Background For fit patients (pts), combining kinase inhibitors (midostaurin, quizartinib) with intensive chemotherapy is standard of care for FLT3mutated AML. New studies also suggest a role for kinase inhibitors (e.g. quizartinib) in modifying disease history in pts with FLT3wild-type AML (Montesinos et al, EHA 2023). Clonal evolution of kinase-activating mutations, including FLT3-ITDis an important mechanism of treatment failure in pts with non-adverse (NON-ADV) cytogenetic risk AML receiving frontline therapy with azacitidine and venetoclax (AZA-VEN) (DiNardo and Tiong et al, Blood 2020). Incorporating kinase inhibitors (e.g. gilteritinib) into less-intensive VEN-based regimens in unfit, older pts has been challenging, with cumulative myelosuppression a dominant issue (Short et al, JCO 2024). Gilteritinib is known to have a long half-life (~113 hrs), which may contribute to its negative impact on marrow recovery when directly combined with chemotherapy and VEN. We hypothesized that myelosuppression could be lessened by using a FLT3 inhibitor with a short half-life (e.g. midostaurin, MIDO) administered sequentiallyafter, rather than concurrently with chemotherapy. The phase 1b/2 INTERVENE study was conducted across 19 sites in Australia and New Zealand to establish the safety and efficacy of a triplet regimen involving low dose cytarabine (LDAC), VEN and MIDO (LVM) in older unfit pts with NON-ADV AML. A phase 1b dose-finding study confirmed safety in 18 pts and determined the recommended phase 2 dose (RP2D) of LVM to be LDAC 20 mg/m2 SQ D1-10, VEN D1-28 with dose ramp-up to 600mg D and MIDO 50 mg BD D11-28 (Chua et al, ASH 2022). INTERVENE was designed to seamlessly transition to a randomized phase 2 expansion comparing the RP2D of LVM with LDAC+VEN (LV), the focus of the current report. Methods Pts aged ≥60 years (y) unfit for intensive chemotherapy with treatment naïve NON-ADV cytogenetic risk AML (excluding APL or core-binding factor) were randomised 2:1 to receive LVM or LV. Posaconazole antifungal prophylaxis was permitted with dose adjustment of VEN to 50 mg daily and MIDO to 50 mg daily due to increased risk of cardiac toxicities in older populations. The primary endpoint was CR/CRi by end of C4 utilizing dual criteria for proof of concept. Secondary endpoints included other measures of response, duration of response and survival. Exploratory endpoints included FLT3-ITD measurable residual disease (MRD) by PCR-NGS (sensitivity 10-5). First patient was 10MAR2021, with data cut-off 31MAY2024. Results 124 pts were randomized with 117 commencing study therapy (76 LVM, 41 LV). Median follow up was 13.4 months. Median age was 74y (range 60-89, 81% ≥70y) and 66% were male. In the LVM vs LV arms, secondary AML was present in 38% vs 26%, prior AZA exposure in 14% vs 3%, adverse European LeukemiaNet 2022 risk in 53% vs 56%, FLT3-ITD in 27% vs 23% and FLT3-TKD in 3% vs 3%, respectively. Overall, 30-day mortality was 6% (4% LVM vs 10% LV): 4 due to infections, 3 due to progressive AML. In the LVM and LV arms, the most frequent non-hematological adverse events in cycle 1 (C1) were nausea (47% vs 22%, G3+ 1% vs 0%), constipation (29% vs 0%, no G3+) and cardiac symptoms (7% vs 5%, G3+ 4% vs 0%). G3 febrile neutropenia occurred in 28% vs 17%. Regarding deliverability, the median number of cycles received was 5.5 (range 1-24) for LVM and 4 (range 1-31) for LV. For pts achieving CR/CRi/CRh, the proportion receiving >6 cycles in the LVM and LV arms was 62% vs 56%, including 38% vs 36% receiving >12 cycles, respectively. For pts in remission, the proportion with delayed treatment (commencing next cycle >day 42) in LVM vs LV were 24% vs 7% at the end of C1, declining to 13% vs 13% at the end of C6, and 11% vs 9% at the end of C12, respectively, with no evidence of cumulative delays after successive treatment cycles. Of 96 patients dosed and completing at least 4 cycles of therapy or off therapy prior to C4, overall response rates (ORR: CR/CRi) were 70% in the LVM arm and 64% in the LV arm. The final analysis of response will be presented at the conference. Among pts with FLT3mutation, ORR in the LVM and LV arms were 86% vs 50%, respectively. FLT3-ITD MRD clearance was observed in 9/15 (60%) in the LVM arm and 1/4 (25%) in the LV arm. Conclusion In unfit, older pts ≥60 years with newly diagnosed NON-ADV cytogenetics AML, LDAC-VEN-MIDO, with sequential delivery of MIDO after LDAC was well tolerated, with promising clinical and molecular responses in FLT3 mutated AML.

Background Adaptive resistance mechanisms leading to treatment failure in older patients (pts) receiving frontline venetoclax (VEN) combination therapies include emergence/expansion of kinase activating mutations (principally, FLT3-ITD) in pts with non-adverse (NON-ADV) karyotype AML. In both the RATIFY and QuANTUM-First trials, the FLT3 inhibitor was scheduled sequentially after chemotherapy, with no evidence that this approach augmented marrow toxicity. We conducted the Phase 1b/2 study, INTERVENE, to explore the deliverability and preliminary efficacy of a novel triplet regimen incorporating the FLT3/multi-kinase inhibitor midostaurin (MIDO) delivered sequentially after low dose cytarabine (LDC), in tandem combination with VEN. We hypothesized that this triplet schedule would suppress emergence of kinase-activating mutations on VEN-based therapy. MethodsEligibility: Pts with treatment naïve AML (excluding APL), aged ≥60 years and unfit for intensive chemotherapy. Prior hypomethylating agents for antecedent myeloid neoplasms were permitted. Pts with NON-ADV karyotype were enrolled to receive VEN-LDC-MIDO. Treatment (safety run-in): LDC 20 mg/m2 SC D1-10, MIDO 50 mg BD commenced on D11 and continued to D28. After a dose ramp-up in cycle 1 (C1), VEN D1-28 was administered at two dose levels (1= 400 mg, 2= 600 mg). Azole antifungals were prohibited during C1 but allowed from C2 with VEN dose reduced to 100 mg. Molecular studies: NGS using a custom 48-gene Roche KAPA HyperCap panel (sensitivity 1%) and FLT3-ITD PCR-NGS (sensitivity 10-4-10-5) were performed on serial bone marrow samples. RT-qPCR was used to detect NPM1 measurable residual disease (MRD) (sensitivity 10-6). Results First pt enrolled: 7SEP2020. Data cut-off: 31MAY2022. Eighteen pts were enrolled to receive VEN-LDC-MIDO in the run-in phase. Median age was 77 years (range 73-87, 84% age≥75). 44% had secondary AML, including 11% t-AML. 2 pts withdrew consent within the first 7 days due to non-therapy related reasons and were not response evaluable. 30-day mortality was 0%. There were no dose limiting toxicities observed. With a median follow up of 18 months (m), median overall survival (OS) was not reached (95% CI 8.02-NR). Overall response rate (CR+CRi+CRh) by intention-to-treat was 77.8%, with 44.4% CR. Median relapse free survival was 11.7m. To assess treatment deliverability, we evaluated the inter-cycle time for the first 12 cycles among pts achieving CR/CRi/CRh (Fig 1A). The median inter-cycle times ranged from 28-35 days, with majority commencing the next cycle in <42 days. There was no evidence of cumulative lengthening of inter-cycle time with successive treatment cycles, suggesting that MIDO did not promote marrow toxicity when combined with VEN-LDC. Pts received a median number of 9 cycles (range 1-16), with 7 completing ≥12 cycles (Fig 1B). Of these, 3 electively ceased therapy (at C12, C14, C16), with a treatment-free remission (TFR) lasting 3.3-5.7m, from which one relapsed. For pts opting for therapy continuation (TC) >12m (4/7), one has relapsed. Of the remaining 5 (2 TFR, 3 TC) pts with durable remissions, 4 had either NPM1 or IDH2 mutations at enrolment and all achieved undetectable MRD in CR. One additional patient with NPM1 mutationat diagnosis relapsed after C5 with NPM1 negative AML. Molecular data and treatment outcomes are shown in Fig 1B. Two of 16 response-evaluable pts had FLT3 mutations at baseline (1=FLT3-ITD, 1=TKD). Using PCR-NGS to enhance detection of FLT3-ITD microclones, an additional 3 FLT3-ITDs were identified in 2 pts. During VEN-LDC-MIDO, all 4 FLT3-ITDs were suppressed to undetectable levels by PCR-NGS. No pts relapsed with FLT3 variants at the time of progression. Other kinase variants extinguished by VEN-LDC-MIDO include NRAS G12D and KRAS G12D. In contrast, 4 pathways of adaptive resistance were observed at time of treatment failure: emergence of 1) TP53 hotspot variants (N=2), 2) PPM1D frameshift variants (N=2), 3) BAX inactivating variant (N=1), and 4) evolution of KRAS Q61 mutations (N=2), Conclusion After a median follow up of 18m, we report that VEN-LDC-MIDO, with MIDO delivered sequentially after LDC, is tolerable with excellent treatment deliverability. Preliminary efficacy of this triplet is promising, with molecular evidence of FLT3-ITD suppression in all cases so far. A randomized phase 2 comparison of VEN-LDC-MIDO versus VEN-LDC is ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract BACKGROUND Survival rates in multiple myeloma (MM) have significantly improved in recent decades with the advent of high-dose chemotherapy conditioned autologous stem cell transplantation (ASCT) and the availability of novel agents for induction therapy (Kumar SK et al. Blood 2008). Failure to respond to front-line bortezomib-based induction therapy remains a significant clinical challenge in transplant eligible (TE) newly diagnosed multiple myeloma (NDMM), and is associated with poor outcomes with shortened progression free survival (PFS) and overall survival (OS) (Lee SE et al. Ann Hematol. 2014). In combination with immunomodulatory agents (IMiDs), carfilzomib, a second generation proteosome inhibitor, has been shown to be highly effective in the context of MM induction with high rates of negativity for minimal residual disease (MRD) and few dose limiting toxicities (Langren O et al. Leukemia 2019). The ALLG MM17 trial is a multicentre single arm study of carfilzomib-thalidomide-dexamethasone (KTd) in TE NDMM patients refractory or with suboptimal response to bortezomib-based induction therapy, designed to evaluate the efficacy of early response adaption with a switch to an intensive salvage strategy. METHOD Eligible patients included those with TE NDMM, aged 18 years and older, demonstrating sub-optimal response to bortezomib-based induction therapy (failure to achieve a minimal response after 2 cycles, partial response [PR] after 4 cycles, or disease progression within 60 days of completing induction). Salvage therapy consisted of 100mg daily oral thalidomide, with 20 mg of oral dexamethasone and 20mg/56mg of IV carfilzomib on days 1, 2, 8, 9, 15, and 16, with of each 28-day cycle. Following 4 cycles, patients in stringent complete response (sCR) proceeded to melphalan conditioned ASCT whereas those in less than sCR received a further 2 cycles of KTd prior to ASCT. Consolidation therapy consisted of a further 2 cycles of KTd, followed by maintenance 100mg daily thalidomide and 40mg weekly dexamethasone (Td) continuing until progressive disease, unacceptable toxicity, or 12 months of therapy. Primary objectives were to determine the overall response rate (ORR) and safety profile of treatment with KTd salvage therapy, with secondary objectives to determine the maximal depth of response, progression free survival (PFS), and overall survival (OS) achieved with sequential treatment with KTd salvage, ASCT, post-ASCT consolidation, and maintenance Td therapy. Efficacy assessments were performed via serum protein electrophoresis, serum free light chain and bone marrow evaluation. Next generation flow (NGF) cytometry MRD evaluation of bone marrow aspirate was undertaken pre-ASCT, at day 100 post-ASCT, after 2 cycles of consolidation KTd, and following completion of Td using standardized 8-colour EuroFlow platform. RESULTS 50 patients were recruited across 6 Australian sites between September 2016 and April 2018. Overall response rate to KTd salvage was 78% (Credible Interval 95%: 64.4-87.1%), with dual proof of concept criteria met (observed ORR ≥ 50% and posterior probability that the true ORR exceeds 30% is ≥ 0.90). Response rates included 12% sCR, 6% CR, 38% VGPR, and 22% PR. Sixteen patients discontinued treatment (32%) including 10 cases (20%) of progressive disease, and 2 patient deaths without progression. NGF MRD negativity was found to be 32%, 36% and 55% at the pre-ASCT, post-ASCT and post-consolidation time-points. At the cut-off date, estimated median follow-up for disease status was 38.6 months and median PFS and OS had not been reached. At 36 months PFS and OS were 63.9% (95%CI: 49.0 - 75.5%) and 79.9% (95%CI: 65.8 - 88.6%) respectively (Figure 1). KTd was found to be well tolerated with 44% of patients experiencing a grade 3 of higher adverse event (AE). Most common AEs included upper respiratory infection (48%), peripheral neuropathy (36%), musculoskeletal pain (32%), dyspnoea (28%), fatigue or lethargy (28%), and constipation (28%). Significant cardiac toxicity was not observed at this higher dose level of carfilzomib. CONCLUSION Results demonstrate that response-adaptive utilisation of KTd salvage, ASCT, and consolidation therapy induces high response rates, improving depth of response with high levels of sequential MRD negativity, and durable responses with an acceptable toxicity profile in TE NDMM patients failing bortezomib-based induction therapy. Figure 1 Figure 1. Disclosures Quach: Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen/Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Antengene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy, 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, Research Funding; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kalff: Amgen: Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Pfizer: Honoraria; Janssen: Honoraria; Roche: Honoraria; CSL: Honoraria; Sandoz: Honoraria. Bergin: Amgen: Other: Travel to workshop; Celgene: Consultancy. Reynolds: Novartis AG: Current equity holder in publicly-traded company; Alcon: Current equity holder in publicly-traded company; Abbvie: Research Funding. Spencer: Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Takeda: Honoraria, Research Funding, Speakers Bureau; STA: Honoraria.

Abstract Background: Adaptive resistance mechanisms leading to treatment failure have been identified in older patients receiving venetoclax (VEN) in combination with either azacitidine or low dose cytarabine (LDAC) as frontline therapy for acute myeloid leukemia (AML). These include the expansion or secondary emergence of kinase activating mutations, including FLT3-ITD in patients with non-adverse karyotype (NON-ADV), as well as TP53 mutations among patients with adverse karyotype (ADV)(DiNardo & Tiong et al, Blood 2020). INTERVENE is a phase 2 study evaluating the safety and efficacy of the "risk-stratified" addition of a novel third agent to VEN-LDAC, delivered in tandem to LDAC to minimize the risk of myelotoxicity (Figure 1A). To mitigate VEN resistance associated with activated kinases in NON-ADV risk AML, midostaurin (MIDO), a FLT3/multi-kinase inhibitor, was incorporated in combination with VEN. To address VEN resistance associated with TP53 defects in ADV risk AML, a HDAC inhibitor pracinostat (PRAN) was incorporated in accordance with pre-clinical studies suggesting synergistic induction of TP53 independent cell death with VEN plus HDAC inhibition (Salmon et al, ASH 2018). We hereby report the results of the dose-finding safety run-in phase of the study. Methods: Eligibility: Patients with treatment naïve AML (excluding APL), aged ≥60 years and unfit for intensive chemotherapy were included. Prior hypomethylating agents for antecedent myeloid neoplasms were permitted with a 14-day washout. Patients were stratified according to cytogenetic risk, as per Medical Research Council 2010 criteria. Treatment: VEN D1-28 (with dose ramp-up in cycle 1) was combined with LDAC (20mg/m 2 SC D1-10), with the third agent starting after/on the last day of LDAC (Fig 1A). Each cycle was 28 days. In the NON-ADV stratum (VEN-LDAC-MIDO), 2 dose levels were explored: (L1) VEN 400mg + LDAC + MIDO 50mg BD D11-28; (L2) VEN 600mg + LDAC + MIDO 50mg. In the ADV stratum (VEN-LDAC-PRAN), 3 dose levels were tested: (L1) VEN 400mg + LDAC + PRAN 45mg starting D10 and given 3x/week orally for a total of 9 doses; (L2) VEN 600mg + LDAC + PRAN 45mg; (L3) VEN 600mg + LDAC + PRAN 60mg. Azole antifungals were prohibited in cycle 1 but allowed from cycle 2 with VEN dose modification. Endpoints (safety run-in): Primary: occurrence of dose-limiting toxicities (DLT) during cycle 1 and determination of recommended phase 2 doses (RP2D) using a Bayesian Logistic Regression Model. Secondary: Preliminary response rates. Molecular studies: Next generation sequencing using a custom 48-gene Roche KAPA HyperCapture myeloid panel and FLT3-ITD targeted amplicon sequencing were performed on baseline bone marrow samples. First patient enrolled: 7SEP2020. Data cut-off: 29JUN2021. Results: 32 patients were enrolled: 18 in NON-ADV and 14 in ADV strata, respectively. Two patients in the NON-ADV stratum withdrew within the first 7 days due to non-therapy related reasons (1=personal, 1=incidental lung lesion) and were not DLT/response evaluable. Median age was 77 years (68-87; 69% ≥75 years). 43.8% (14/32) had secondary/therapy related AML. Although gastrointestinal adverse events (AE) during cycle 1 were more common in VEN-LDAC-PRAN arm with nausea (57 vs 17%), vomiting (36% vs 6%) and diarrhea (50% vs 22%), grade 3+ toxicities were uncommon (0-7%)(Table 2). Occurrence of febrile neutropenia was similar between the two arms. 30-day mortality was 0% and 14% (2/14: 1=infection, 1=disease progression) for NON-ADV and ADV strata, respectively. No DLTs were observed in either stratum across all dose levels, thus the RP2D was the highest dose level explored for both triplet combinations. The intention-to-treat overall response rate CR+CRi+CRh was 72.2% (13/18) in the NON-ADV arm and 57.1% (8/14) in ADV arm. The expanded response rate including PR and MLFS was 77.8% (14/18) and 71.4% (10/14) in the NON-ADV and ADV strata, respectively. Median time to best response was 1 cycle (range 1-6). Updated response and survival outcomes will be presented at the meeting. Conclusion: The addition of MIDO or PRAN to VEN-LDAC was tolerable in older/unfit patients with treatment naïve AML. Preliminary efficacy with this risk-stratified approach compared favorably to prior studies with VEN-LDAC alone (Wei et al Blood 2020: CR+CRi 56% in NON-ADV, 28% in ADV). The randomized phase 2 part of this tandem triplet strategy with the goal of preventing adaptive resistance is underway. Figure 1 Figure 1. Disclosures Chua: Abbvie: Other: Conference travel and accommodation . Reynolds: Alcon: Current equity holder in publicly-traded company; Abbvie: Research Funding; Novartis AG: Current equity holder in publicly-traded company. Enjeti: Astra Zeneca: Honoraria; Sanofi: Honoraria; AbbVie: Honoraria; Roche: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hiwase: AbbVie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Marlton: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: 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; F. Hoffmann-La Roche Ltd: 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; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Queensland Health: Current Employment; BeiGene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bajel: Abbvie, Amgen, Novartis, Pfizer: Honoraria; Amgen: Speakers Bureau. Grove: Astellas: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Cooney: Amgen: Other: Travel, accommodation, expenses ; Roche: Other: Travel, accommodation, expenses ; Novartis: Other: Online conference registration . Beligaswatte: Astellas: Membership on an entity's Board of Directors or advisory committees. Anstee: Walter and Eliza Hall Institute: Patents & Royalties: Dr Anstee was a former employee of the Walter and Eliza Hall Institute and is eligible for a fraction of the royalty stream related to Venetoclax. Perera: Abbvie: Speakers Bureau; BMS: Speakers Bureau. Ritchie: Takeda: Research Funding; BMS: Research Funding; Novartis: Honoraria; CRISPR Therapeutics: Research Funding; Amgen Inc: Honoraria, Research Funding; CSL: Honoraria. Wei: Genentech: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees; Macrogenics: Membership on an entity's Board of Directors or advisory committees; Roche: 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; Astellas: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Amgen: 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, Research Funding, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: This presentation will focus on the ALLG INTERVENE clinical trial combining venetoclax+LDAC+midostaurin or venetoclax+LDAC+pracinostat. Although venetoclax and midostaurin are individually FDA-approved in some indications, the combinations examined in this clinical trial have not been approved by FDA.

Abstract Background Data from the Australian and New Zealand (ANZ) Myeloma and Related Diseases Registry (MRDR) shows that 85% of newly diagnosed multiple myeloma (NDMM) patients (pts) in ANZ are induced with bortezomib(V)-containing therapies, predominantly triplets of V-cyclophosphamide-dexamethasone (VCD). Of these, 15% demonstrate treatment failure - either a sub-optimal response ( Methods MM17 was a multi-centre single arm study sponsored by the ALLG. Eligible pts were TE NDMM undergoing pre-autologous stem cell transplant (ASCT) induction with V-based therapy and demonstrating either SOR (defined as Results Fifty pts were recruited from 6 Australian sites between September 2016 and April 2018. EMC92 stratification was successful in 21 pts with 10 (48%) being high-risk and with cfDNA successfully obtained from 49 pts and currently undergoing baseline TAS. Data cut-off date was July 18 2018 with 39 pts evaluable for the primary end-point with the reverse-Kaplan-Meier estimate of the median potential follow-up for survival being 10.9 months (95% CI: 6.0 - 13.1 months). Median age was 50 years (36-71) with 72% males. Disease status at study entry was SOR in 26 (66%) (< MR n = 13, < PR n = 13) and 1REF in 13 (33%). The median number of pre-ASCT KTd cycles was 6 (6 cycles, n = 27 [69%]; 5 cycles, n = 1 [3%], 4 cycles, n = 5 [13%]; ≤ 3 cycles, n = 6 [15%]). Two pts were withdrawn due to treatment related toxicity - pulmonary arterial hypertension (n=1) and acute renal failure (n=1). One pt died of sepsis on treatment and one was withdrawn and subsequently died due to a second primary malignancy. ORR was 72% (95% CI: 56-83%)* - sCR 13%, CR 5%, VGPR 36% and PR 18%. Euroflow confirmed MRD negativity in 36% (14 of 39) of pts pre-ASCT and in 43% (12 of 28) at day 100 post-ASCT. Eight pts have progressed, 7 with highly aggressive extra-medullary disease. Neither median PFS (left panel) nor OS (right panel) have been reached. Conclusions This preliminary analysis of the ALLG MM17 trial demonstrates that early response adaptive escalation to KTd results in high response rates, including MRD negativity, in patients failing V-based induction therapy. *95% Credible interval from the posterior distribution. Bayesian updating based on observed data and a minimally informative prior for ORR with a median of 35%. Figure. Figure. Disclosures Spencer: Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen-Cilag: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Research Funding; BMS: Research Funding; Takeda: Honoraria, Research Funding, Speakers Bureau; STA: Honoraria. Quach:Sanofi Genzyme: Research Funding; Janssen Cilag: Consultancy; Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Kalff:Janssen: Honoraria; Amgen: Other: travel to preceptorship; Celgene: Honoraria; Takeda: Honoraria. Bergin:AMGEN: Other: Travel to education meeting; Celgene: Consultancy. Reynolds:Novartis: Equity Ownership, Other: former employee of Novartis AG and holds stock in the company. .

Tumor necrosis factor (TNF) is a key cytokine in the effector phase of graft-versus-host disease (GVHD) after bone marrow transplantation, and TNF inhibitors have shown efficacy in clinical and experimental GVHD. TNF signals through the TNF receptors (TNFR), which also bind soluble lymphotoxin (LTalpha3), a TNF family member with a previously unexamined role in GVHD pathogenesis. We have used preclinical models to investigate the role of LT in GVHD. We confirm that grafts deficient in LTalpha have an attenuated capacity to induce GVHD equal to that seen when grafts lack TNF. This is not associated with other defects in cytokine production or T-cell function, suggesting that LTalpha3 exerts its pathogenic activity directly via TNFR signaling. We confirm that donor-derived LTalpha is required for graft-versus-leukemia (GVL) effects, with equal impairment in leukemic clearance seen in recipients of LTalpha- and TNF-deficient grafts. Further impairment in tumor clearance was seen using Tnf/Lta(-/-) donors, suggesting that these molecules play nonredundant roles in GVL. Importantly, donor TNF/LTalpha were only required for GVL where the recipient leukemia was susceptible to apoptosis via p55 TNFR signaling. These data suggest that antagonists neutralizing both TNF and LTalpha3 may be effective for treatment of GVHD, particularly if residual leukemia lacks the p55 TNFR.

Donor treatment with granulocyle-colony stimulating factor (G-CSF) is known to modulate immune function, characterized by the generation of regulatory myelogenous and T cell populations and Th2 differentiation. Recently, these effects have been shown to be enhanced by pegylation of the G-CSF molecule, which also improves graft-versus-leukemia (GVL) via activation of invariant natural killer (iNK) T cells. We have compared G-CSF bound to a single PEG molecule (monopeg-G-CSF) as used clinically to a G-CSF molecule bound to multiple PEG molecules (multipeg-G-CSF) in major histocompatibility complex (MHC) disparate and matched models of graft-versus-host disease (GVHD) and GVL. We demonstrate that multipeg-G-CSF induces greater levels of progenitor cell, myelogenous, and iNKT cell expansion than monopeg-G-CSF, while inducing similar protection from GVHD. Despite this, multipeg-G-CSF enhanced CTL function in vivo and improved iNKT cell-dependent leukemia clearance. Thus, GVL and GVHD can be further separated after allogeneic stem cell transplantation by mobilization with a multiple-pegylated G-CSF molecule.

Granulocyte colony-stimulating factor (G-CSF) is used to accelerate neutrophil engraftment in bone marrow transplant (BMT) recipients to reduce bacterial infections but may also enhance the risk of graft-versus-host disease (GVHD). Morris et al . now show that total body irradiation increases the expression of the G-CSF receptor on recipient dendritic cells, resulting in the activation of donor natural killer T cells and enhanced GVHD when G-CSF is administered shortly after BMT ( pages 363–364 ).

Invariant natural killer T cells (iNKT cells) have pivotal roles in graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects. iNKT cells are activated through their T-cell receptors by glycolipid moieties (typically the alpha-galactosylceramide [alpha-GalCer] derivative KRN7000) presented within CD1d. We investigated the ability of modified alpha-GalCer molecules to differentially modulate alloreactivity and GVL. KRN7000 and the N-acyl variant, C20:2, were administered in multiple well-established murine models of allogeneic stem cell transplantation. The highly potent and specific activation of all type I NKT cells with C20:2 failed to exacerbate and in most settings inhibited GVHD late after transplantation, whereas effects on GVL were variable. In contrast, the administration of KRN7000 induced hyperacute GVHD and early mortality in all models tested. Administration of KRN7000, but not C20:2, was found to result in downstream interleukin (IL)-12 and dendritic cell (DC)-dependent natural killer (NK)- and conventional T-cell activation. Specific depletion of host DCs, IL-12, or donor NK cells prevented this pathogenic response and the induction of hyperacute GVHD. These data demonstrate the ability of profound iNKT activation to modulate both the innate and adaptive immune response via the DC-NK-cell interaction and raise concern for the use of alpha-GalCer therapeutically to modulate GVHD and GVL effects.

G-CSF is often used to hasten neutrophil recovery following allogeneic bone marrow transplantation (BMT), but the clinical and immunological consequences invoked remain unclear. We examined this in murine models and found that administration of both standard G-CSF and pegylated G-CSF early after BMT significantly increased graft-versus-host disease (GVHD). This effect was seen in the B6 → B6D2F1, BALB/c → B6 and C3H.SW → B6 systems of GVHD to either MHC or multiple minor histocompatibility antigens. This effect was dependent on total body irradiation (TBI) rendering host dendritic cells (DC) responsive to G-CSF by up-regulating their expression of the G-CSF receptor as determined by real-time PCR. This induction of G-CSFR expression was not seen following busulfan (Bu), cyclophosphamide (Cy) or fludarabine. The enhanced GVHD was present when G-CSF was administered to both WT and G-CSFR−/− donors but not G-CSFR−/− recipients, confirming that host signalling was critical for this effect. G-CSF administration after BMT had no effect on inflammatory cytokine generation but enhanced in vivo CTL activity after BMT when administered to WT but not G-CSFR−/−, CD1d−/−, IFNgR−/− or CD40−/− recipients. Furthermore, donor iNKT cell activation was absent in CD11c Diptheria Toxin Receptor recipient transgenic mice depleted of dendritic cells (DC) by diphtheria toxin and treated with G-CSF. Thus, stimulation of host DC by G-CSF subsequently unleashed a cascade of events characterized by CD1d dependent donor iNKT cell activation, IFNg secretion and CD40-dependent amplification of donor CTL function during the effector phase of GVHD. Critically, the detrimental effects of G-CSF on GVHD were present when administered early following TBI conditioning and at a time when residual host APC were still present (day +1), but had no effect when administered at day +8 when host DC were not detectable by phenotypic or functional analysis. This is consistent with the inefficient cross presentation of host Ag within MHC class I by donor DC after BMT. In addition, the administration of G-CSF after Bu/Cy conditioning had no effect, perhaps explaining the conflicting and somewhat controversial clinical studies from the large European and North American BMT registries since TBI conditioning predominated only in the positive European study. These data have major implications for the use of G-CSF in disease states where NKT cell activation may have important effects on outcome and suggest a guide to the safe use of G-CSF after allogeneic BMT.

TNF is a key cytokine in the effector phase of both graft-versus-host disease (GVHD) and the graft-versus leukemia (GVL) effect after bone marrow transplantation (BMT). TNF neutralizing antibodies are now established as effective therapeutic adjuncts for the treatment of severe acute GVHD. TNF signals through the p55 and p75 TNF receptors (TNFR), which are also receptors for the soluble lymphotoxin homotrimer (LTα3). The membrane-bound lymphotoxin heterotrimeric molecule (LTα1β2) signals through the LTβ receptor. The function of these molecules in GVHD remains unknown. Pharmacological agents are available which block either TNF alone, or both TNF and LTα3 and elucidating the roles of these molecules in GVHD is essential for the design of rational therapeutic strategies.

Allogeneic stem cell transplantation (SCT) remains the definitive immunotherapy for malignancy. However, morbidity and mortality due to graft-vs.-host disease (GVHD) remains the major barrier to its advancement. Emerging experimental data highlights the immuno-modulatory roles of diverse cell populations in GVHD, including regulatory T cells, natural killer (NK) cells, NK T cells, gammadelta T cells, and antigen presenting cells (APC). Knowledge of the pathophysiology of GVHD has driven the investigation of new rational strategies to both prevent severe GVHD and treat steroid-refractory GVHD. Novel cytokine inhibitors, immune-suppressant agents known to preserve or even promote regulatory T-cell function and the depletion of specific alloreactive T-cell sub-populations all promise significant advances in the near future. As our knowledge and therapeutic options expand, the ability to limit GVHD whilst preserving anti-microbial and tumour responses becomes a realistic prospect.

Antigen-presenting cells (APCs) are critical for the initiation of graft-versus-host disease (GVHD), although the responsible APC subset and molecular mechanisms remain unclear. Because dendritic cells (DCs) are the most potent APCs and the NF-kB/Rel family member RelB is associated with DC maturation and potent APC function, we examined their role in GVHD. Within 4 hours of total body irradiation, RelB nuclear translocation was increased and restricted to CD11c(hi) DCs within the host APC compartment. Furthermore, the transient depletion of CD11c(hi) donor DCs that reconstitute in the second week after transplantation resulted in a transient decrease in GVHD severity. By using RelB(-/-) bone marrow chimeras as transplant recipients or RelB(-/-) donor bone marrow, we demonstrate that the induction and maintenance of GVHD is critically dependent on this transcription factor within both host and donor APCs. Critically, RelB within APCs was required for the expansion of donor helper T cell type 1 (Th1) effectors and subsequent alloreactivity, but not the peripheral expansion or function of donor FoxP3(+) regulatory T cells. These data suggest that the targeted inhibition of nuclear RelB translocation within APCs represents an attractive therapeutic strategy to dissociate effector and regulatory T-cell function in settings of Th1-mediated tissue injury.

There are very few reports in the literature of successful pregnancy following allogeneic transplantation for Fanconi anaemia (FA). Despite attenuation of conditioning, transplantation causes significant germ cell injury and is generally considered to induce secondary infertility. Goi et al (2006) recently reported a patient presenting with menorrhagia aged 19 years, subsequently found to be pancytopenic due to FA. She received an allogeneic transplantation from her human leukocyte antigen (HLA)-matched sister and re-started regular menstruation at 6 months post-transplant. She went on to conceive naturally 48 months post-transplant and delivered a healthy child. The one other case described is of a patient with FA transplanted from her HLA-matched sibling at aged 17 years (Dalle et al, 2004). The patient had experienced spontaneous menarche aged 11 years, followed by regular periods. Secondary amenorrhoea was evident by 18 months post-transplant, although it is unclear what replacement therapy was instigated. She subsequently carried two natural pregnancies and delivered two healthy children. We report a female patient initially diagnosed with FA aged 8 years. She underwent HLA-matched allogeneic bone marrow transplantation from her sibling aged 10 years, conditioned with Cyclophosphamide 20 mg/kg total dose, total body irradiation (TBI) over 4 d (4 Gy total dose) and antithymocyte globulin (60 mg/kg total dose). The procedure was tolerated extremely well and she did not develop graft-versus-host disease. She entered puberty spontaneously aged 12 years with normal menstrual cycles. She became pregnant naturally 13 years and 16 years post-transplant and delivered two healthy children after uncomplicated pregnancies monitored in collaboration with the Late Effects and Obstetric clinics at our institution. Clearly, despite the significant doubts that transplant and reproductive specialists may harbour, successful natural pregnancy is realistic in patients with FA following allogeneic haemopoietic transplantation, even when myeloablative (TBI-containing) conditioning is used. Significantly, this is the first report of successful pregnancies in a patient with FA transplanted in the pre-pubertal period with preservation of fertility. We recommend patients transplanted for FA should be appropriately counselled in relation to family planning and contraception, and ideally managed within a multidisciplinary Late Effects clinic.

Although proinflammatory cytokines are key mediators of tissue damage during graft-versus-host disease (GVHD), IFNgamma has previously been attributed with both protective and pathogenic effects. We have resolved this paradox by using wild-type (wt), IFNgamma(-/-), and IFNgammaR(-/-) mice as donors or recipients in well-described models of allogeneic stem cell transplantation (SCT). We show that donor-derived IFNgamma augments acute GVHD via direct effects on (1) the donor T cell to promote T helper 1 (Th1) differentiation and (2) the gastrointestinal (GI) tract to augment inflammatory cytokine generation. However, these detrimental effects are overwhelmed by a protective role of IFNgamma in preventing the development of idiopathic pneumonia syndrome (IPS). This is the result of direct effects on pulmonary parenchyma to prevent donor cell migration and expansion within the lung. Thus, IFNgamma is the key cytokine differentially controlling the development of IPS and gastrointestinal GVHD after allogeneic SCT.

A 4-year-old boy was diagnosed in 2001 with high-risk precursor-B acute lymphoblastic leukaemia (ALL) (CD 10/19 = 97%, CD20 = 90%) without central nervous system (CNS) disease, according to the National Cancer Institutes classification and treated on regimen B of the UK Medical Research Council (MRC) trial, UKALL 2003 (http://www.ctsu.ox.ac.uk/projects/leuk/ukall2003). He had a slow early response at day 8 of induction (>25% lymphoblasts) and was transferred to the more intensive Regimen C of the protocol. After achieving complete remission (CR) at the end of induction, the remainder of his treatment was uneventful and he completed 3 years of maintenance therapy in December 2004. Seven months later, he presented with a combined bone marrow and testicular relapse and was re-treated on the UK MRC R3 relapse protocol. Although he achieved rapid marrow and CNS remission, treatment was complicated by significant adverse events including appendicitis, pancreatitis and shingles. The second remission was consolidated with a 10/10 human leucocyte antigen (HLA)-matched unrelated donor peripheral blood stem cell transplant from a female donor in January 2006 [conditioning Treosulphan, Alemtuzumab and Cyclophosphamide; graft-versus-host disease (GVHD) prophylaxis Ciclosporin A plus short-course Methotrexate]. He suffered minimal toxicity with rapid engraftment (100% donor chimerism at 1 month) and mild steroid-responsive acute GVHD (maximum grade I). Three months post-transplant, he presented with an isolated testicular relapse, which was treated with systemic and intra-thecal chemotherapy as well as bilateral orchidectomy and scrotal radiotherapy. After approximately 3 months of maintenance therapy persistent thrombocytopenia was noted. Although bone marrow examination was morphologically and karyotypically normal, donor chimerism (XY fluorescence in situ hybridization) had fallen to approximately 17%. Peripheral blood counts fell shortly after and further investigation demonstrated frank bone marrow and CNS relapse. Chimerism analysis demonstrated complete loss of donor chimerism. As the leukaemic blasts continued to show significant CD20 expression (60%), and because of the significant toxicity experienced with previous chemotherapy, we elected to treat with a weekly regimen of Rituximab (375 mg/m2). For CNS-directed treatment, he received fortnightly intrathecal liposomal cytarabine (Ara-C) (DepoCyte 35 mg with oral dexamethasone cover for prevention of arachnoiditis) with complete clearance of blasts from the CSF after the first dose. Bone marrow blasts fell rapidly from 97% pretreatment to 48%, 25% and 0% after three, five and seven infusions of Rituximab respectively. Concomitantly, a progressive increase in donor chimerism was observed, reaching 80% after seven infusions. However, reassessment after the 8th infusion demonstrated a fall in chimerism to 12%, associated with a small increase in morphologically abnormal blasts (4%). Subsequent intensification with a modified ADE regimen (Ara-C 100 mg/kg 12 hourly for 5 d, Etoposide 100 mg/kg/d for 3 d and Daunoxome 90 mg/kg single dose) achieved a morphological CR but with 100% host chimerism. At the time of writing, he is 21 d postsecond unrelated donor bone marrow transplant following total body irradiation-based myeloablative conditioning. Treatment of multiply relapsed acute leukaemia represents a major challenge, particularly in heavily pretreated patients. Up to 1/3 of precursor B-cell ALL blasts express CD20 (Gokbuget & Hoelzer, 2006). Rituximab (Rituxan® or Mabthera®) is a chimaeric murine/human anti-CD20 monoclonal antibody, widely utilized in the therapy of both aggressive and indolent non-Hodgkin Lymphoma (Marcus & Hagenbeek, 2007). Despite extensive experience in other haematological malignancies (and increasingly non-malignant disorders), there is very limited data regarding the use of Rituximab either as a single agent or as part of a combination regimen for the treatment of precursor-B ALL. There have been a small number of case reports describing the use of Rituximab to successfully treat minimal residual disease following haemopoietic transplantation (Jandula et al, 2001; Ozsahin et al, 2002). Koren-Michowitz et al (2006) recently reported the successful treatment of two adults with precursor-B ALL who developed severe hepatic toxicity during induction therapy. Both patients had significant bone marrow infiltration at the time of suspension of therapy. Administration of single agent Rituximab allowed recovery of hepatic function, although only minor reductions in the percentages of bone marrow blasts were observed. Greater success has been reported in mature B-ALL. Corbacioglu et al (2003) described successful salvage therapy using single agent Rituximab in a 12-year-old male with bone marrow relapse immediately following completion of induction therapy. Five doses of Rituximab (375 mg/m2) were administered over 4 months, with achievement of morphological remission after the first treatment and molecular remission after five treatments. Unfortunately, isolated CNS relapse occurred prior to the next planned dose and the patient proceeded to salvage cytotoxic therapy and autologous transplantation. A further case report described successful treatment of a 3-year-old boy with isolated testicular relapse of B-ALL with four infusions of Rituximab followed by orchidectomy and autologous stem cell transplant (de Vries et al, 2004). The present case is the first report of significant therapeutic activity of single agent Rituximab in resistant CD-20 positive precursor-B ALL. Perhaps of the greatest interest was the ability of Rituximab to suppress the leukaemic clone sufficiently to allow at least temporary resurgence of donor chimerism. Our experience should provide encouragement to investigators exploring the therapeutic potential of Rituximab in combination with conventional chemotherapy in clinical trials of precursor-B ALL.