Spliceosome mutations in myeloid neoplasms Mario Cazzola Department of Hematology Oncology, Fondazione IRCCS Policlinico San Matteo Department of Molecular Medicine, University of Pavia Pavia, Italy

WHO classification of myeloid neoplasms Myeloproliferative neoplasms Myelodysplastic syndromes Myelodysplastic/myeloproliferative neoplasms Acute myeloid leukemia

2008 WHO classification of MDS Ineffective hematopoiesis RCUD RARS Progression to AML (25-30%) RCMD RAEB-1 RAEB-2 MDS del(5q) Peripheral blood cytopenia Cazzola M. File image 2013

Outcome of MDS according to WHO classification 943 MDS pts Kaplan-Meier survival curves of 943 patients diagnosed with MDS according to the 2008 WHO criteria at the Department of Hematology Oncology, Fondazione IRCCS Policlinico San Matteo & University of Pavia, Pavia, Italy. Patients grouped according to the WHO classification. Patients classified as RA or RARS according to the FAB classification (previous slide) are split here into two subgroups with different survival based on the presence of unilineage [RCUD or RARS, including also MDS with del(5q)] or multi-lineage dysplasia (RCMD). Moreover, patients with RAEB are also split here into two subgroups according to their blast percentage (5–9% in RAEB-1, 10–19% in RAEB-2). Cazzola. Haematologica. 2011 Mar;96(3):349-52.

Clonal cytogenetic abnormality Diagnosis of MDS is currently reliant on bone marrow morphology and cytogenetics Peripheral cytopenia Dysplasia Clonal cytogenetic abnormality

Clonal cytogenetic abnormality Diagnosis of MDS is currently reliant on bone marrow morphology and cytogenetics Peripheral cytopenia Dysplasia Clonal cytogenetic abnormality • lack of specificity • poor inter-observer reliability

Clinical relevance of the molecular basis of disease Philadelphia-negative MPN: - 2005: JAK2 (V617F) - 2008: WHO diagnostic criteria - 2010: safety & efficacy of ruxolitinib in PMF - 2012: FDA & EMA approval of ruxolitinib for treatment of PMF

Chronic Myeloid Disorders Working Group of the International Cancer Genome Consortium Research project on the molecular basis of MDS started in 2010 with whole exome sequencing Cazzola et al. Blood. 2003 Mar 1;101(5):1996-2000 - Pellagatti et al. Blood. 2006 - Della Porta et al. Leukemia. 2006 Apr;20(4):549-55 - Malcovati et al. J Clin Oncol. 2005 Oct 20;23(30):7594-603 RARS RARS

RNA splicing machinery RNA polymerase II DNA Spliceosome Pre-mRNA U1 U2 Cazzola M. File image 2013

SF3B1 mutations are present in the dominant clone Papaemmanuil et al. N Engl J Med. 2011 Oct 13;365(15):1384-95 10

Mutant genes in MDS Gene Frequency SF3B1 ~25% STAG2 3% TET2 ~20% JAK2 <5% SRSF2 ~10% MPL RUNX1 ATRX TP53 <10% NPM1 ASXL1 IDH1- IDH2 NRAS/KRAS NADPH EZH2 CEBPA CBL CKIT <2% U2AF1 6% CSF1R ZRSR2 PTPN11 BCOR GNAS <1% Chronic Myeloid Disorders Working Group of the International Cancer Genome Consortium, 2013

Targeted pathways in MDS Chronic Myeloid Disorders Working Group of the International Cancer Genome Consortium, 2013

Potential outcomes of spliceosome mutations Normal splicing Exon 1 Exon 2 Exon 3 Decreased (or increased) transcription of normal pre-mRNA (co-transcriptional RNA splicing) Exon skipping Exon 1 Exon 3 Intron retention Exon 1 Exon 2 Exon 3 Intron Cryptic splice site Exon 1 Exon 3 Exon 2 (truncated exon) Stem cells Hematopoietic precursors

Clinical significance of SF3B1 mutations in MDS Malcovati et al. Blood. 2011 Dec 8;118(24):6239-46

Clinical significance of SRSF2 mutations in MDS Thol et al. Blood. 2012 Apr 12;119(15):3578-84

Refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T) as the paradigmatic example of MDS/MPN Anemia & thrombocytosis Ring sideroblasts (myelodysplastic feature) Abnormal megakaryocyte proliferation (myeloproliferative feature)

RARS-T: molecular basis Normal hematopoietic cell Ring sideroblasts and ineffective erythropoiesis (myelodysplastic features of RARS) Ring sideroblasts and thrombocytosis (myelodysplastic & myeloproliferative features of RARS-T) Somatic mutation of SF3B1 determining mitochondrial iron overload, ineffective erythropoiesis and anemia Somatic mutation of JAK2 or MPL determining gain of signaling and thrombocytosis Cazzola et al. Blood. 2013 Jan 10;121(2):260-9

TET2 +/- SRSF2 & ASXL1 Courtesy of R. Solary

International Cancer Genome Consortium Chronic Myeloid Disorders Working Group Elli Papaemmanuil Peter Campbell Mike Stratton Luca Malcovati Matteo Della Porta Anna Gallì Ilaria Ambaglio Cristiana Pascutto Mario Cazzola Eva Hellstrom-Lindberg David Bowen Jaqueline Boultwood James S Wainscoat Andrea Pellagatti Mike Groves Anthony Green Carlo Cambacorti-Passerini Paresh Vyas Nick Cross AGIMM – AIRC-Gruppo Italiano Malattie Mieloproliferative