The Ninth International Symposium on Myelodysplastic Syndromes

1. The Ninth International Symposium on Myelodysplastic Syndromes Florence May 16th -19th 2007 Dr Dominic Culligan Aberdeen Royal Infirmary

2. The del 5q story so far Cytogenetics of MDS and detection of del 5q-. Ghulam Mufti, KCH, London: >50% of MDS patients have a clonal cytogenetic abnormality at presentation Partial or complete deletion of 5q is the commonest identified abnormality in de-novo MDS (10-15%) There is a hierarchy of prognosis relating to del 5q: Isolated 5q-/ < 5% blasts >> additional cytogenetic abnormalities & increased blasts

3. Mufti, continued Numerous ways of detecting del 5q: FISH Multicolour FISH/spectral karyotyping Whole chromosome painting Loss of heterozygosity Comparative genomic hybridisation Gene expression profiling by the Affymetrix array Important because of the therapeutic implications with the availability of lenalidomide UPD identified as a mechanism in some del 5q cases

4. Molecular analysis of the 5q- syndrome, Jackie Boultwood, University of Oxford The Oxford group have defined the commonly deleted region (CDR) of 5q- syndrome: 1.5Mb interval at 5q31-32, flanked by D5S413 and the GLRA1 gene. CDR is gene rich & contains 48 genes Including putative tumour suppressor genes MEGF1 & SPARC and several microRNA genes

5. Boultwood, continued Mutation analysis of all 48 genes is now complete No mutations on the retained allele identified! Using gene expression profiling in CD34 + cells, the majority of the 48 genes mapping to CDR show reduced expression, consistent with loss of one allele RBM22 significantly down regulated <50% expression Disrupts actin cytoskeleton signalling pathway and apoptotic pathways These data support the theory that haploinsufficiency ( a gene dosage effect) for one or more of the genes in CDR is the pathogenic basis of MDS with sole del5q. Could aberrant methylation of the retained allele add to the gene insufficiency?

6. Boultwood continued Compared CD 34 gene expression profiles of: Del 5q alone v normal individuals v RA with a normal karyotype. Many down regulated genes map to CDR Some differentially expressed genes map to other chromosomes: SPAG6, WIG-1, BM11 (up-regulated in 5q-) DPH5 (down-regulated in 5q-)

7. Boultwood continued Gene expression profiling in response to lenalidomide Cultured erythroblasts from del 5q patients Day 7 cultures in the presence of lenalidomide shows up regulation: SPARC & Activin A genes However, lenalidomide seems to kill del 5q cells and promote normal erythropoiesis & hence the interpretation of these day 7 culture data is difficult. SPARC is a tumour suppressor gene with antiproliferative & anti-angiogeneic properties and is located within the CDR.

8. The treatment of MDS with lenalidomide.Alan List, H. Lee Moffitt Cancer Centre, Florida 20-35% of all anaemic low/int-1 MDS patients respond to erythropoietic stimulating proteins Response is worse for del 5q patients GFM trial del 5q had poor erythroid responses compared to other karytopic abnormalities and shorter response duration (12 v 24 months) Only 15% had an erythroid response to thalidomide

9. Alan List, continued To date 168 patients with del 5q have been treated on 4 phase II trials with lenalidomide MDS-001, 002, 003, PK-002 71% required = 2 units per month & had failed prior ESP therapy Overall 114/168 (68%) achieved transfusion independence with 128 (76%) having an erythroid response. Time to response is rapid-median interval of 4.7 weeks TI was durable with a median duration of 2.2 years

10. Alan List, continued No difference in TI response between IPSS groups or cytogenetic patterns However TI duration differed: Longer TI for 5q- syndrome, lower IPSS score & lower cytogenetic complexity ( 1 additional abnormality v complex abnormalities) A cytogentic response was observed in 75% of evaluable patients. 48% achieving a cytogenetic CR after 16-24 weeks 36% achieved a pathological CR, all of whom achieved a cytogenetic CR 24 (14%) have experienced disease progression Advanced FAB type in 11 cases & AML in 13 cases.

11. Cytogenetic response to lenalidomide is associated with improved survival in patients with chromosome 5q deletion. AF List, et al. Data from 168 patients from 4 trials with max follow up of >5yrs 27 (16%) del5q + 1 additional abnormality 12(7%) complex (=3 abnormalities) 128 (76%) isolated del5q ECOG score, baseline transfusion requirement & 5q- syndrome were independent variables for associated with longer TI Median overall survival was 96 months for sole del5q v 67 months for = 1 additional abnormality. Complete & partial cytogenetic responders (90) had better OS than non-cytogentic responders/not evaluable (78) median not reached v 28 months (p< 0.0001) Ten year predicted OS 84% V 4% Multivariate analysis of survival co-variants identified marrow blast percentage, baseline transfusion burden and cytogenetic response to be independent variables associated with extended survival with lenalidomide therapy

12. Cytopenias correlate with response to lenalidomide in del 5q MDS patients. MA Sekeres et al. Commonest toxicities with lenalidomide are neutropenia and thrombocytopenia Commoner in del 5q than non del 5q Retrospective analysis of 003 study All had del 5q & treated with lenalidomide 10mg 21/28 days 147 patients, 40% thrombocytopenia at baseline, 40% neutropenia at baseline, 80% one or other.

13. Sekeres et al, continued 99 (67%) achieved TI Regardless of baseline count a fall in plats of = 50% during first 8 weeks correlated with higher chance of red cell TI For patients whose neuts fell = 75% from a normal baseline (>2) 82% achieved red cell TI v 56% for those in whom neuts fell by <75%

14. Treating non-del 5q patients with lenalidomide. CA Schiffer, Karmanos Cancer Institute, Detroit, Michigan 214 patients with low/int 1 MDS Red cell transfusion requirement of = 2 units/8 weeks No del 5q Well tolerated at 10mg od 21/28 days 56/214 (26%) became red cell transfusion independent Median increase in HB 3.3g/dl (1.0-9.8) Median duration of TI of 43.1 weeks 19% had a cytogenetic response! Much less neutropenia and thrombocytopenia

15. Dameshek�s riddle! �The immune pathophysiology of bone marrow failure and the emergence of clonal hematologic diseases�-NS Young, AJ Barrett and EM Sloand Many bone marrow failure syndromes appear to share an immune pathophysiology Aplastic anaemia-stem cell destruction follows FAS- mediated apoptosis by T-cell attack and type 1 cytokines (TNF & gamma-interferon) Why are stem cells attacked & what are the molecular pathways involved in the aberrant immune response? Why do PNH &MDS clones emerge on the background of aplasia?

16. Monosomy 7, trisomy 8 and PNH after AA 15% of AA evolves to MDS Most commonly monosomy 7 Next commonest trisomy 8 50% of AA and 20% of MDS show expanded PNH clones These evolutions are explained by survival/escape mechanisms possessed by the abnormal clone within the aplastic marrow

17. Young et al, continued In Japan there is a high incidence of monosomy 7 in children with congenital neutropenia & some adults with AA given prolonged G-CSF. Sloand et al PNAS 2006 Cultured MDS patients progenitors (normal cytogenetics) with exogenous G-CSF Monosomy 7 clone appeared in culture with over expression of a short isoform of G-CSF receptor-signals for proliferation and not differentiation Failure of this isoform to internalise and up regulation of the anti-apoptotic gene AKT gives the selected growth advantage Socie et al � confirmed the same in European patients Monosomy 7 clone is selected out for expansion by exogenous or endogenous G-CSF acting on the G-CSF isoform of the monosomy 7 clone-autonomous

18. Young et al, continued All trisomy 8 cases (de- novo & secondary) have a biased VB T-repertoire to WT1. Trisomy 8 overexpress WT1 T-cells recognise aneuploid cells & specifically antigens encoded by the WT1 Trisomy 8 cells have up regulation of c-myc and survivn (both anti-apoptotic) Trisomy 8 clone therefore survives the immune attack and expands whilst bystander death of normal progenitors explains the cytopenias of the MDS/aplasia

19. Paroxysmal nocturnal hemoglobinuria. L Luzatto, R Notaro, G Gianfaldoni. University of Firenze. PNH cells originate from clonal expansion of a stem cell harbouring a somatic mutation in the X-linked gene PIG-A Small PNH clones exist in some healthy people PNH expansion is common in AA and MDS This expansion seems to result from two processes � damage to normal stem cells & sparing of the PNH clone from the resultant immune attack

20. Luzzatto et al, continued Several case reports of expanded CD8+CD57+ LGL T-cells in PNH TCR-B repertoire is very limited �oligoclonal Different patients show the identical clonotypic TCR-B restrictions, not seen in 25 healthy controls 11/16 patients shared at least one of five clonotypes and a set of closely related clonotypes was present in 9/16 These patients had severe haemolytic PNH-marked PNH clonal expansion secondary to an autoreactive immune process driven by the same or similar antigens However,the patients sharing clonotypes do not have identical HLA antigens-suggesting the antigenic target is not a peptide

21. Luzzatto et al, continued The following mechanisms to explain these findings were speculated: CD 1D presents lipid antigens. CD 1D is present on haemopoietic stem cells. CD 1D is GPI anchored. Possibility 1 is a common lipid antigen presented and attacked on normal stem cells but not presented on PNH stem cells Possibility 2 is GPI anchor itself is the antigenic target

22. Iron chelation in MDS The launch of the oral iron chelator deferasirox has re-ignited enthusiasm for exploring the role of iron chelation in MDS. This was highlighted by a plenary session symposium in Florence sponsored by Novartis & The MDS Foundation entitled: �Iron overload in MDS: Clinical consequences and management strategies�

23. Iron overload in MDS Luca Malcovati firstly reviewed the impact of transfusion dependency & secondary iron overload on survival in MDS: >90% of MDS patients become anaemic Red cell transfusion remains the basis of therapy for most symptomatic patients Transfusion dependence is associated with poorer overall survival & greater risk of leukaemic progression This is most noticeable in �low risk� patients & correlates with the number of units transfused. Of course this in part reflects the negative impact of more aggressive disease. However, transfusion dependence is also associated with a higher risk of death from cardiac failure & the hazard for overall survival increases by ~ 30% for every increase in ferritin of 500ng/ml above a threshold of 1000ng/ml. This apparent effect of iron overload is most noticeable in patients with WHO type RA with a median survival of over 100 months, i.e. patients with a good survival from the underlying disease process.

24. �Improved survival in MDS patients receiving iron chelation therapy� Heather A Leitch, Columbia Vancouver The second presentation in this symposium proved to be rather controversial. This was a retrospective study of 178 MDS patients seen between 1981-2006. Patients receiving iron chelation received desferioxamine at 0.5-3.0 g per 12 hrs 5 days per week Serum ferritin = 2000ug/ml in 28 patients Clinical evidence of �iron overload� was documented in 28 patients, 22 cardiac failure, 5 liver disease, 18 endocrine dysfunction, 4 �other.� Biopsy or imaging evidence of iron overload only present in 6 cases. 18 patients received iron chelation for the following reasons, elevated ferritin, n=13, clinical evidence of iron overload n=3, no of transfusions received n=2

25. Heather Leitch, continued In these 18 patients mean serum ferritin pre-chelation was 4,215ug/l & post chelation was 2,659 ug/l. In the non-chelated majority the median first raised ferritin was 1,647ug/l rising to 3,188ug/l on follow up. There was a trend to higher initial ferritins & significantly lower follow up ferritins in chelated patients (p<0.003). In multifactorial analysis, factors significant for overall survival were IPSS score (P<0.008), and iron chelation therapy (P<0.002). For patients with IPSS low & int-1 median OS for chelated patients was not reached at 160 months v 40.1 months for non-chelated patients

26. Heather Leitch, continued The discussion of this study was lively & controversial. Bearing in mind the limitations of a small retrospective study there were some points of significant criticism: The author stated that the policy for selecting patients for iron chelation in Vancouver included a predicted survival of 5 years. This led to criticism that the results were self-fulfilling. However, it remained unclear whether these criteria were applied during the years of the retrospective study or only recently. The OS was calculated from diagnosis rather than from the point of starting chelation, raising the possibility of a time bias effect in the survival data. Despite these valid concerns this remains the only significant attempt to identify a real benefit from iron chelation therapy and if nothing else highlights the need for prospective studies, perhaps made easier in the future by the availibility of newer oral iron chelators.

27. Guidelines on iron chelation therapy in patients with MDS and transfusional iron overload. Norbert Gatterman, Dusseldorf, Germany Norbert Gatterman outlined the presently available guidelines which include statements on the management of iron overload in MDS patients Italian Society of Hematology, Haematologica, 2002;87:1286-306 British Committee for Standards in Haematology, British Journal of haematology, 2003;120:187-200 National Comprehensive Cancer Network (NCCN), 2007, available at www.nccn.org. Consensus Conference in Nagasaki, Hematology/Oncology Clinics of North America, 2005;19 Suppl 1:18-25

28. Gattermann, continued Taken together the suggested target population for iron chelation would include: IPSS score low or int-1 Usually this would correspond to WHO types RA, RARS, 5q- syndrome & some patients with RCMDS Candidates for allograft in whom there is a significant delay until the procedure. Ferritin should be 1000-2000 ng/ml or clinical or radiological evidence of iron loading at the start of chelation This would often correlate with 20-30 units of red cells transfused Currently a LeukemiaNet working group are producing therapeutic guidelines for MDS which will include further recommendations on iron chelation More studies of the role of iron loading in the morbidity and mortality of MDS and of the benefits of chelation are needed.

29. Novel treatment options in transfusional iron overloaded patients, Stuart L Goldberg, Hackensack, USA The final presentation in this symposium looked at the potential role of oral Deferasirox in MDS Deferasirox is a tridentate iron chelator Selective for iron in ferric (Fe3+) state Binds to iron in a 2:1 stoichiometry Lipid soluble but highly protein bound with a long plasma half-life of 8-16 hours compatible with once daily dosing Deferasirox comes as a dispersible tablet to take in water or juice.

30. Goldberg, continued Registration studies including various bone marrow failure syndromes have demonstrated a dose dependent decrease in serum ferritin and liver iron concentration (LIC) Among 47 MDS patients treated in trial 108, the mean decrease in serum ferritin was 268ng/ml with an iron excretion to intake ratio of 1.7 In a subset analysis of 28 patients with MDS the mean decrease of LIC was 5.7mg iron per gram dry weight of liver over one year 10mg/kg dosing led to stable markers of iron stores whilst 30 and 30mg/kg dosing led to reduced hepatic iron The clinical impact of deferasirox, like iron chelation in general in MDS needs clarifying. Given the data suggesting increased cardiac death in iron loaded MDS patients, the role of cardiac iron in these patients is being explored by studies of cardiac MRI. In thalassaemia deferasirox has been shown to improve cardiac T2* after one year of therapy

31. Goldberg, continued The side effect profile of deferasirox seems clinically manageable The commonest side effects are mild GI disturbances (nausea and diarrhoea) An important observation is a non-progressive rise in serum creatinine in one third of patients. If the creatinine exceeds 33% of the baseline dose adjustment is advised. Given the elderly age of MDS patients and the frequent co-morbidities such as hypertension and diabetes this aspect of the side effect profile will need careful observation as the numbers of MDS patients treated increases.