Update on long-term outcomes of a cohort of patients with TCF3::HLF positive acute lymphoblastic leukemia treated with blinatumomab and stem cell transplantation

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Genetic determinants of risk in pulmonary arterial hypertension:international genome-wide association studies and meta-analysis

Background: Rare genetic variants cause pulmonary arterial hypertension, but the contribution of common genetic variation to disease risk and natural history is poorly characterised. We tested for genome-wide association for pulmonary arterial hypertension in large international cohorts and assessed the contribution of associated regions to outcomes. Methods: We did two separate genome-wide association studies (GWAS) and a meta-analysis of pulmonary arterial hypertension. These GWAS used data from four international case-control studies across 11 744 individuals with European ancestry (including 2085 patients). One GWAS used genotypes from 5895 whole-genome sequences and the other GWAS used genotyping array data from an additional 5849 individuals. Cross-validation of loci reaching genome-wide significance was sought by meta-analysis. Conditional analysis corrected for the most significant variants at each locus was used to resolve signals for multiple associations. We functionally annotated associated variants and tested associations with duration of survival. All-cause mortality was the primary endpoint in survival analyses. Findings: A locus near SOX17 (rs10103692, odds ratio 1·80 [95% CI 1·55–2·08], p=5·13 × 10 –15 ) and a second locus in HLA-DPA1 and HLA-DPB1 (collectively referred to as HLA-DPA1/DPB1 here; rs2856830, 1·56 [1·42–1·71], p=7·65 × 10 –20 ) within the class II MHC region were associated with pulmonary arterial hypertension. The SOX17 locus had two independent signals associated with pulmonary arterial hypertension (rs13266183, 1·36 [1·25–1·48], p=1·69 × 10 –12 ; and rs10103692). Functional and epigenomic data indicate that the risk variants near SOX17 alter gene regulation via an enhancer active in endothelial cells. Pulmonary arterial hypertension risk variants determined haplotype-specific enhancer activity, and CRISPR-mediated inhibition of the enhancer reduced SOX17 expression. The HLA-DPA1/DPB1 rs2856830 genotype was strongly associated with survival. Median survival from diagnosis in patients with pulmonary arterial hypertension with the C/C homozygous genotype was double (13·50 years [95% CI 12·07 to &gt;13·50]) that of those with the T/T genotype (6·97 years [6·02–8·05]), despite similar baseline disease severity. Interpretation: This is the first study to report that common genetic variation at loci in an enhancer near SOX17 and in HLA-DPA1/DPB1 is associated with pulmonary arterial hypertension. Impairment of SOX17 function might be more common in pulmonary arterial hypertension than suggested by rare mutations in SOX17. Further studies are needed to confirm the association between HLA typing or rs2856830 genotyping and survival, and to determine whether HLA typing or rs2856830 genotyping improves risk stratification in clinical practice or trials. Funding: UK NIHR, BHF, UK MRC, Dinosaur Trust, NIH/NHLBI, ERS, EMBO, Wellcome Trust, EU, AHA, ACClinPharm, Netherlands CVRI, Dutch Heart Foundation, Dutch Federation of UMC, Netherlands OHRD and RNAS, German DFG, German BMBF, APH Paris, INSERM, Université Paris-Sud, and French ANR. </p

GWAS meta-analysis of intrahepatic cholestasis of pregnancy implicates multiple hepatic genes and regulatory elements

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder affecting 0.5–2% of pregnancies. The majority of cases present in the third trimester with pruritus, elevated serum bile acids and abnormal serum liver tests. ICP is associated with an increased risk of adverse outcomes, including spontaneous preterm birth and stillbirth. Whilst rare mutations affecting hepatobiliary transporters contribute to the aetiology of ICP, the role of common genetic variation in ICP has not been systematically characterised to date. Here, we perform genome-wide association studies (GWAS) and meta-analyses for ICP across three studies including 1138 cases and 153,642 controls. Eleven loci achieve genome-wide significance and have been further investigated and fine-mapped using functional genomics approaches. Our results pinpoint common sequence variation in liver-enriched genes and liver-specific cis-regulatory elements as contributing mechanisms to ICP susceptibility

PAX5 Is a Frequently Altered Transcription Factor In Genetically Unclassified Childhood Common/B-Cell Precursor Acute Lymphoblastic Leukemia

Abstract Abstract 1458 Introduction: Genetic lesions define treatment and prognosis in childhood common/B cell-precursor Acute Lymphoblastic Leukemia (c/pre-B ALL) (Pui CH et al., J Clin Oncol, 2011). However, up to 40% of childhood pre-B ALL cases, lack the presence of prognostically significant abnormalities; and relapses are common in this group (Mejerink J et al., Semin Hem, 2009). Molecular genetic characterization of genetically unclassified ALL is critical for a better understanding of the biology of this disease, to determine new prognostic markers and to optimize treatment for this group of patients. Several transcription factors, including EBF1, IKZF1 and PAX5, which regulate B-cell development, have recently been shown often to be altered in B-ALL (Mullighan CG et al., Nature, 2007; den Boer ML et al., Lancet, 2009). We decided to investigate the incidence of abnormalities involving B-cell transcription factors, in a cohort of patients with childhood c/pre-B ALL lacking significant prognostic genetic aberrations. Materials and methods: 60 patients diagnosed with c/pre-B ALL in Great Ormond Street Hospital (London, UK) from 1994 to 2011 were included in the study. The median age was 3.43 (range: 1.14–12.24), with a median white blood cell count of 14.0 (range: 1.36–213.2). All patients showed an absence of high-hyperploidy/hypodiploidy, BCR-ABL1, MLL, ETV6-RUNX1 and E2A rearrangements, and also i(amp)21 by karyotype, fluorescence in-situ hybridization (FISH) and/or reverse transcriptase-polymerase chain reaction (RT-PCR) investigations. We performed FISH using commercially available probes and BAC clones, in parallel with RT-PCR, to detect deletions/rearrangements of the EBF1, IKZF1 and PAX5 gene regions. Direct sequence analysis was performed for all exons of the PAX5 gene. Real-time PCR was used to determine the level of expression of PAX5 and its transcriptional targets, mostly components of the pre-B cell receptor (pre-BCR) complex including CD19, CD79α, VpreB, BLNK and BLK genes. Western blotting was further performed for the identification of an abnormal PAX5 protein. Results: 25/60 patients (42%) showed abnormalities involving PAX5, 2/60 patients (3%) showed deletions of IKZF1. No deletions/rearrangements of EBF1 were detected. Several forms of PAX5 deletions were observed: the entire loss of one PAX5 allele (32%), loss of 5'PAX5 (20%), loss of 3'PAX5 (12%) and loss of 3'PAX5 due to unbalanced rearrangements (8%). Balanced PAX5 rearrangements comprise 12% of the PAX5 alterations. Sequence analysis revealed 9 somatic mutations of PAX5 in 8/60 patients (13%): 6 frameshifts, 2 splice-site and 1 non-stop mutations. The mutations were located in functional exons of PAX5 and resulted in the initiation of premature stop-codons, exon skips, and the formation of abnormal transcripts. Interestingly, 2 patients with an identical splice site mutation, showed the same karyotypic abnormality of an isochromosome of the long arm of chromosome 9 leading to loss of the second PAX5 allele. Moreover, further investigation DNA and mRNA from remission samples demonstrated that in one case the mutation was acquired, and that in the second case the mutation was a germ-line variant. In total, 20% of patients with PAX5 abnormalities had bi-allelic alterations of the PAX 5 gene. PAX5, BLNK and BLK gene-expression levels were lower (p&lt;0.05) in the group of patients with loss of one entire PAX5 allele. There were no significant changes in expression levels of the tested genes in patients with partial PAX5 deletions, rearrangements or mutations. Expression of an abnormal PAX5 protein was confirmed by western blot analysis. Conclusion: The high incidence of PAX5 alterations in childhood c/pre-B ALL, without prognostically significant genetic abnormalities, suggests a pathogenetic role for PAX5 in these acute lymphoblastic leukemia cases. Down-regulation of expression of the BLNK and BLK genes could be one of the mechanisms by which abnormal PAX5 blocks B-cell differentiation and contributes to the initiation of leukemia. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Kaposiform Haemangioendothelioma and Kasabach-Merritt Phenomenon: Safe and Effective Treatment With Sirolimus

Abstract Kaposiform Haemangioendothelioma (KHE) is a rare vascular tumour of infancy that can be life-threatening from haemorrhage, high output cardiac failure or local tumour effects when associated with Kasabach-Merritt Phenomenon (KMP). The precise pathogenesis of KHE triggered thrombocytopenia and consumptive coagulopathy remains unclear, but treatment with vincristine and steroids is reported as effective and has recently been recommended as first-line therapy in consensus derived practice standards. Therapeutic targets in cases that fail to respond remain empirical and potentially hazardous. Sirolimus is a mammalian target of Rapamycin (mTOR) inhibitor that targets protein synthesis downstream of the Akt pathway. Here we describe the case of an infant girl who presented at birth with an extensive right arm vascular lesion associated with thrombocytopenia, platelet count 12 x 109/L, hypofibrinogenaemia and raised D-dimers, 8336 ug/L. She was anaemic and jaundiced, from a microangiopathic haemolytic anaemia. Tissue biopsy confirmed the histological diagnosis of KHE with characteristic morphology and expression by lesional endothelial cells of D2-40 immunostaining. She commenced steroids and vincristine within the first week of life, but both lesion and thrombocytopenia were resistant. As alternative therapeutic options were limited, she received two protracted courses of vincristine, weekly for 38 weeks, and then every two to six weeks for three years and ten weeks, maintaining a mean platelet count of 25.18 x 109/L, range 9- 46 x 109/L. Severe rectal bleeds with normal histology, epistaxis and a painful enlargement of the KHE toward the wrist mandated an urgent clinical intervention. Additional immunohistochemistry staining demonstrated lesional cells to strongly express anti- ribosomal protein S6 (pS6) and weaker anti- eukaryotic translation initiation factor 4E – binding protein (4E-BPI), both mTOR pathway markers. There was no staining for Vascular derived Endothelial Growth Factor Receptor 3 (VEGFR3). These results plus information from published case reports provided rationale for off-licence use of sirolimus. Within 21 days of commencing sirolimus, with a modest target trough level of&lt;10 ng/ml, the platelet count had risen to 84 x 109/L, and after three months was within the normal range, 164 x 109/L (Figure 1). After six months of the treatment there was remarkable resolution in the size of the lesion and apart from an episode of chicken pox that resolved without complication she had no other side effects or toxicity. This is the first case report of safe and effective treatment of KMP associated with a KHE with confirmed expression of mTOR pathway markers. This provides a rational use for sirolimus in KHE and KMP and that it should be considered early in life-threatening cases resistant to first line therapy. Further research focusing on the mTOR pathway may provide key insight into the pathogenesis of KMP associated with KHE and elucidate if excess production of VEGF or events downstream of the tyrosine kinase receptor/PI3K/Akt/ mTOR pathway are the initiating mechanism. The lack of VEGFR staining in our patient's histology and lack of anecdotal reports of response with VEGF inhibition suggest a VEGF independent mechanism. Disclosures: Off Label Use: Sirolimus is a mammalian target of rapamycin inhibitor. </jats:sec

Periodontal status in children with primary immunodeficiencies

Objective: This study aimed to assess associations between neutrophil-related primary immunodeficiencies (PIDs) and the presence of periodontal disease and other oral diseases and response to periodontal treatment. Background: Presence of neutrophil-related PIDs is thought to be a major risk factor for development of periodontitis. Methods: This study had both a cross-sectional and cohort design. Twenty-four children (age 4–16) with PIDs and 24 age-matched systemically healthy subjects received a dental clinical examination, including measures of probing pocket depths (PPD), clinical attachment loss (CAL) and bleeding on probing (BOP). Those found to be affected by periodontal disease were offered periodontal treatment and reassessed 6 months later. Results: Diagnosis of PIDs was associated with increased odds of presence of periodontal disease (p =.008 adjusted for age, gender, plaque, OR = 10.0, 95% CI = 1.83–54.38) and with continuous measures of periodontal disease such as number of PPDs &gt;4 mm, mean PPD and mean CAL (all p &lt;.001) and BOP (p =.001). However, only 7 out of 24 children were diagnosed with periodontitis. PIDs were also associated with a history of oral ulcers (p =.001, OR 12.47, 95% CI 2.71–57.29). An improvement in periodontal parameters (PPD and CAL) was detected following oral hygiene instructions and non-surgical periodontal therapy. Conclusion: Although children affected by neutrophil-associated PIDs exhibited a higher prevalence of periodontal disease compared with systemically healthy children, severe periodontitis was rarely seen. This suggests that good systemic control of the PIDs may reduce their impact on the periodontium.</p

Mitoxantrone, Not Idarubicin, Significantly Improves the Outcome of Children with Relapsed ALL – Result of the International, Randomised ALLR3 Study.

Abstract Abstract 3390 Poster Board III-278 Introduction: While the outcome of children with acute lymphoblastic leukemia (ALL) has steadily improved, the prognosis for those who relapse (rALL) remains poor. Partly this has been due to the lack of a standardised approach to children with rALL. The international collaborative trial ALLR3 stratified patients into standard (SR), intermediate (IR) and high risk (HR) groups based on the time from first diagnosis to relapse, site of relapse and immunophenotype. All patients received 3 blocks of therapy, with a minimal residual disease (MRD) assessment at the end of blocks 1 and 3. Allogeneic stem cell transplantation (allo-SCT) was offered to all HR and those IR who had a MRD of ≥10-4 at week 5. In children in whom MRD was unavailable, allo-SCT was offered to those who relapsed within 24 months of stopping therapy. All other patients continued on chemotherapy for 24 months. A day 1 and 2 randomisation was performed between mitoxantrone and idarubicin. Results: 239 patients were enlisted, of whom 216 were randomised and 109 idarubicin and 103 mitoxantrone patients are analysable. There were non-significant differences between the groups with regards to time to relapse, site of relapse and cytogenetic subtypes.108 (51%) were in CR2 at a median follow up of 36 months (range 1-70) and Progression Free Survival (PFS) at 3 years was 50.3% (95% CI 42.9%, 57.3%). Forty four percent of idarubicin and 40% of mitoxantrone patients had disease levels of ≥10-4 at week 5 (p=0.90) at the end of block 1. The PFS at 3 years for those who received idarubicin was 35.9% (25.9%, 45.9%) and mitoxantrone 64.6% (54.2%, 73.2%) (p=0.0004). Adjusted for differences in risk group, country, age, gender and cytogenetic subtype, this difference continues to be significant (p=0.003). Overall mitoxantrone was better tolerated and less toxic than idarubicin. A competing risks model showed that the difference between the two drugs is primarily related to disease control (p=0.02), rather than toxicity of treatment (p=0.09). In the patients who were transplanted, 35% and 5% of patients who received idarubicin (n = 48) or mitoxantrone (n = 44) respectively have relapsed. Based on the intention to treat analyses, there were non significant differences in patients treated without an allo-SCT in both arms. IR patients who were transplanted did worse (p = 0.01) in the idarubicin group but had comparable results in the mitoxantrone group to those who received chemotherapy. As a result the randomisation in ALLR3 has now been closed, though the study continues to accrue to better answer the MRD stratification question. Conclusions: Mitoxantrone is a well tolerated and highly effective drug for the treatment of children with rALL. While MRD at week 5 failed to predict the differences in outcome for the two randomised groups, it identified that children in the IR group with &lt;10-4 could be treated without an allo-SCT. Disclosures: Off Label Use: Although Idarubicin and Mitoxantrone have been shown to be effective in ALL, they are not licensed for use in children in the UK. . </jats:sec