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LBA-2 The Genomic Landscape of Myeloproliferative Neoplasms: Somatic CALR Mutations in the Majority of JAK2-Wildtype Patients

Program: General Sessions
Session: Late-Breaking Abstracts Session
Tuesday, December 10, 2013, 7:30 AM-9:00 AM
Hall F (Ernest N. Morial Convention Center)

Jyoti Nangalia, MBBChir FRCPath1,2,3*, Charles E Massie, PhD1,3*, E Joanna Baxter, PhD3*, Francesca L Nice, MRes3*, Gunes Gundem, PhD1*, David Wedge, PhD1*, Edward Avezov, PhD4*, Juan Li, PhD3*, Karoline Kollmann, PhD3*, David G Kent, PhD3*, Athar Aziz, PhD3*, Anna L Godfrey, PhD FRCPath2,3*, Jonathan Hinton, MSc1*, Inigo Martincorena, PhD1*, Peter Van Loo, PhD1*, Amy V Jones, PhD5*, Paola Guglielmelli, PhD MD6*, Patrick Tarpey, PhD1*, Heather P Harding, PhD4*, John D Fitzpatrick, BA3*, Calum T Goudie, BA3*, Christina A Ortmann, MD3*, Stephen J Loughran, PhD3*, Keiran Raine, MSc1*, David Jones, MSc1*, Adam Butler, MSc1*, Jon W Teague, MSc1*, Sarah O'Meara1*, Stuart McLaren1*, Michele Bianchi2*, Yvonne Silber, MSc3*, Danai Dimitropolou, BSc3*, David Bloxham, MSc FRCPath2*, Laura J Mudie, BSc1*, Mark Maddison, BSc1*, Ben Robinson, BSc1*, Clodagh Keohane, MD7*, Cathy MacLean, MSc2*, Kate Hill, BM BS8*, Kim H. Orchard, PhD9, Sudhir Tauro, PhD10*, Ming-Qing Du, MB, PhD11, Mel Greaves, PhD, FRS12*, David Bowen, MD, PhD13, Brian Huntly, MBChB FRCPath PhD2,3*, Claire N Harrison, DM, FRCP, FRCPath7, Nicholas C.P. Cross, PhD, FRCPath5, David Ron, MD4*, Alessandro M. Vannucchi, MD14, Elli Papaemmanuil, PhD1*, Peter J. Campbell, PhD1,2* and Anthony R. Green, FRCPath, FMedSci2,3

1Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, United Kingdom
2Department of Haematology, Addenbrookes Hospital, Cambridge, United Kingdom
3Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom
4Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, University of Cambridge, Cambridge, United Kingdom
5Faculty of Medicine, University of Southampton, Southampton, United Kingdom
6Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy
7Guy's and St Thomas' NHS Foundation Trust, London, United Kingdom
8Department of Haematology, University Hospital Southampton, Southampton, United Kingdom
9University Hospital Southampton, Southampton, United Kingdom
10Dept of Haematology, Ninewells Hospital, Dundee, United Kingdom
11Dept. Pathology, Div. Histopathology, University of Cambridge, Cambridge, United Kingdom
12Haemato-Oncology Research Unit, Division of Molecular Pathology, The Institute of Cancer Research, Sutton, United Kingdom
13St. James's Institute of Oncology, Leeds, United Kingdom
14University of Florence, Florence, Italy

BCR-ABL-negative myeloproliferative neoplasms (MPNs), such as polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) are chronic myeloid malignancies characterized by overproduction of hematopoietic cells. JAK2 mutations are found in most patients with PV, and in only 50-60% of patients with ET and MF. JAK2 mutation testing has greatly simplified MPN diagnosis, but distinguishing JAK2-wildtype ET from reactive thrombocytosis remains a diagnostic challenge. Mutations in signalling pathways (MPL, LNK) and epigenetic regulators (TET2, DNMT3A, IDH1/2, EXH2, ASXL1) have been found in a minority of MPNs. However genome-wide data are lacking and the pathogenesis of MPNs that do not harbor JAK2 or MPL mutations remains obscure.

METHODS. Exome sequencing was performed in 151 MPN patients on matched tumor and constitutional samples. CALR status was assessed in 3412 samples using Sanger sequencing and analysis of exome/genome sequencing data. Presence of CALR mutations in hematopoietic stem and progenitor cells was assessed by flow sorting and sequencing. Phylogenetic trees were established using hematopoietic colonies. Calreticulin cellular localisation was assessed in patient samples and cell lines expressing CALR variants by flow cytometry and immunofluorescence.

RESULTS. Exome sequencing identified 1498 somatic mutations with a median of 6.5 mutations in PV and ET, and 13 in MF (MF vs ET, P=0.0002; MF vs PV, P=0.008). JAK2V617F was found in all cases of PV (n=48), 56% of ET (35/62), and 69% of MF (27/39), and MPL mutations in 7 ET and MF cases.  Mutations in epigenetic regulators TET2, DNMT3A, ASXL1, EZH2, and IDH1/2 were identified in 22, 12, 12, 4, 3 patients respectively, and components of the splicing machinery (U2AF1, SF3B1 or SRSF2) were mutated in 9 patients.  Mutations in rare genes reported to be mutated in MPNs were found in four patients (1 CBL; 2 NFE2; 1 SH2B3/LNK). We found novel somatic mutations in CHEK2 (1 PV, 1 ET and 1 MF) which have not been previously reported in MPNs.  The mutation spectrum showed a predominance of C>T transitions. Pairwise associations between MPN genes demonstrated that ASXL1 and SRSF2 mutations were positively correlated with mutations in epigenetic modifiers.

Novel somatic mutations in calreticulin (CALR) were identified by exome sequencing in the majority (26/31) of JAK2 or MPL unmutated patients. CALR and JAK2/MPL mutations were mutually exclusive, and 97% of patients harbored a mutation in 1 of these 3 genes. In an extended follow up screen of 1345 hematological malignancies, 1517 other cancers and 550 controls we found CALR mutations in 71% of ET (80/112), 56% of idiopathic MF (18/32), 86% of post ET-MF (12/14) and 8% of myelodysplasia (10/115), but not in other myeloid, lymphoid or solid cancers. Compared to JAK2-mutated MPNs, those with CALR mutations presented with higher platelet counts (Wilcoxon rank-sum, P=0.0003), lower hemoglobin levels (Student’s t test, P=0.02) and showed a higher incidence of transformation to MF (Fishers exact, P=0.03).

All CALR mutations were insertions or deletions affecting exon 9, with 2 common variants L367fs*46 (52 bp deletion) and K385fs*47 (5 bp insertion). Loss of heterozygosity over CALR was seen in a minority of patients. Of 148 CALR mutations identified, there were 19 distinct variants. Remarkably, all generated a +1 basepair frameshift, which results in loss of most of the C-terminal acidic domain of the protein as well as the KDEL Golgi-to-endoplasmic reticulum (ER) retrieval signal, raising the possibility of compromised ER retention. Mutant proteins were readily detected in transfected cell lines and localised to the ER in the same manner as wildtype CALR, without Golgi or cell surface accumulation. These results are consistent with studies reporting KDEL-independent mechanisms of ER retention.

Mutation of CALR was detected in highly purified hematopoietic stem/progenitor cells. Clonal analyses demonstrated CALR mutations in the earliest phylogenetic node in 5/5 patients, consistent with it being an initiating mutation in these individuals.

CONCLUSIONS. We describe the mutational landscape of BCR-ABL negative MPNs and demonstrate that somatic mutations in the endoplasmic reticulum chaperone CALR are found in the majority of patients with JAK2-unmutated MPNs. These results reveal a novel biological pathway as a target for tumorigenic mutations and will simplify diagnosis of MPN patients.

Disclosures: Bowen: Celgene: Honoraria . Harrison: S Bio: Honoraria , Membership on an entity’s Board of Directors or advisory committees ; Shire: Speakers Bureau ; Celgene: Honoraria ; YM Bioscience: Honoraria , Membership on an entity’s Board of Directors or advisory committees ; Sanofi: Honoraria , Membership on an entity’s Board of Directors or advisory committees , Speakers Bureau ; Novartis: Honoraria , Membership on an entity’s Board of Directors or advisory committees , Research Funding , Speakers Bureau ; Gilead: Honoraria , Membership on an entity’s Board of Directors or advisory committees . Vannucchi: Novartis: Honoraria , Membership on an entity’s Board of Directors or advisory committees .