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Decreased neutrophil alkaline phosphatase (NAP) synthesis is a classical feature of Philadelphia (Ph) positive chronic phase chronic myeloid leukemia (CML). Whether this aberration is an integral leukemic property of the cell or results from mediation by other factors is unclear. During a-interferon (α-IFN) based therapy the relationship between Ph chromosome suppression and NAP synthesis was examined. Four categories of response were observed in 19 patients studied sequentially. Significantly, persistent low NAP activity was observed in one patient in complete cytogenetic remission, while a second group of 7 patients demonstrated normal NAP activity in spite of persistence of the Ph chromosome in 100% of metaphases. In the absence of various clinical influences that can modulate NAP activity in chronic phase CML, the results reinforce the observation that the BCWABL fusion gene product is not a key factor influencing NAP activity in CML.

Using X-linked clonal analysis, mechanism of action of all- transretinoic acid (ATRA) was sought in a 16-year-old female with relapsed clonally evolved acute promyelocytic leukemia (APL), who achieved complete remission. On ATRA, metamorphosis of peripheral blood leukemic promyelocytes to mature neutrophils was observed, despite the persistence of t(15;17) in 100% of bone marrow metaphases. DNA was extracted from fractionated serial blood specimens, collected at diagnosis, in first complete remission (CR), relapse, and during ATRA treatment. Using a phosphoglycerokinase (PGK) probe, the patient was heterozygous for both BglI and BstXI PGK polymorphisms. Methylation analysis showed monoclonal leukemic promyelocytes with a polyclonal first CR achieved by standard chemotherapy. Subsequent exami-nation, in relapse, of granulocytes appearing during ATRA treatment showed these to be monoclonal, proving these were derived from the neoplastic clone. The X-linked clonal analysis methodology has provided in vivo evidence of cellular differentiation as the mechanism of action of ATRA. Parallel studies of cytogenetic and clonal analysis showed a regression of the t(15;17) cytogenetic abnormality and return of a polyclonal PGK methylation pattern in 5 weeks, indicating a repopulation of marrow by normal stem cells. As standard cytogenetic techniques are inappropriate for nondividing cells, X-linked clonal analysis provides a marker system to allow insight into mechanism of drug action in malignant hematologic disease.

To report two cases of chronic neutropenic states successfully treated with granulocyte colony stimulating factor (G‐CSF). A 23‐year‐old man with severe congenital cyclic neutropenia causing lifelong recurrent infections and consequent debilitation presented with intractable infected leg ulcers. A 56‐year‐old man with acquired idiopathic neutropenia presented with severe perianal infection and sepsis. Both patients were successfully treated with recombinant G‐CSF. G‐CSF is an agent with major clinical potential for the therapy of primary neutropenic states.

DeAngelo: Incyte Corporation: Consultancy; Celgene Corporation: Consultancy; Pfizer, Inc.: Consultancy; GlycoMimetics: Research Funding; AbbVie, Inc.: Research Funding; Takeda Pharmaceuticals: Consultancy; Shire: Consultancy; Amgen: Consultancy; Blue Print Medicines: Consultancy, Research Funding; Jazz Pharmaceuticals, Inc.: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding. Jonas:AbbVie, Accelerated Medical Diagnostics, AROG, Celgene, Daiichi Sankyo, Esanex, Forma, Genentech/Roche, GlycoMimetics, Incyte, LP Therapeutics, Pharmacyclics: Research Funding; AbbVie, Amgen, Celgene, GlycoMimetics, Jazz, Pharmacyclics, Tolero: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie, Amgen, GlycoMimetics: Other: Travel expenses. Liesveld:Abbvie: Membership on an entity's Board of Directors or advisory committees; Onconova: Other: Data safety monitoring board. Advani:Pfizer: Honoraria, Research Funding; Glycomimetics: Consultancy, Research Funding; Amgen: Research Funding; Kite Pharmaceuticals: Consultancy; Abbvie: Research Funding; Macrogenics: Research Funding. Marlton:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. O'Dwyer:Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; GlycoMimetics Inc: Research Funding; BMS: Research Funding. Fogler:GlycoMimetics Inc: Employment, Equity Ownership. Magnani:GlycoMimetics Inc: Employment, Equity Ownership. Chen:GlycoMimetics: Employment. Feldman:GlycoMimetics Inc: Employment. Thackray:GlycoMimetics Inc: Employment. Becker:AbbVie, Amgen, Bristol-Myers Squibb, Glycomimetics, Invivoscribe, JW Pharmaceuticals, Novartis, Trovagene: Research Funding; Accordant Health Services/Caremark: Consultancy; The France Foundation: Honoraria.

Cull: Amgen: Other: Travel, accommodation ; AbbVie: Other: Travel, accommodation; Glycomimetics: Other: Travel, accommodation. Simpson:Amgen: Research Funding; BeiGene: Research Funding; AbbVie: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Celgene: Research Funding; MSD: Research Funding; Acerta: Research Funding; Pharmacyclics: Research Funding; Sanofi: Research Funding; GSK: Research Funding; Jannsen: Honoraria. Opat:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Epizyme: Research Funding; CSL: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Research Funding; Novartis: Consultancy; BMS: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Beigene: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Amgen: Research Funding; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Burger:Gilead Sciences: Research Funding; Pharmacyclics, an AbbVie company: Research Funding; Janssen Pharmaceuticals: Consultancy, Honoraria; BeiGene: Research Funding; AstraZeneca: Honoraria; Aptose Biosciences, Inc: Research Funding. Trotman:Roche: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; Janssen: Research Funding; BeiGene: Research Funding. Marlton:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. Gottlieb:AbbVie: Consultancy; Gilead: Consultancy; Merck: Consultancy; Haemalogix P/L: Membership on an entity's Board of Directors or advisory committees, Research Funding; University of Sydney: Employment; Novartis: Consultancy. Munoz:Incyte: Research Funding; Portola: Research Funding; Celgene: Research Funding; Seattle Genetics: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Speakers Bureau; AstraZeneca: Speakers Bureau; Pharmacyclics LLC an AbbVie Company: Consultancy, Research Funding, Speakers Bureau; Kite Pharma: Consultancy, Research Funding, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Fosunkite: Speakers Bureau; Kyowa: Consultancy, Honoraria, Speakers Bureau. Seymour:Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Honoraria; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Acerta: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria. Roberts:AbbVie: Other: Unremunerated speaker for AbbVie, Research Funding; Janssen: Research Funding; Australasian Leukaemia and Lymphoma Group: Membership on an entity's Board of Directors or advisory committees; Walter and Eliza Hall Institute: Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.; BeiGene: Research Funding. Wu:BeiGene: Employment, Equity Ownership. Atwal:BeiGene: Employment, Equity Ownership. Novotny:BeiGene: Employment, Equity Ownership. Huang:BeiGene: Employment, Equity Ownership. Tam:BeiGene: Honoraria; AbbVie: Honoraria, Research Funding; Novartis: Honoraria; Roche: Honoraria; Janssen: Honoraria, Research Funding.Zanubrutinib is an investigational agent and has not yet been approved in the US

Winkler: GlycoMimetics: Patents & Royalties. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Marlton:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlycoMimetics: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Perkins:Novartis Oncology: Honoraria. Levesque:GlycoMimetics: Equity Ownership, Patents & Royalties.

Greil: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Honoraria, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sandoz: Honoraria, Research Funding. Fraser:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria. Leber:Novartis Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees. Marks:BMS: Honoraria; Servier: Honoraria; Merck: Honoraria. Middeke:Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees. Schary:AbbVie: Employment. Boyer:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Breuleux:Roche: Employment, Equity Ownership; Gilead: Equity Ownership; Basilea: Patents & Royalties; Novartis: Patents & Royalties. Crompton:Roche: Employment, Equity Ownership. Humphrey:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Marlton:Pfizer: Membership on an entity's Board of Directors or advisory committees; GlycoMimetics: Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

DeAngelo: Amgen: Consultancy, Research Funding; BMS: Consultancy; Immunogen: Honoraria, Research Funding; Celgene: Research Funding; ARIAD: Consultancy, Research Funding; Blueprint Medicines: Honoraria, Research Funding; Incyte: Consultancy, Honoraria; Glycomimetics: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; Takeda Pharmaceuticals U.S.A., Inc.: Honoraria; Shire: Honoraria; Pfizer Inc.: Consultancy, Honoraria, Research Funding. Jonas: Celgene: Membership on an entity's Board of Directors or advisory committees; AbbVie, Celgene, Daiichi Sankyo, Pharmacyclics, Genentech/Roche, Glycomimetics, Esanex, Kalobios: Research Funding; Rigel: Consultancy. Liesveld: Seattle Genetics: Honoraria; Onconova: Honoraria. Advani: Pfizer: Consultancy; Takeda/ Millenium: Research Funding. O'Dwyer: Onkimmune Ltd: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; GlycoMimetics Inc: Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Magnani: GlycoMimetics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Thackray: GlycoMimetics, Inc.: Employment, Equity Ownership. Becker: GlycoMimetics, Inc.: Research Funding.

Introduction

Gill: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Mollee: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Nilelse: Research Funding.

Tam: janssen: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees. Opat:Roche: Consultancy, Honoraria, Other: Provision of subsidised drugs, Research Funding. Gottlieb:Indee: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees. Simpson:Celgene, Roche, Janssen: Honoraria; Amgen Pharmaceuticals: Research Funding. Anderson:Walter and Eliza Hall Institute of Medical Research: Other: Walter and Eliza Hall Institute of Medical Research receives milestone payments for the development of venetoclax. Kirschbaum:Beigene: Employment. Wang:Beigene: Employment. Xue:Beigene: Employment. Yang:BeiGene: Employment. Hedrick:Beigene: Employment. Seymour:Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Roberts:Genentech: Patents & Royalties: Employee of Walter and Eliza Hall Institute of Medical Research which receives milestone payments related to venetoclax; Janssen: Research Funding; Genentech: Research Funding; AbbVie: Research Funding; Servier: Research Funding.

Szer: Ra Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alnylam: Honoraria, Membership on an entity's Board of Directors or advisory committees. Marlton:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: 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; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Wei:Novartis: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; CTI: Consultancy, Honoraria; Abbvie: Honoraria, Research Funding; Servier: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Cartwright:ROCHE: Consultancy, Membership on an entity's Board of Directors or advisory committees. Roberts:Servier: Research Funding; Janssen: Research Funding; Genentech: Research Funding; AbbVie: Research Funding. Mills:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Meeting attendance sponsorship. Gill:Janssen: Membership on an entity's Board of Directors or advisory committees. Seymour:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Gill: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Tam: Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; janssen: Honoraria, Research Funding. Opat:Roche: Consultancy, Honoraria, Other: Provision of subsidised drugs, Research Funding. Simpson:Amgen Pharmaceuticals: Research Funding; Celgene, Roche, Janssen: Honoraria. Anderson:Walter and Eliza Hall Institute of Medical Research: Other: Walter and Eliza Hall Institute of Medical Research receives milestone payments for the development of venetoclax. Kirschbaum:Beigene: Employment. Wang:Beigene: Employment. Xue:Beigene: Employment. Yang:BeiGene: Employment. Hedrick:Beigene: Employment. Seymour:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Roberts:AbbVie: Research Funding; Servier: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Genentech: Patents & Royalties: Employee of Walter and Eliza Hall Institute of Medical Research which receives milestone payments related to venetoclax.

(1) Validate and establish a WT1expression assay from peripheral blood and bone marrow. (2) Investigate prognostic relevance of WT1expression levels at: diagnosis, post-induction and post-consolidation.

Hiwase: Celgene Corporation: Research Funding.

Zelenetz: Gilead Sciences: Research Funding. Off Label Use: Idelalisib for relapsed/refractory CLL. Robak:MorphoSys AG: Consultancy , Honoraria , Research Funding ; Janssen: Consultancy , Research Funding ; Gilead Sciences: Research Funding. Coiffier:CELLTRION, Inc.: Consultancy , Honoraria ; Gilead Sciences: Research Funding. Delgado:Gilead Sciences: Research Funding. Marlton:Gilead Sciences: Research Funding. Adewoye:Gilead Sciences: Employment , Equity Ownership. Kim:Gilead Sciences: Employment , Equity Ownership. Dreiling:Gilead Sciences: Employment , Equity Ownership. Hillmen:Gilead Sciences: Honoraria , Research Funding ; Abbvie: Honoraria , Research Funding ; Janssen: Honoraria , Research Funding ; Roche: Honoraria , Research Funding ; Novartis: Honoraria , Research Funding ; Pharmacyclics: Honoraria , Research Funding ; Acerta Pharma BV: Research Funding.

Grigg: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Szer:Alexion Pharmaceuticals Australasia Pty Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer Australia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire Australia: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Mollee: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Onyx: Membership on an entity's Board of Directors or advisory committees. Wei:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees.

No relevant conflicts of interest to declare.

Off Label Use: Obinutuzumab is a type II CD20 monoclonal antibody which is licensed for use in combination with chlorambucil in untreated patients with CLL but is not currently approved for use in follicular lymphoma.. Grigg:Roche: Consultancy. Dreyling:Roche: Honoraria, Research Funding. Rule:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Lei:Roche Products Ltd.: Employment. Wassner-Fritsch:Roche: Employment. Fingerle-Rowson:F. Hoffmann–La Roche: Employment. Marlton:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees.

CBF abnormalities remain a robust prognostic indicator of relatively favorable outcome in AML. Several groups have demonstrated greater responsiveness to HiDAC and studies at MD Anderson established the feasibility of omitting anthracycline when using fludarabine to favourably modulate the intracellular accumulation of cytarabine (Borthakur Cancer 2008). This study further explored that approach by assessing the clinical outcome and molecular responses of patients (pts) treated with fludarabine and HiDAC in the absence of anthracycline.52 eligible pts with previously untreated newly diagnosed CBF AML were enrolled from 13 centres between 02/05 and 06/09 (protocol registration ANZCTRN012605000152628). One enrolled pt with non-CBF AML was excluded from analysis. Therapy consisted of induction with fludarabine 30 mg/m2/d followed by cytarabine 2 g/m2/d for 5 days (FA) and post-remission treatment with 3 cycles of HiDAC (2 g/m2 q 12 h Days 1, 3 and 5) followed by 3 cycles of FA for 4 days. No patient was transplanted in first complete remission (CR). Quantitative PCR (QPCR) for CBF-specific fusion transcripts was conducted at diagnosis, post induction, post consolidation cycles and 3 monthly during 2 years of post-treatment follow-up. The primary endpoints were failure free survival (FFS) at 2 and 4 years.Median age was 39 (17 – 62) years. 22 pts had t(8;21) and/or AML1-ETO and 30 had inv(16)/t(16;16) and/or CBFB-MYH11, of which 21 had Type A, 5 type D, 3 Type E and 1 Type G transcripts. CR was achieved in 48 of 52 pts (92%): 2 of 4 non-CR pts were responding (post-induction blast counts of 6 and 7%), 1 had residual extra-medullary disease and only 1 pt had refractory AML. 13 pts relapsed (3 AML1-ETO and 10 CBFB-MYH11 p=0.17; median time to relapse 12.4 (range 9.1 – 25.1) months) and 3 died in CR (sepsis). At a median follow-up of 4.6 (range 2.4 – 6.8) years, FFS was 62% (95% CI 50–77%) and 60% (47–75%) at 2 and 4 years respectively. Overall survival (OS) was 83% at 2 years (95% CI 73–94%) and 78% at 4 years (68–91%) reflecting the relative success of salvage (10 of 13 relapsed pts underwent allogeneic transplant). There was no statistical difference between the 2 molecular subtypes for either PFS (p=0.27) or OS (p=0.5). OS was similar to that achieved in our previous trial (Bradstock Blood 2005) utilizing ICE induction followed by either one further ICE or two IcE consolidation cycles (81% at 2 years). Time to neutrophil recovery (>0.5 x 109/L on 2 consecutive measures) was longer for FA cycles compared to HiDAC (mean 10 vs 6 days; p = 0.001). The rate of grade 3,4 infection was high (88% of pts had at least 1 episode) with 17% coming off protocol for toxicity and only 29 pts (56%) completing all 6 cycles of post-remission therapy with no difference in cycles received between pts who relapsed and those who did not (median cycles delivered was 6 in both groups). Detectable QPCR levels in remission were significantly associated with risk of relapse (10 of 13 relapses were preceded by MRD positivity at a median of 13 weeks; p=0.001). All 10 patients with levels >250 copies in PB or BM at any post induction time point subsequently relapsed. LFS was significantly different for patients with MRD positivity (>10 copies on 2 consecutive measurements or >250 at any time) during follow-up compared to those who remained negative (Fig 1). Multivariate analysis did not identify any significant associations between recognized clinical variables and either PFS or OS. LFS was not associated with c-kit mutational status for the subset of 33 pts with results available (p = 0.6).The high CR rate and relatively favourable FFS and OS of pts with CBF AML treated with non-anthracycline containing fludarabine-modulated HiDAC based therapy, are similar to published results achieved with standard anthracycline combination treatments. Short-term infectious toxicity of this regimen, particularly during the prolonged post remission therapy, remained significant and the lack of association between relapse and number of cycles delivered suggests the duration may be shortened. QPCR monitoring was confirmed to be clinically useful in the prediction of relapse. Results will be updated after the planned final analysis.Off Label Use: Fludarabine in the treatment of AML.

The optimal reduction of IST in renal transplant patients with PTLD is uncertain. As chemotherapy used to treat PTLD is inherently immunosuppressive, IST may be able to be stopped during this time without compromising graft function. Subsequent long-term reduction of IST is important to reduce PTLD relapse, but may increase long-term risk of graft rejection.We performed a retrospective matched cohort study of adult renal transplant patients where IST was ceased during chemotherapy and then resumed at lower dose (calcineurin inhibitor at 50%, prednisolone <6mg daily, no third agent). Outcomes were compared to renal transplant patients without PTLD, matched for creatinine at the equivalent time post-transplant that PTLD was diagnosed in the cases, as well as age, gender, year of transplant and age at transplant. The primary endpoint of time to renal deterioration, defined as the time from PTLD diagnosis (cases) or study entry (controls) to a 25% increase in creatinine, was analysed using competing risk survival analyses. Additional endpoints were time to renal allograft failure requiring dialysis or re-transplant and overall survival.24 cases were identified: median age at PTLD diagnosis 45yrs; 75% male; median time to onset of PTLD post-transplant was 9 years. 22 cases received CHOP-like chemotherapy and 2 received primary CNS lymphoma protocols, 7 received concurrent rituximab and 4 had consolidation radiation therapy. 88% attained complete remission. Five patients have relapsed. The 5 yr overall survival for the cases was 71% with a median follow-up of survivors of 11.9 years.The 24 PTLD cases were compared to 84 well matched controls. There were 11 deaths in the cases and 5 in the controls. Three cases recommenced dialysis, compared to 3 controls (HR 2.5, p=0.27). The 5 yr rate of 25% increase in creatinine was 36% in cases and 20% in controls (HR 1.8, p=0.098). Of the 11 cases with ≥25% increase in creatinine, only 1 of these occurred within 6 months of IST cessation in the setting of rapidly progressive PTLD and early death. Only 2 of these 11 cases received rituximab. In contrast 5 of the 7 patients given rituximab did not develop a ≥25% deterioration in renal function.IST can be safely ceased during chemotherapy for PTLD in renal transplant patients. Whilst long-term reduction in IST is necessary to reduce PTLD relapse, this is associated with a trend to increased risk of chronic allograft nephropathy. Prospective trials are needed to address the ideal reduction of IST in PTLD.No relevant conflicts of interest to declare.

Dyer: Roche: Consultancy, Research Funding. Off Label Use: Obinutuzumab (GA101) in Combination with Cyclophosphamide, Doxorubicin, Vincristine and Prednisone (CHOP) or Bendamustine in Patients with Previously Untreated Follicular Lymphoma (FL). Grigg:Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding. Dreyling:Roche: Honoraria, Support of (other) clinical trials and Scientific Advisory Boards Other. Rule:Roche: Consultancy, Research Funding. Lei:Roche: Employment. Wassner-Fritsch:Roche: Employment. Wenger:Roche: Employment. Marlton:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 5119

Cardiac failure is the leading cause of death in patients with thalassaemia major. However, the introduction of new monitoring techniques, such as cardiac iron Magnetic Resonance Imaging (MRI), may lead to the improvement of patient care and prolong survival. Although the impact of cardiac siderosis in patients with thalassaemia major has been extensively studied, further examination of the effect of specific iron chelators such as deferasirox are important in the development of treatment protocols, while the clinical consequences of cardiac iron accumulation in patients with myelodysplastic syndromes (MDS) is still controversial.The primary objective of this study is to investigate the effect of single agent deferasirox on cardiac iron levels and cardiac function, in a cohort of patients receiving regular red blood cell transfusions.Patients with transfusional siderosis were enrolled in this study and received deferasirox for 53 weeks. Doses up to 40 mg/kg/day were allowed according to transfusional requirement, response to chelation therapy and safety markers. Cardiac siderosis (cardiac T2* measured by linear fitting to the decay of logarithmic magnetic resonance signal) and left ventricular ejection fraction (LVEF) were measured at baseline and at 53 weeks. The sample size was originally calculated based on the standard deviation (SD) of the change in log(T2*) (0.47), according to previously published reports. A planned sample size reassessment as per protocol was undertaken when 42 patients had follow-up data. For the sample size reassessment, log(T2*) was calculated for each result and the change in log(T2*) estimated for each patient. Results from the interim sample size reassessment and the analysis of cardiac iron and function are described below.Results were available for 36 patients with haemoglobinopathies (mainly represented by thalassaemia major), 5 patients with MDS and one post-bone marrow transplant patient. The SD observed for change in log(T2*) with the current data was 0.26 (original assumption 0.47). Assuming the SD for the final data does not increase above 0.34 the current cohort should be sufficient to detect a change in log(T2*) of the pre-specified 0.14. Safety assessments were consistent with previous studies on deferasirox. An overall increase in cardiac T2* from 23.0 to 26.1 ms was observed (p<0.001). Further analysis was performed based on the T2* baseline values: 5–10 ms, no significant change (n=6; 8.5 to 7.9 ms; p=0.6; non-significant); 10–20 ms, improvement of 26% (n=8; from 14.8 to 18.7 ms; p=0.001); >20 ms, increase of 10% (n=28; 32.3 to 35.6 ms; p=0.04). Overall, an increase in LVEF of 2.1% was observed, just below statistical significance (from 60.9% to 63.0%, p=0.076). The largest increase in LVEF was observed in patients with moderate cardiac siderosis (baseline T2* 10–20 ms; statistically significant increase in LVEF of 5.7%, from 56.8% to 62.5%, p=0.045). Descriptive preliminary analyses of the MDS subgroup showed that all 5 patients had baseline cardiac T2* >20 ms (median 32 ms; range: 29.7 to 34 ms). Similarly to the overall patient pool, deferasirox induced a 12% increase in cardiac T2* although not statistically significant (32.2 to 35.9 ms, p=0.13). In this subgroup of MDS patients, a statistically significant increase in LVEF was also observed (56.4% to 63.6%, p=0.04). Results on liver iron concentration will also be presented.The interim results from this study showed for the first time that once-daily deferasirox induced a simultaneous and statistically significant increase in both cardiac T2* levels and cardiac function in patients with moderate cardiac siderosis. Although the small number of patients in the MDS subgroup had cardiac T2* levels above 20 ms, chelation therapy induced an increase in LVEF in all 5 patients enrolled to date. Taken together, these results also confirm previous reports showing that single agent deferasirox is an effective therapy for maintenance and reduction of cardiac iron levels, especially for patients with cardiac T2* >10 ms. Recruitment has been halted based on the recommendations of the interim sample size re-estimation in consultation with the Data Monitoring Committee. Six patients are ongoing, and will be included in the final analysis of the study.Ho: Novartis: Honoraria, Invited speaker, Membership on an entity's Board of Directors or advisory committees. Tay:Novartis: Membership on an entity's Board of Directors or advisory committees. Teo:Novartis: Research Funding. Marlton:Novartis: Membership on an entity's Board of Directors or advisory committees. Grigg:Novartis: Invited speaker, Membership on an entity's Board of Directors or advisory committees. Pierre:Resonance Health: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau. Brown:Novartis: Consultancy, Invited speaker. Gervasio:Novartis: Employment. Bowden:Novartis: Honoraria, Research Funding, Speakers Bureau.

Aya-Bonilla: Cancer Council of Queensland: Research Funding.

Acute Myeloid Leukaemia (AML) has a worldwide incidence of approximately 3.5 per 100,000 population per year with most cases occurring in adults. Survival at 12 months is less than 30% and at 5 years is less than 10% (NCI, 2010). AML is a complex disease that demonstrates marked heterogeneity morphologically, cytogenetically and molecularly. The largest cytogenetic subgroup of AML has normal cytogenetics and accounts for approximately 45% of de novo AML cases. Furthermore, the response to treatment and survival outcomes of cytogenetically normal AML (cn-AML) is remarkably heterogeneous.– To undertake whole-genome and exome sequence analysis of a cn-AML case at diagnosis and at relapse to identify subtle, potentially oncogenic mutations at the molecular level that may initiate AML and those that may be responsible for drug resistance or relapse.i. –Comparison of the AML genomes at diagnosis, remission and relapse to identify genomic regions with changes in copy number and loss of heterozygosity, using single nucleotide polymorphism (SNP) microarray analysis. ii. Identify chromosomal rearrangements such as small insertions, duplications, inversions and deletions, (“indels”) and translocations that may contribute to AML initiation or relapse, using massively parallel, ultra-high throughput (“next generation”), paired-end sequencing of total genomic DNA at low depth. iii. Identify DNA point mutations potentially affecting protein function that may contribute to AML initiation or relapse by using next generation sequencing at high depth of the captured exomes (putative coding regions) of the AML and control genomes.The PwC (Price-Waterhouse-Coopers) Leukaemia and Lymphoma Tissue Bank, a joint initiative of the Australasian Leukaemia and Lymphoma Group and the Leukaemia Foundation, provided samples from an AML patient with normal cytogenetics and a blast count at diagnosis of 70% and at relapse of 85%. Genomic DNA extracted from autologous mesenchymal stem/stromal cells (MSCs) was used to represent non-leukaemic, germline, control DNA. Primary cell culture of MSCs from cryopreserved bone marrow aspirate cells (50 × 106) of our test patient at remission was achieved with standard tissue culture methods. High molecular weight DNA was extracted from the patient's MSCs and marrow cells at diagnosis, remission and relapse samples. Sonication (Covaris) and libraries appropriate for paired-end high-throughput sequencing (Illumina Genome Analyzer II instrument) were prepared from gel purified DNA fragments approximately 200 bp in size.Primary cell culture of MSCs from bone marrow aspirates proved to be a robust source of germline genomic DNA with several advantages over skin. Firstly, tissue banked samples, will not include biopsies of normal skin. Furthermore, skin can be contaminated by circulating leukaemic cells, which is problematic with low-depth genomic sequencing. The homogenous immunophenotype of the cultured MSCs indicate their purity. Preliminary SNP microarray analysis identified a large region of uniparental disomy (copy number neutral loss of heterozygosity) involving most of chromosome 13q, which was not identified by standard cytogenetic analysis. Low-pass or shallow paired-end genomic DNA sequencing has generated the following outputs. MSC genome: 10.7 Gb (3.6X haploid genome coverage) resulting from 152 × 106 paired sequence reads, AML_diagnosis genome: 21.6 Gb (7.2X) resulting from 142 × 106 paired sequence reads, AML_relapse genome: 26.8 Gb (8.9X) resulting from 177 × 106 paired sequence reads. The capture baits representing the human exome span 26,225,870 bp (approximately 0.9% of haploid human genome). Exome capture libraries were prepared from the three sources of DNA (MSC, AML_diagnosis and AML_relapse) and sequenced using a single lane for each library. The outputs for all three were very similar, approximately 23 × 106 paired sequence reads, representing 1.3 Gb (49X haploid exome coverage). The reads for whole genome or exome capture were of high quality and more than 98% could be unambiguously aligned to the human reference genome in the correct orientation and interval distance. Here we will present MSC enrichment results and sequencing output results, quality control and preliminary analysis of genomic alterations and exonic mutations.No relevant conflicts of interest to declare.

As wt1 expression is elevated in the majority (80%) of acute myeloid leukemias at diagnosis and relapse, a validated wt1 assay would be a powerful adjunctive tool for evaluation and monitoring of patients during their treatment. Monitoring of disease status with wt1 is applicable to more AML patients in a single assay than any other leukemic marker and includes patients that are cytogenetically normal and may not otherwise be amenable to molecular MRD evaluation. We aimed to analyse wt1 expression in the peripheral blood and bone marrow of patients with de novo AML, when measured at diagnosis, post-induction and post-consolidation; and to correlate these wt1 expression levels with patient characteristics and outcome.This is a prospective, longitudinal study in which the levels of wt1 expression from 99 patients with de novo AML are measured from bone marrow (59 patients) and peripheral blood (94 patients) specimens at the time of diagnosis and at various time-points along their treatment and clinical courses. The specimens were prospectively stored in the PwC Australasian Leukaemia and Lymphoma Group (ALLG) Tissue Bank. Samples analysed included: 59 bone marrow (bm-wt1) and 94 peripheral blood (pb-wt1) at diagnosis; 54 bm-wt1 and 57 pb-wt1 post-induction; and 46 bm-wt1 and 41 pb-wt1 post-consolidation. 90 of the 99 patients were treated with standard induction chemotherapy on a variety of protocols. RNA was extracted from 107 leukocytes purified from blood and bone marrow using TRIzol® (Invitrogen) extraction. Reverse transcription was performed using Superscript ® (Invitrogen) and RQ-PCR was performed using multiplexed 5' nuclease assay with Black Hole Quencher labelled probes according to published methods. Copy number of wt1 was normalised to 104 copies of ABL. Baseline patient characteristics were correlated with baseline wt1 expression levels and the impact of baseline and post-treatment wt1 expression on leukemia-free survival (LFS) was assessed.99 AML patients were studied: median age 55years, 53% male. Cytogenetic prognostic groups (SWOG) were favourable – 12%, intermediate – 59% and poor – 29%. 90 patients were treated with curative intent and 83% achieved CR, 6% had refractory disease and 11% died during induction. With a median follow-up of 33 months, the median LFS was 12 months and median OS was 24 months. Median pb-wt1 levels at diagnosis were 5122 (range, 1 to 36125) and varied significantly according to: cytogenetic risk group (good risk 12745, intermediate risk 2632, poor risk 4554 (P = 0.009)) and marrow blast percentage (P = 0.02). Diagnostic bm-wt1 showed similar correlations with baseline parameters, but did not reach statistical significance. Median pb-wt1 at relapse was 3781. One of 21 relapsed patients with elevated pb-wt1 expression at diagnosis did not express wt1 at relapse. In a multivariate Cox regression analysis model including age and cytogenetic risk-group, increased expression of wt1 from peripheral blood at diagnosis is predictive of decreased LFS (P=0.04). This correlation was not seen with wt1 measured from bone marrow aspirates. The wt1 expression levels were reduced following induction chemotherapy with a median 2.6 log reduction (range, 0 to 4.2) seen in marrow aspirate and 3.2 log reduction (range, 0 to 4.3) seen in peripheral blood. There was a trend for lower post-induction peripheral blood wt1 to correlate with better LFS when measured as a continuous variable (P=0.067) or undetected vs detected (P=0.051). Lower post-consolidation pb-wt1 significantly correlated with better LFS when measured as a continuous variable (P=0.002) or undetected vs detected (P=0.02) and continued to be significant in multivariate analysis. Bone marrow aspirate wt1 levels post-induction or post-consolidation did not correlate with LFS.This study demonstrates a correlation between wt1 expression levels and known risk factors for early relapse. In the MRD setting, detectable wt1 levels in peripheral blood post-induction and post-consolidation predicted very poor leukaemia free survival. Analysis is ongoing to validate these findings in a uniformly treated cohort of patients enrolled on the current ALLG clinical trial.No relevant conflicts of interest to declare.

Treatment Of Acute Promyelocytic Leukemia (APML) In The Jehovah's Witness (JW) Population.

CD123 (IL-3Rα) is a phenotypic marker of putative leukemic stem cells (LSC) in AML (Jordan, Leukemia2000;14:1777). We and others have found that CD34+38− cells from AML patients (pts) express high levels of CD123, in contrast to absence of expression on CD34+38− cells in normal individuals. Binding of CD123 by monoclonal antibody (mAb) 7G3 inhibits IL-3-dependent signalling and proliferation in vitro. In a NOD-SCID xenograft model 7G3 inhibits human AML engraftment, but not normal human hematopoiesis (Lock ASH2007; Abs161). CSL360, a recombinant chimeric IgG1 mAb derived from 7G3, binds the same epitope. CSL360 concentrations ≥ 0.1μg/mL in vitro inhibited 90% AML cell growth in the presence of supraphysiological IL-3 levels. Preclinical toxicology studies with doses up to 100 mg/kg weekly × 4 in cynomolgus monkeys showed no CSL360-related effects in clinical signs, hematology, chemistry, urinalysis, gross pathology or histopathology. A Phase 1 study of safety, pharmacokinetics (PK) and bioactivity of CSL360 in relapsed, refractory or high risk AML began in March 2007. Pts receive 12 weekly iv infusions if not withdrawn early due to treatment-related toxicity or disease progression. Additional treatments may be given to pts who achieve a response. Bone marrow aspirates/trephine samples are obtained at screening, after dose 3 and before doses 5 and 11. More than 180 infusions have been administered to 26 AML pts (21 M, 5 F; 17 de novo, 8 MDS-related, 1 treatment-related AML) in 5 dose level cohorts: 0.1, 0.3, 1.0, 3.0 and 10 mg/kg. There is no intra-patient dose escalation. PK parameters over the dose range, estimated in 19 pts over 7 days after doses 1 and 4, were linear with dose-proportional increases in the AUC and Cmax; dose 1 Cmax ranged from 0.62 – 287.33 μg/mL and dose 4 Cmax from 1.02 – 178.22 μg/mL. CSL360 mean plasma half-life (dose 1, 83 ± 33 h; dose 4, 117 ± 59 h) appears to be independent of dose and treatment number. Dose 1 systemic clearance (0.21 ± 0.16 L/h) and volume of distribution (0.39 ± 0.22 L/kg) were relatively low, consistent with this size IgG. In all pre-treatment samples anti-CSL360 antibody titers were negative, determined by enzyme immunoassay. Anti-CSL360 antibodies were detected post-treatment in 8/12 pts; these antibodies have not been fully quantified or characterised. CSL360 has been well tolerated; a MTD has not been defined. Seven pts received all 12 doses, 13 pts were withdrawn due to progressive disease or investigator’s decision, 3 pts were withdrawn in association with infections, 2 pts withdrew consent, and 1 pt is ongoing. Three serious adverse events have been considered possibly related or related to CSL360: 1 invasive fungal infection (Gr 5), and 2 infusion reactions (Gr 2; hospitalised). Other adverse events are consistent with expectations for the disease population. Of 8 pts in the 3 mg/kg and 10 mg/kg cohorts who are evaluable for response after ≥ 4 doses, 1 complete response (CR) has been observed. A 22 yr old male, de novo FAB M1 cytogenetically normal AML, who had relapsed post-2 allogeneic SCT, achieved a morphologic leukemia free state after 3 doses (3.0 mg/kg) and CR after 12 doses, sustained for > 9 weeks. The pt received 17 doses before withdrawal to treat co-morbidities. Flow cytometry studies with anti-CD123 antibodies demonstrated dose-dependent CSL360 coating of both AML blasts and LSC. Saturation of target antigen on marrow and blood cells was observed 1 day after dosing at 0.3mg/kg, associated with decreased expression of CD123 detected by an antibody to a different epitope. At higher dose levels saturation of CD123 was maintained 7 days post dosing, associated with ongoing reduction in surface CD123 expression. In a representative sample, plasma from a pt treated at 10 mg/kg specifically inhibited IL-3-induced proliferation of AML blasts ex vivo, indicating sufficient circulating concentration of CSL360 to inhibit IL-3 mediated effects in vivo. Effects of CSL360 on proliferation and apoptosis of AML cells in treated patients are being investigated. These preliminary results show anti-CD123 mAb therapy with CSL360 is safe and tolerable; biological effects have been demonstrated; a sustained CR was achieved in 1 advanced, refractory AML pt. The study is continuing, with 20 evaluable patients to be accrued and treated at 10 mg/kg weekly; at this dose level the PK and correlative assays predict that complete blockade of IL-3 signalling through CD123 can be achieved in vivo.

Aim. The optimal management of IST in renal transplant patients with PTLD is uncertain. As chemotherapy regimens used for PTLD are in themselves immunosuppressive, IST may not be required during this phase of treatment. Subsequent long-term reduction in IST is important to prevent relapse. We examined whether a protocol (instituted in 1994) of ceasing IST during chemotherapy for PTLD and recommencing IST at reduced doses after chemotherapy (calcineurin inhibitor at 50%, prednisolone <6mg daily and no third agent) was associated with deleterious effects on renal graft function. Methods. We performed a retrospective audit of adult renal transplant patients with PTLD requiring chemotherapy who were managed according to this protocol. Outcomes were compared (matched 1 case to 2 controls) to renal transplant patients without PTLD matched for age, gender, diabetes, graft type (cadaveric or live donor) and year of transplant. Matching was performed blinded to outcome. Results. 20 cases were identified: median age at PTLD diagnosis 39yrs; 85% male; 17 late onset PTLD; median time to onset of PTLD post-transplant 110 months (range, 5–276). 18 patients received prior triple combination IST (cyclosporine/tacrolimus plus azathioprine/mycophenolate mofetil plus prednisone). All patients received anthracycline-based chemotherapy with additional therapies of rituximab in 6 and radiation therapy in 4. 85% attained complete remission. 4 patients have relapsed. The 5yr OS was 75% with a median follow-up of 67 months from PTLD diagnosis. 6 cases (30%) had died (5 due to PTLD, 1 unknown) compared to 1 control (2.5%). Of the surviving 14 cases, 2 patients had failed renal allografts at 14 and 19yrs post-transplant and recommenced haemodialysis compared to 18 controls; neither case was retransplanted compared to 8 controls. 2 cases had >25% increment in serum creatinine but not requiring dialysis (assumed secondary to chronic allograft nephropathy) compared to 4 controls. 10 cases had normal functional allografts with no significant decrement in renal function. The cumulative rate of renal allograft failure requiring change of treatment to dialysis at 22yrs post-transplant was 34% vs 63% for cases and controls, respectively (p<0.05). Figure Figure Conclusions. IST can be safely ceased during chemotherapy for PTLD in renal transplant patients and recommenced at reduced doses after chemotherapy, without deleterious effects on renal graft function.

Background: Ocrelizumab is a new humanised anti-CD20 antibody with the potential for enhanced efficacy in non-Hodgkin’s lymphoma (NHL) compared with rituximab due to increased binding affinity for the low-affinity variants of the FcγRIIIa receptor. An open-label, multicentre, dose-escalation study was conducted to evaluate the safety, efficacy, pharmacokinetics and pharmacogenetics of ocrelizumab in patients (pts) with relapsed/refractory follicular NHL following prior rituximab-containing therapy. Methods: A total of 47 pts with a response (complete response [CR], unconfirmed CR [CRu], partial response [PR]) or stable disease (SD) of ≥6 months’ duration following prior rituximab-containing treatment were enrolled into 3 sequential dose cohorts. Pts received infusions of ocrelizumab q3w at 200 mg/m2 (cohort A), 375 mg/m2 (cohort B) and 750 mg/m2 (cohort C) for up to 8 doses. Response was assessed after 4 doses and after the end of treatment. Results: Fifteen pts were enrolled in cohort A, 16 in cohort B and 16 in cohort C; 60% were male; mean age was 57 years; 78% had stage III/IV disease. At study entry 30%, 50% and 67% had intermediate or high FLIPI score in cohorts A, B and C, respectively. Median number of prior therapies was 2 (range 1–7); time to progression after last rituximab-containing therapy of <12 months: 47% (cohort A), 31% (cohort B) and 62% (cohort C) of pts. The majority of pts reported ≥1 adverse event (AE) [80–100%, cohorts A-C]; most of these were grade 1/2, with only 6 pts experiencing a grade 3 AE and no grade 4 AEs observed. The most common AEs were infusion-related reactions (IRR; 73%, 75% and 69% in cohorts A, B and C, respectively); only one was grade 3. Two pts discontinued treatment due to toxicity (dyspnoea, IRR). A total of 17 pts responded to ocrelizumab for a response rate (RR) of 36% (13% CR/CRu) across all cohorts. RR by cohort was 27% (13% CR/CRu) for A, 50% (25% CR/CRu) for B and 31% (no CR/CRu) for C. In pts relapsing after prior response (CR/PR) to rituximab-containing therapy (n=40), RRs were 23% (15% CR/CRu), 62% (31% CR/CRu) and 33% (no CR/CRu) in cohorts A, B and C, respectively. Of 6 pts with SD after prior rituximab-containing therapy, 1 pt had a PR, 4 pts had SD and 1 pt progressed. Conclusion: Ocrelizumab is well tolerated at doses up to 750 mg/m2 given q3w; AEs consist mainly of grade 1/2 IRRs. Severe IRRs (grade 3/4) following ocrelizumab occur less frequently than with rituximab. In this heavily and rituximab-pretreated pt population, the RR of 36% is encouraging.

Background: ET carries a significant risk of thrombotic complications which are difficult to predict, with a past history of thrombosis being the main predictor of subsequent events. The JAK2V617F mutation (JAK2) and spontaneous erythroid colony growth (SEC) have been inconsistently associated with increased thrombosis in these patients. We aimed to assess the predictive value of these assays for the development of thrombotic events post-diagnosis in patients with ET. Methods: A retrospective review of consecutive patients with ET diagnosed according to WHO criteria between January 1998 and August 2006 was performed. Data was abstracted from the medical record regarding baseline demographic characteristic, vascular risk factors, therapy, occurrence of thrombo-haemorrhagic events, and transformation to AML or myelofibrosis. Thrombotic events were defined as arterial or venous events occurring at or 12 months prior to diagnosis (prior thrombosis) and at any time post diagnosis of ET (subsequent thrombosis). Investigators were blinded to JAK2 and SEC results at the time of data collection. SECs were performed at the time of diagnosis in 53 patients. JAK2 analysis was performed by an allele specific PCR method on peripheral whole blood (n=32) or archived bone marrow samples (n=29). Results: 62 patients with ET were identified: median age 60yrs; 53% female; median platelet count at diagnosis 873 × 109/L; 10,8 and 44 being low, intermediate and high clinical risk, respectively. 67% (41/61) had a positive JAK2 test and 47% (25/53) had a positive SEC assay. 8 (13%) and 6 (10%) had a history of prior arterial and venous thrombotic complications. Median follow up was 39 months (range, 1 to 137 months). At diagnosis JAK2 positive patients had higher white cell counts (12.3 vs 8.9 ×109/L, p=0.01) and neutrophil counts (9.1 vs 5.7 ×109/L, p=0.01), and a trend to older age (62 vs 57yrs, p=0.3) and higher haemoglobin (139 vs 133 g/L, p=0.12), but not platelet count (870 vs 892 ×109/L, p=0.8). There was a trend for increased rates of prior thrombosis among JAK2 positive patients (27% vs 5%, p=0.08). Survival was not affected by JAK2 status (5yr OS 71% vs 72%, p=0.7). The presence of the JAK2 mutation predicted for the development of subsequent thrombosis (5yr event rate 32% vs 0%, p=0.01) which persisted when the analysis was stratified for a prior history of thrombosis (p=0.01). Further analysis demonstrated the JAK2 mutation to predict subsequent arterial thrombosis (5yr event rate 22% vs 0%, p=0.05) but not venous thromboembolism (5yr event rate 11% vs 0%, p=0.18). The increased risk of arterial thrombosis in JAK2 positive patients persisted when corrected for a prior history of vascular events (p=0.04). The SEC assay was not found to be predictive of any thrombotic events. Conclusions: Patients with ET who are JAK2 positive are at increased risk of thrombotic complications, particularly arterial thrombosis. This increased risk persists after correction for a history of prior thrombosis.

Hyper-CVAD is a dose-intensive regimen which has been employed in the last decade to treat patients with a range of hematologic malignancies, with impressive efficacy. This regimen has been associated with considerable short-term hematologic toxicity, which has been mostly described as transient and reversible. However, recent reports have highlighted a concerning early rate of MDS or AML on medium-term follow-up. While cases of MDS/AML may be the most extreme end of the spectrum of hematopoietic stem cell damage, in the current retrospective study we describe other prolonged hematologic sequelae of this regimen. All patients were previously untreated and received Hyper-CVAD for the treatment of NHL or ALL. 125 patients (pts) with a median age of 47 (range, 15–76) were followed up for a median of 23 months (range, 1–84). The median number of Hyper-CVAD cycles was 6 (range, 2–9). Follow-up for blood counts was censored at the next cytotoxic therapy. At 3 months post therapy, 78 patients were evaluable. Thirty-two (41%) had achieved full blood count recovery and 45 (59%) had persisting cytopenias. Dose intensity delivered was significantly associated with persisting cytopenias (p = 0.028), but age (p = 0.61), underlying disease (0.49) and bone marrow involvement at diagnosis (p = 0.81) were not. Of the patients who had not normalised all counts by 3 months, anemia was present in 56% (median 106 g/L, range 76–119), neutropenia in 47% (median 1.27 × 109/L, range 0.03–1.75) and thrombocytopenia in 76% (median 96 × 109/L, range 9–120). One, two and three lineage cytopenias were present in 42%, 42% and 16% of pts, respectively. The median time to normalisation of counts for those with post-treatment cytopenias in the respective lineages was 9 months (range, 6–12) for Hb, 6 months (range, 6–30) for neutrophils, and 6 months (range, 6–30) for platelets. The median time to normalisation of the lowered counts was 9 months (n = 18) in those with multi-lineage cytopenias and 6 months (n = 12) in those with one lineage cytopenia. At 1 year following Hyper-CVAD with no further therapy, 49 patients were evaluable and 3 (6%), 7 (14%), and 9 (18%) still had anemia, neutropenia or thrombocytopenia respectively. MDS/AML was diagnosed in 3 patients at 4, 21, and 21 months after therapy. Of these patients, one received an autologous stem cell transplant 2 months after Hyper-CVAD, one was cytopenic at 3 months, and one was lost to follow-up until re-presenting with AML. These results indicate a considerable rate of prolonged hematologic toxicity after Hyper-CVAD, and a minor rate of MDS at this limited follow-up. These findings likely reflect cumulative damage to hematopoietic stem cells.

Background: Wilms’ tumour gene 1 (WT-1) is frequently over-expressed in adult AML and may be of prognostic value. We examined the prognostic utility of WT-1 expression in the peripheral blood, measured by RQ-PCR on diagnostic specimens, and characterised this compared to other clinical and biological predictors of survival. Methods: RQ-PCR was performed in triplicate on samples from newly diagnosed AML patients, using the Corbett Rotorgene 3000. Values were averaged and levels expressed relative to 104 ABL copies. Multivariate stratified Cox regression, with the stratification variable cytogenetic risk group and WT-1 levels analysed as a continuous variable, was used to model the outcomes of overall survival (OS) and event free survival (EFS). Results: Samples were collected from 58 patients with AML. The median age was 66 years (range 16–82) and 30 were male. Seven had antecedent haematological disorders and 9 had received prior chemo/radiotherapy. Induction therapy was with cytarabine based regimens in 18, high dose cytarabine regimens in 20, ATRA/chemotherapy in 6 and supportive care in 14. Complete remission was achieved in 64% with 18% induction mortality. Median OS was 4 months (17 months in responders) and 27% at 2 years. The median WT-1 transcript level in diagnostic AML samples was 5122 (range 9–36125), while in normal control samples it was 3 (n=11, mean 3.6, SD 1.6). In multivariate Cox regression, stratified by cytogenetic risk group, age (p<0.001), LDH (p<0.001) and performance status (p=0.04) retained prognostic significance for OS and EFS. Elevated peripheral blood WT1 levels were significantly associated with impaired OS (HR 1.24 per log10 transcript levels p=0.022) and EFS (HR 1.31 per log10 transcript levels p=0.0019). WT1 levels correlated with cytogenetic risk group (p=0.002 with higher levels seen in the favourable risk group), antecedent haematological disorder (p=0.05), peripheral blood blast percentage (p=0.05) and bone marrow blast percentage (p=0.01). WT1 levels were significantly higher in acute promyelocytic leukaemia (p=0.0001) compared to other AML subtypes. WT1 levels did not correlate with age (p=0.16), sex (p=0.12), prior chemo/radiotherapy (p=0.96), performance status (p=0.33) or LDH (p=0.91). Conclusions: WT1 RQ-PCR levels are frequently elevated in peripheral blood from patients with AML at diagnosis and are associated with subtype, cytogenetic risk group, blast percentage and antecedent haematological disorder. Within specified cytogenetic risk groups, elevated WT1 levels may predict AML patients at high risk of relapse and subsequent mortality.

Background: CBF AML, with t(8;21)(q22;q22), inv(16)(p13q22) or t(16;16)(p13;q22) and the associated fusion proteins AML1/ETO or CBFß/MYH11, has a favourable clinical prognosis although significant numbers of patients still relapse. We examined the prognostic utility of MRD monitoring by RQ-PCR and propose a simple model for prediction of impending haematological relapse. Methods: Patients with CBF AML had samples collected at diagnosis, after induction and consolidation chemotherapy and at routine regular intervals thereafter. RQ-PCR, using the Applied Biosystems 7700 Sequence Detection System, was performed in triplicate and the final result was calculated by averaging 3 values, expressed relative to PGK levels. Stratified Cox regression was used to assess the impact of predictor variables (diagnostic, post-induction, post-consolidation RQ-PCR levels and presence of a 1 log10 increase in sequential RQ-PCR levels) on leukaemia-free survival (LFS). Results: Of 46 patients identified with CBF AML, 29 had diagnostic, regular longitudinal samples and clinical follow up allowing further evaluation; 12 AML1-ETO and 17 CBFß-MYH11. The median age was 39 years (range 7–68) with 52% male. Median follow up was 34 months (range 1–106) and median sample number was 6 (2–15). Twelve relapses occurred at a median of 11 months (range 4–17) from diagnosis. There were significant differences between transcript levels at diagnosis (median 1.9), post-induction (8.96*10-04), post-consolidation (5.01*10-05), in remission (1*10-06) or relapse (0.15) (p=0.01). Diagnostic, post-induction and post-consolidation RQ-PCR levels did not predict outcome. A log10 rise in a remission bone marrow sample correlated with adverse LFS and imminent risk of haematological relapse (HR 8.6). Relapses occurred a median of 60 days (range 45–272) after a log10 rise. RQ-PCR levels Hazard Ratio (HR) LFS 95% CI P-value Diagnosis 0.22 0.02–2.9 0.25 Post-induction 1.3 0.8–2.1 0.36 Post-consolidation 0.8 0.5–1.2 0.27 1 log10 increase 8.6 1.8–42 0.008 Conclusions: A 1 log10 rise in transcript levels was a highly significant predictor of haematological relapse. Transcript levels at early post-treatment time points did not predict long term outcome, cautioning against de-escalation protocols based on these results. Prospective identification of high risk patients will enable clinical trials to address the efficacy of treatment initiated at molecular progression.

Background: Wilms' tumour gene 1 (WT-1) is frequently over-expressed in adult AML and may be of prognostic value. We examined the prognostic utility of WT-1expression in the peripheral blood, measured by RQ-PCR on diagnostic specimens, and characterised this compared to other clinical and biological predictors of survival.

Aim A prospective study to determine the safety of the accelerated delivery of rituximab. Methods A pilot study investigating the safety of the accelerated delivery of rituximab was commenced in September 2004. First doses of rituximab were delivered as per prescribing guidelines. Patients without infusion related toxicity were eligible for accelerated rituximab in second or subsequent infusions. In the first 4 patients the infusion was administered at 100mg/hr, increasing to 400mg/hr after 15 minutes in the absence of a reaction. All subsequent patients commenced the infusion at 400mg/hr. All patients received premedication with paracetamol, promethazine and hydrocortisone unless contra-indicated. Patients with a previous grade III/IV infusion related toxicity were excluded. Results 23 patients have been enrolled to date. Data is available for 62 infusions - median 3 infusions per patient (range: 1–6). Median age was 64 years (range: 28–85). 65% of patients were male. Diagnoses included non-Hodgkin’s Lymphoma - 20 patients (Diffuse Large Cell Lymphoma - 8, Follicular Lymphoma - 9, Mantle Cell Lymphoma - 1, Lymphoplasmacytic Lymphoma - 2), Immune Thrombocytopenic Purpura - 2 and Dermatomyositis-1. 44% of patients received single agent rituximab and 56% received it in combination with chemotherapy. The median dose of rituximab was 700mg (range: 600–800mg). There were 2 adverse events with no grade III/IV infusion related toxicity. One patient experienced grade I hypothermia. A second patient experienced grade II fevers and rigors which required interruption of the infusion. The patient was subsequently found to have pneumonia. The infusion was completed without further incident. Excluding data from the latter patient, median infusion time was 1 hour and 55 minutes and 76% of infusions were completed within 2 hours. Conclusion For second or subsequent infusions, the accelerated delivery of rituximab is safe and well tolerated allowing shorter outpatient stays and improving the efficiency of resource utilisation. The study has been extended to include patients with bulky lymphoma and Chronic Lymphocytic Leukaemia.

Aim Pegfilgrastim (Neulasta®) has proven efficacy as supportive therapy in a variety of chemotherapy regimens, but has not been studied in dose intensive, rapidly cycling regimens such as hyper-CVAD. We examined whether pegfilgrastim leads to similar kinetics of neutrophil recovery as daily G-CSF. Methods Using retrospective analysis, patients receiving pegfilgrastim were matched with controls (G-CSF) for cycle of chemotherapy, prior chemotherapy, dose of cytarabine received, age (<60 or >60), diagnosis (ALL or NHL) and bone marrow involvement. The primary endpoint was duration of grade IV neutropenia (ANC <500/ul). Secondary endpoints included time to neutrophil recovery, incidence of febrile neutropenia, positive blood cultures and delay in subsequent chemotherapy. Results Between 01/1999 – 06/2005 we identified 124 pegfilgrastim supported cycles in 43 patients. Cases were successfully matched to 124 G-CSF supported cycles from 38 controls. There were no significant differences between cases and controls with regards to prior chemotherapy, age, sex, cytarabine dose, diagnosis or bone marrow involvement. The median duration of grade IV neutropenia was 4 days in both groups (p =0.55). Pegfilgrastim (95% CI) G-CSF controls (95% CI) P value Grade IV neutropenia - All cycles 4 days (0–11) 4 days (0–10) 0.55 - A cycles 2 days (0–7) 2 days (0–6) 0.65 - B cycles 6 days (2–12) 6 days (0–10) 0.70 Time to ANC >500/ul 13 days (10–19) 14 days (8–18) 0.75 Time to ANC >2000/ul 14 days (11–29) 14.5 days (9–22) 0.27 Febrile Neutropenia 29% 38% 0.16 Positive Blood Cultures 11% 12% 0.85 Delay in next cycle 44% 47% 0.75 Conclusions A single dose of pegfilgrastim is as effective as daily G-CSF for supporting the hyper-CVAD chemotherapy regimen.

The Hyper-CVAD chemotherapy regimen (JCO2000;18:547) is being increasingly applied to a number of haematological malignancies. We have previously demonstrated Hyper-CVAD to be a highly stem cell toxic regimen which adversely affects peripheral blood stem cell (PBSC) mobilisation and that mobilising efficiency can be significantly improved by collecting PBSC earlier in the treatment course. We now examine the optimal timing of PBSC collection when using this regimen. A retrospective analysis was performed at two centres of consecutive patients (pts) treated with Hyper-CVAD in whom an attempt was made to collect PBSC. The regimen consists of alternating cycles of: (A) cyclophosphamide 300mg/m2 q12hrs for 6 doses d1-3, vincristine 2mg d4&11, doxorubicin 50mg/m2 d4 and dexamethasone 40mg daily d1-4 and d11-14; and (B) methotrexate 1g/m2 over 24 hours d1 and ara-C 1–3g/m2 q12hrs for 4 doses d2&3. PBSCs were collected on recovery from chemotherapy using G-CSF in a daily dose of 10mcg/kg. Target cell number for optimum engraftment was defined as CD34 ≥5 x106/kg, while the minimum threshold for proceeding to ASCT was CD34 ≥2 x106/kg. Pts were analysed according to the timing of mobilisation. 74 pts were identified: median age 44yrs; 69% male; 53% with initial marrow involvement; diagnoses (ALL n=14, lymphoblastic lymphoma n=7, Burkitt’s/Burkitt-like lymphoma n=8, aggressive NHL n=34, mantle cell lymphoma n=11). 28 pts were mobilized off the back of cycle A, 32 pts off the back of cycle B, and 14pts after other mobilising regimens. Overall 66% and 42% of pts obtained a minimum and optimal PBSC collection, respectively. Only 3 of 17 patients (18%) collected beyond cycle 3B successfully mobilised. Two patients mobilised prior to 3B but receiving alternate mobilising chemotherapy were not analysed. The results of stem cell collection on the 55 remaining patients are shown below. Stem cell collection results following hyper-CVAD chemotherapy Timing of stem cell mobilisation Cycle 1B 2A 2B 3A N 13 21 14 7 Total CD34+ (range) 21.36 (4.2–69.1) 3.41 (0–11.18) 5.1 (0–22.3) 4.6 (1.9–8.6) Day of 1st apheresis 12 (11–13) 13.5 (11–16) 13.5 (12–19) 15 (13–19) % with CD34+≥2 x106/kg All patients 100% 81% 64% 86% Obtained in 1 apheresis 92% 19% 36% 14% % with CD34+≥5 x106/kg All patients 92% 38% 50% 39% Obtained in 1 apheresis 77% 0% 21% 0% Cycle 1B compared to 2A was not significantly better in obtaining a minimum graft (100% vs. 81%, p=0.14), but was superior in terms of total CD34+ yield (21.4 vs. 3.4x106/kg, p<0.001) and in achieving both a minimum (92% vs. 19%, p<0.001) and optimal (77% vs. 0%, p<0.001) PBSC collection with a single apheresis. There were no significant differences in PBSC yields following cycles 2A, 2B and 3A. The dose of ara-C used in cycle B had no effect on the subsequent stem cell yield. Hyper-CVAD is a highly stem cell toxic regimen. The mobilizing efficiency can be significantly improved by collecting PBSC earlier in the treatment course with cycle B appearing to be the superior mobilising arm. Mobilisation following cycle 1B allows the most predictable timing of PBSC collection and provides a significantly better PBSC graft and should be considered in those in whom marrow involvement has cleared.