In Utero Cltc-ALK Fusion In Hematopoietic Progenitor Cells As a First Step Of Leukemogenesis In Blastic Plasmacytoid Dendritic Cell Neoplasm

Introduction Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a subtype of myeloid leukemia mainly affecting the elderly and often accompanied by cutaneous legions. It is a rare disease, and neither the genetic nor clonal origin of the disease is known. We report the first case of BPDCN with clathrin heavy chain (CLTC)-anaplastic lymphoma kinase (ALK) fusion gene. We performed a detailed analysis to understand the origin of the tumor cells and the leukemic process involved. Samples and Results Samples were collected from a female infant who was admitted under the diagnosis of hemophagocytic lymphohistiocytosis (HLH) at 1 month of age. One month later, leukemic blasts appeared in the peripheral blood showing karyotypic abnormality 46,XX,t(2;17;8)(p23;q23;p23). Fluorescence in situ hybridization with break apart probes covering the ALK gene revealed translocation of the 3’-ALK signal to der(17) and loss of the 5’ ALK signal on der(2). CLTC-ALK fusion was identified by direct sequencing of the RT-PCR product obtained from the peripheral blood specimen. Although HLH symptoms improved after one course of chemotherapy, blast cells re-appeared in the peripheral blood and bone marrow after 3 courses of chemotherapy, with a karyotype of 45, XX, t(2;17;8)(p23;q23;p23), -7. Multicolor flow cytometry showed the blast cells were weakly positive for CD4 and negative for CD3, and expression of CLTC-ALKwas confirmed in these cells. Some of the blasts were highly positive for CD123 and CD303, indicating the plasmacytoid dendritic cell phenotype and leading to the diagnosis of BPDCN. The rest of the blasts were positive for CD56 and weakly positive for CD123. Nearly half of this CD4+CD56+ population was also positive for monocytic marker, CD14. The possibility of in utero origin of the leukemic cells was tested by analyzing the presence of CLTC-ALK fusion in the Guthrie card. The genomic breakpoint of the CLTC-ALKfusion was determined by inverse PCR, and then 24 pieces of the Guthrie card containing the neonatal blood were tested for the existence of the cells carrying the same fusion breakpoint. The testing revealed the prenatal origin of the fusion gene. To explore the origin of leukemic transformation in the patient, the presence of the CLTC-ALK fusion gene was assessed in genomic DNA extracted from subpopulations sorted from the patient’s peripheral blood. As well as leukemic CD4+CD3- cells, most of the monocytes possessed the CLTC-ALK fusion gene, and a small portion of T cells, B cells and neutrophils were also positive for genomic CLTC-ALK fusion. Immature cells with high CD34 expression but without lineage markers separated from the peripheral blood were also positive for CLTC-ALKfusion. Conclusions The CLTC-ALK fusion gene was identified for the first time as the leukemia-promoting abnormality in an infant case of myeloid neoplasm BPDCN, indicating the tumorigenic potential of CLTC-ALK in myeloid progenitor cells. In addition, activated monocytes with the CLTC-ALK fusion might be responsible for the occurrence of HLH in the patient. Formation of the CLTC-ALK fusion was suggested to have occurred in a hematopoietic progenitor cells in utero, and the subsequent acquisition of monosomy 7, one of the myeloid lineage-oriented abnormalities, might have determined the cell fate to a myeloid neoplasm in this patient. Disclosures: No relevant conflicts of interest to declare.

Neonatal screening for severe primary immunodeficiency diseases using high-throughput triplex real-time PCR

Severe combined immunodeficiency (SCID) and X-linked agammaglobulinemia (XLA) are inborn errors of immune function that require prompt diagnosis and treatment to prevent life-threatening infections. The lack of functional T or B lymphocytes in these diseases serves as a diagnostic criterion and can be applied to neonatal screening. A robust triplex PCR method for quantitation of T-cell receptor excision circles (TRECs) and κ-deleting recombination excision circles (KRECs), using a single Guthrie card punch, was developed and validated in a cohort of 2560 anonymized newborn screening cards and in 49 original stored Guthrie cards from patients diagnosed with SCID, XLA, ataxia-telangiectasia, Nijmegen-breakage-syndrome, common variable immunodeficiency, immunoglobulin A deficiency, or X-linked hyper-IgMsyndrome. Simultaneous measurement of TREC and KREC copy numbers in Guthrie card samples readily identified patients with SCID, XLA, ataxia-telangiectasia and Nijmegen-breakage-syndrome and thus facilitates effective newborn screening for severe immunodeficiency syndromes characterized by the absence of T or B cells.

Use of HbA2 As a Discriminator for S/Beta Thalassaemia in a Nigerian Setting,

Abstract 4206 An integral part of an EU-UNDP funded pilot sickle cell screening project, was the installation, in Abuja (Federal Capital Territory) Nigeria, June 2010, of a High Performance Liquid Chromatography (HPLC) instrument. Prior to the installation, haemoglobinopathy screening was carried out using only unstained paper electrophoresis. Minor bands were difficult to visualise and the proportions of haemoglobins were not measured. Full blood count (FBC) data was also not routinely available. Therefore awareness of both alpha and beta thalassaemia was low as was the implications of coinheritance of beta thalassaemia with haemoglobin (Hb) S. Stratified community surveys were carried out with samples collected as Guthrie card blood spots; children aged less than 6 months, and whole blood; children aged between 6 months and 5 years. Blood spot samples were analysed using newborn sickle cell screening reagents and whole blood samples using beta thalassaemia short reagents, the later allowing accurate Hb A2 and Hb F measurement. Samples were processed on a Biorad™ Classic (instrument and reagents, Biorad, Hercules, CA.). Reporting algorithms were defined for both reagent sets. Using the beta thalassaemia reagents, Hb S/beta thalassaemia was considered when Hb S was predominant, Hb A absent or significantly reduced and the Hb A2>5. Over 10,000 samples were analysed, 410 had sickle cell disease, of which 370 were transported to London for further analysis, 70 of these had an Hb A2>5%, mean and range 5.9(5.1 – 9.1)%. To validate the algorithm 31 samples (Hb A2 3.5 – 7.4%), were selected for beta gene sequencing (Mai et al, 2004) and/or PCR analysis for the 7 common alpha gene deletions (3.7kb, 4.2kb, SEA, MED, THAI, FILL, 20.5kb and triplicated alpha gene locus, anti 3.7kb), (Chong et al, 2000, Wang et al, 2003). Five samples had an Hb A2of 3.5 – 4.9%, 2 with Hb A present (23 and 25%), all were Hb SS, 4 negative for alpha thalassaemia deletions and 1 heterozygous for the alpha 3.7kb deletion. Sixteen samples had an Hb A2 of 5.0 – 5.9%, all were Hb SS, 5 negative for alpha thalassaemia deletions and 11 heterozygous for the alpha 3.7kb deletion. Seven samples had an Hb A2 of 6.0 – 6.9%, 6 were Hb SS, 1 negative for alpha thalassaemia deletions, 1 heterozygous and 4 homozygous for the alpha 3.7kb deletion. One, Hb A2 6.9%, was a compound heterozygote for Hb S/ beta zero codon 106/107(+G) mutation. Three samples had an Hb A2 of 7.0 – 7.4%, review of chromatograms indicated that all showed poor chromatography due to lack of separation between Hb A2 and Hb S or shoulders to the left of the Hb A2 peak. All were Hb SS and heterozygous for the alpha 3.7kb deletion, with 1 also positive for the triplicated alpha globin gene, anti 3.7kb. Four other samples with Hb A2 >7% also showed poor chromatography, but were insufficient for molecular testing. The results indicate that Hb A2values >5% were mainly due to co-existing alpha thalassaemia with 76% of those tested, positive for the 3.7kb deletion. Poor chromatography was also a contributor particularly at the higher Hb A2 levels. During the initial stages of the project air conditioning failure caused major temperature fluctuations on overnight runs, this problem was resolved, improving chromatography. One case of S/beta zero thalassaemia was detected confirming the presence in this population. The results suggest that the Hb A2 may be considered as a discriminator for S/beta thalassaemia screening in this setting, increasing the algorithm Hb A2 level to >6.0% may improve specificity and reduce the number of false positives, this warrants further investigation. Future aims of the project for children over 6 months of age, include initiating routine FBC analysis and testing parental samples as a cost effective means of confirming suspected cases. Disclosures: No relevant conflicts of interest to declare.