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Unravelling the Cellular Origin and Clinical Prognostic Markers of Infant Published Ahead of Print on January 24, 2019, as doi:10.3324/haematol.2018.206375. Copyright 2019 Ferrata Storti Foundation. Unravelling the cellular origin and clinical prognostic markers of infant B-cell acute lymphoblastic leukemia using genome-wide analysis by Antonio Agraz-Doblas, Clara Bueno, Rachael Bashford-Rogers, Anindita Roy, Pauline Schneider, Michela Bardini, Paola Ballerini, Gianni Cazzaniga, Thaidy Moreno, Carlos Revilla, Marta Gut, Maria G Valsecchi, Irene Roberts, Rob Pieters, Paola De Lorenzo, Ignacio Varela, Pablo Menendez, and Ronald W Stam Haematologica 2019 [Epub ahead of print] Citation: Antonio Agraz-Doblas, Clara Bueno, Rachael Bashford-Rogers, Anindita Roy, Pauline Schneider, Michela Bardini, Paola Ballerini, Gianni Cazzaniga, Thaidy Moreno, Carlos Revilla, Marta Gut, Maria G Valsecchi, Irene Roberts, Rob Pieters, Paola De Lorenzo, Ignacio Varela, Pablo Menendez, and Ronald W Stam. Unravelling the cellular origin and clinical prognostic markers of infant B-cell acute lymphoblastic leukemia using genome-wide analysis Haematologica. 2019; 104:xxx doi:10.3324/haematol.2018.206375 Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors. After having E-published Ahead of Print, manuscripts will then undergo technical and English editing, typesetting, proof correction and be presented for the authors' final approval; the final version of the manuscript will then appear in print on a regular issue of the journal. All legal disclaimers that apply to the journal also pertain to this production process. Unravelling the cellular origin and clinical prognostic markers of infant B-cell acute lymphoblastic leukemia using genome-wide analysis Antonio Agraz-Doblas1,2, Clara Bueno2#, Rachael Bashford-Rogers3#, Anindita Roy4,#, Pauline Schneider5, Michela Bardini6, Paola Ballerini7, Gianni Cazzaniga6, Thaidy Moreno1, Carlos Revilla1, Marta Gut8,9, Maria G Valsecchi10, Irene Roberts4,11, Rob Pieters5, Paola De Lorenzo10, Ignacio Varela1,$,*, Pablo Menendez2,12,13,$,*, Ronald W Stam5,. 1Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria-CSIC, Santander, Spain. 2Josep Carreras Leukemia Research Institute-Campus Clinic, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona. 3Department of Medicine, University of Cambridge, Cambridge Biomedical Campus, Hills Road, Cambridge, UK. 4Department of Paediatrics, University of Oxford, Oxford, UK. 5Princess Maxima Center for Paediatric Oncology, Utrecht, The Netherlands. 6Centro Ricerca Tettamanti, Department of Paediatrics, University of Milano Bicocca, Fondazione MBBM, Monza, Italy. 7Pediatric Hematology, A. Trousseau Hospital, Paris. France. 8CNAG-CRG, Centre for Genomic Regulation, Barcelona, Spain. 9Universitat Pompeu Fabra, Barcelona, Spain. 10Interfant Trial Data Center, University of Milano-Bicocca, Monza, Italy. 11MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.12Instituciò Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain. 13Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), ISCIII, Barcelona, Spain. Running Title: Cellular origin and clinical prognostic markers of infant MLLr B-ALL Key words: infant B-ALL, MLL-AF4, AF4-MLL, HOXA, MLL-AF9, Whole-exome Seq, RNA Seq, RAS mutations. #These authors contributed equally to this work. $These senior authors contributed equally to this work. *Correspondence should be addressed to: Pablo Menéndez, PhD MBA ICREA Research Professor Josep Carreras Leukemia Research Institute. School of Medicine, University of Barcelona. Casanova 143, 08036 Barcelona. Spain. [email protected] Ignacio Varela, PhD Instituto de Biomedicina y Biotecnología de Cantabria Albert Einstein 22. 39011 Santander, Spain [email protected] 1 ABSTRACT B-cell acute lymphoblastic leukemia is the commonest childhood cancer. In infants, B-cell acute lymphoblastic leukemia remains fatal, especially in patients with t(4;11), present in ~80% of cases. The pathogenesis of t(4;11)/KMT2A-AFF1+ (MLL-AF4+) infant B-cell acute lymphoblastic leukemia remains difficult to model, and the pathogenic contribution in cancer of the reciprocal fusions resulting from derivative translocated-chromosomes remains obscure. Here, a “multi-layered” genome-wide analyses and validation was performed on a total of 124 de novo infant B-cell acute lymphoblastic leukemias uniformly diagnosed/treated according to Interfant99/06 protocol. These patients showed the most silent mutational landscape reported so far for any sequenced pediatric cancer. Recurrent mutations were exclusively found in K- and N-RAS, and were subclonal and frequently lost at relapse, despite a larger number of non-recurrent/non-silent mutations. Unlike non-MLL-rearranged B-cell acute lymphoblastic leukemias, B-cell receptor repertoire analysis revealed minor, non-expanded B-cell clones in t(4;11)+ infant B-cell acute lymphoblastic leukemia, and RNA-sequencing showed transcriptomic similarities between t(4;11)+ infant B-cell acute lymphoblastic leukemias and the most immature human fetal liver hematopoietic stem/progenitor cells, confirming a “pre-VDJ” fetal cellular origin for both t(4;11) and RASmut. The reciprocal fusion AF4-MLL was expressed in only 45% (19/43) of the t(4;11)+ patients, and HOXA cluster genes are exclusively expressed in AF4-MLL-expressing patients. Importantly, AF4-MLL/HOXA-expressing patients had a significantly better 4-year event-free survival (62.4% vs 11.7%, p=0.001), and overall-survival (73.7 versus 25.2%, p=0.016). AF4-MLL expression retained its prognostic significance when analyzed in a Cox model adjusting for risk stratification according to Interfant-06 protocol based on age at diagnosis, WBC count and Prednisone response. This study has clinical implications in disease outcome and diagnostic risk-stratification of t(4;11)+ infant B-cell acute lymphoblastic leukemia. 2 INTRODUCTION B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most frequent cancer in children.1 Current 5-year survival rates in pediatric BCP-ALL approach 90%. However, BCP-ALL in infants (iBCP- ALL; <1 year of age) remains clinically challenging with an aggressive early clinical presentation in uniquely vulnerable hosts.2 Approximately 80% of iBCP-ALLs are diagnosed with chromosomal rearrangements involving the mixed-lineage leukemia (KMTA2, also called MLL) gene, located on 11q233–5, which confers a dismal prognosis especially in patients carrying the t(4;11)/KMT2A-AFF1+ (MLL-AF4+).6–8 MLL is a H3K4 histone methyltransferase (HMT) required for normal hematopoiesis and HOX gene expression.9,10 Leukemia transformation by MLL fusions requires the recruitment of the H3K79 HMT Dot1L to the MLL transcriptional complex.11,12 Indeed, an H3K79 methylation profile defines both mouse and human t(4;11)/MLL-AF4+ BCP-ALL.13 Importantly, MLL rearrangements (MLLr) occur prenatally during embryonic/fetal hematopoiesis, and the concordance rate for iBCP-ALL in identical twins with a monochorionic placenta is close to 100%.14–17 This, coupled to the extremely short latency, suggests that MLL fusions might be sufficient for leukemogenesis.4 Accordingly, genome-wide studies using both SNP arrays and whole-genome sequencing revealed that MLLr iBCP-ALL has a very low frequency of somatic mutations with the predominant clone carrying ~1.3 non-silent mutations and 1 copy number alteration (CNA).18–20 Although these studies were performed at low coverage sequencing they reinforce that MLLr iBCP-ALL requires few additional mutations to induce full transformation. In contrast, MLL-AF4-induced leukemogenesis has proven difficult to model.4,9 With the exception of a recent work by Lin et al21,22 who fused human MLL to murine Af4, creating an artificial leukemogenic human-mouse chimeric fusion, current murine and humanized models of MLL-AF4+ BCP-ALL do not faithfully recapitulate the disease pathogenesis/phenotype, suggesting that MLL-AF4 per se is insufficient to initiate leukemogenesis.23–28 3 The few mutations and CNAs present in MLLr iBCP-ALL seem subclonal and not always retained at relapse.20 Intratumor heterogeneity drives clonal evolution in response to microenvironmental cues and cytotoxic treatment and therefore recurrent mutations at diagnosis and relapse may be found in minor but clinically relevant subclones.29 Here we aimed to address the clinical relevance of subclonal mutations and gene expression signatures in a large cohort of iBCP-ALL. For this, we performed deeper exome sequencing along with whole-genome DNA and RNA-sequencing on a large cohort of 50 MLLr and non-MLL iBCP-ALL patients uniformly treated and followed up according to the Interfant treatment protocol.30 Similar to Anderson AK et al 20, we report a silent mutational landscape in iBCP- ALL irrespective of the MLL rearrangement/status. However, strikingly our genome-wide DNA and RNA analysis reveals new, clinically relevant information about disease outcome and cell-of-origin for t(4;11) and RAS mutations. 4 METHODS Patients Bone Marrow (BM) or peripheral blood (PB) samples from 124 infants (<12 months old) diagnosed with either pro-B or pre-B cell ALL were used in this study. The discovery patient
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