1. 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

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

3. 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

4. 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 Mar;96(3):

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

6. 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

7. 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

8. 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 Mar 1;101(5): Pellagatti et al. Blood Della Porta et al. Leukemia Apr;20(4): Malcovati et al. J Clin Oncol Oct 20;23(30):
RARS
RARS

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

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

11. 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

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

13. 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

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

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

16. 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)

17. 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 Jan 10;121(2):260-9

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

19. 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