis. At that stage he had low TERT expression, suggesting that TERT expression can change as the disease progresses. Taken together, our data support the hypothesis that loss of TERT expression correlates with disease severity and poor prognosis. Our observations further suggest that preliminary and periodic evaluation of TERT expression levels in AA patients is likely to serve as a predictor of disease severity and influence the choice of therapy. Disclosures No relevant conflicts of interest to declare.

Determination of the magnitude of body iron stores helps to identify individuals at risk of iron-induced organ damage in Thalassemia patients. The most direct clinical method of measuring liver iron concentration (LIC) is through chemical analysis of needle biopsy specimens.Here we present a noninvasive method for the measurement of LIC in vivo using magnetic resonance imaging (MRI). Twenty-three pediatric Thalassemia major patients undergoing bone marrow transplantation at our centre were studied. All 23 patients had MRI T2* and R2* decay time for evaluation of LIC on a 1.5 Tesla MRI system followed by liver tissue biopsy for the assessment of iron concentration using an atomic absorption spectrometry. Simultaneously, serum ferritin levels were measured by enzymatic assay. We have correlated biopsy LIC with liver T2* and serum ferritin values with liver R2*. Of the 23 patients 11 were males, the mean age was 8.3 ± 3.7 years. The study results showed a significant correlation between biopsy LIC and liver T2* MRI (r = 0.768; p \ 0.001). Also, there was a significant correlation between serum ferritin levels and liver R2* MRI (r = 0.5647; p \ 0.01). Two patients had high variance in serum ferritin levels (2100 and 4100 mg/g) while their LIC was around 24 mg/g, whereas the difference was not seen in T2* MRI. Hence, the liver T2* MRI is a better modality for assessing LIC. Serum ferritin is less reliable than quantitative MRI. The liver T2* MRI is a safe, reliable, feasible and cost-effective method compared to liver tissue biopsy for LIC assessment.

INTRODUCTION EGFR (epidermal growth factor receptor) is important for the proliferation of stem cells across the body including the hematopoietic niche. However, the role of EGFR in aplastic anemia and subsequent responses to standard-of-care therapy is unknown.TWIST is a basic helix-loop-helix transcription factor recently found to regulate the hematopoietic stem cell (HSC) niche. The HSC niche is important for treatment of aplastic anemia. Telomerase and associated gene mutations have been reported in aplastic anemia, but these mutations are not present in all subjects and hence the cellular mechanisms of therapeutic responses observed is not explained by deregulated telomerase or associated genes. OBJECTIVE: To investigate the utility of measuring gene expression levels of EGFR and TWIST on the clinical response to immunosuppressive therapy in acquired Aplastic anemia patients without mutations in telomerase gene. METHODS: This was a single institution analysis of patients with acquired Aplastic anemia, in the age group of 16 to 60 years, who were treated with immunosuppressive therapy between June 2014 to December 2015. 15 patients who did not have homozygous TERT (telomerase catalytic subunit) or DKC (Dyskeratosiscongenita) mutations as determined by sequencing were included in this study. Diagnosis of Aplastic anemia was established with bone marrow aspiration and biopsy with normal cytogenetics. PNH was ruled out in all patients. 7 patients had very severe aplasticanemia (VSAA) and 8 had severe aplastic anemia. There were 11 males and 4 females.Following informed consent, we performed RT-PCR analysis on harvested RNA from each patient and measured levels of TWIST and EGFR expression compared to that of normal samples (n=6).15 patients underwent immunosuppressive therapy with horse ATG at 40mg/kg/day for 4 days followed by oral cyclosporine for at least 3 months. Clinical response was assessed at 3 months and 6 months post ATG administration. Total RNA from healthy donors (6) were used to establish normal baseline gene expression values. RESULTS: Out of the 15 patients that received ATG infusion, 10 patients (66%) had an 8-fold reduced expression of EGFR and TWIST compared to normal control. We grouped the study subjectsintoEGFR/TWIST-low (C1) and EGFR/TWIST-normal(C2). C1 subjects were younger in age (average age 29) compared with C2 (average age 40) andwere more likely to be diagnosed with a more severe form of the disease (VSAA). In the C1 group, all patients responded well to treatment with 4 (80%) patients achieving CR and 1 patient achieving a partial response. In C2, 8 (80%) patients showed no response, with 2 patients showing a partial response at the end of 6 months. CONCLUSION: Our data suggests that EGFR and TWIST may have significant impact on the ability of the hematopoietic stem cell niche to respond to immunosuppressive therapy in aplastic anemia, particularly in the absence of telomerase mutations. Therefore, low expression levels of EGFR/TWIST at diagnosis in may be useful in predicting response to immunosupressive therapy and thereby influence treatment decisions in aplastic anemia patients. Disclosures No relevant conflicts of interest to declare.

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