Search

Summary Based on promising results in older adults with acute myeloid leukaemia (AML), we treated patients with NPM1 mut measurable residual disease (MRD) using off‐label venetoclax in combination with low‐dose cytarabine or azacitidine. Twelve consecutive patients were retrospectively identified, including five with molecular persistence and seven with molecular relapse/progression. All patients with molecular persistence achieved durable molecular complete remission (CRMRD‐) without transplantation. Six of seven patients with molecular relapse/progression achieved CRMRD‐ after 1–2 cycles of venetoclax. This paper highlights the promising efficacy of venetoclax‐based therapy to reduce the relapse risk in patients with persistent or rising NPM1 mut MRD.

Background Acute T-cell mediated rejection (TCMR) is usually indicated by alteration in serum-creatinine measurements when considerable transplant damage has already occurred. There is, therefore, a need for non-invasive early detection of immune signals that would precede the onset of rejection, prior to transplant damage. Methods We examined the RT-qPCR expression of 22 literature-based genes in peripheral blood samples from 248 patients in the Kidney Allograft Immune Biomarkers of Rejection Episodes (KALIBRE) study. To account for post-transplantation changes unrelated to rejection, we generated time-adjusted gene-expression residuals from linear mixed-effects models in stable patients. To select genes, we used penalised logistic regression based on 27 stable patients and 27 rejectors with biopsy-proven T-cell-mediated rejection, fulfilling strict inclusion/exclusion criteria. We validated this signature in i) an independent group of stable patients and patients with concomitant T-cell and antibody-mediated-rejection, ii) patients from an independent study, iii) cross-sectional pre-biopsy samples from non-rejectors and iv) longitudinal follow-up samples covering the first post-transplant year from rejectors, non-rejectors and stable patients. Findings A parsimonious TCMR-signature (IFNG, IP-10, ITGA4, MARCH8, RORc, SEMA7A, WDR40A) showed cross-validated area-under-ROC curve 0.84 (0.77–0.88) (median, 2.5th–97.5th centile of fifty cross-validation cycles), sensitivity 0.67 (0.59–0.74) and specificity 0.85 (0.75–0.89). The estimated probability of TCMR increased seven weeks prior to the diagnostic biopsy and decreased after treatment. Gene expression in all patients showed pronounced variability, with up to 24% of the longitudinal samples in stable patients being TCMR-signature positive. In patients with borderline changes, up to 40% of pre-biopsy samples were TCMR-signature positive. Interpretation Molecular marker alterations in blood emerge well ahead of the time of clinically overt TCMR. Monitoring a TCMR-signature in peripheral blood could unravel T-cell-related pro-inflammatory activity and hidden immunological processes. This additional information could support clinical management decisions in cases of patients with stable but poor kidney function or with inconclusive biopsy results.

Approximately 10% to 15% of patients with essential thrombocythemia (ET) lack the common driver mutations, so-called “triple-negative” (TN) disease. We undertook a systematic approach to investigate for somatic mutations and delineate gene expression signatures in 46 TN patients and compared the results to those with known driver mutations and healthy volunteers. Deep, error-corrected, next-generation sequencing of peripheral blood mononuclear cells using the HaloPlexHS platform and whole-exome sequencing was performed. Using this platform, 10 (22%) of 46 patients had detectable mutations (MPL, n = 6; JAK2V617F, n = 4) with 3 of 10 cases harboring germline MPL mutations. RNA-sequencing and DNA methylation analysis were also performed by using peripheral blood mononuclear cells. Pathway analysis comparing healthy volunteers and ET patients (regardless of mutational status) identified significant enrichment for genes in the tumor necrosis factor, NFκB, and MAPK pathways and upregulation of platelet proliferative drivers such as ITGA2B and ITGB3. Correlation with DNA methylation showed a consistent pattern of hypomethylation at upregulated gene promoters. Interrogation of these promoter regions highlighted enrichment of transcriptional regulators, which were significantly upregulated in patients with ET regardless of mutation status, including CEBPβ and NFκB. For “true” TN ET, patterns of gene expression and DNA methylation were similar to those in ET patients with known driver mutations. These observations suggest that the resultant ET phenotype may, at least in part and regardless of mutation type, be driven by transcriptional misregulation and may propagate downstream via the MAPK, tumor necrosis factor, and NFκB pathways with resultant JAK-STAT activation. These findings identify potential novel mechanisms of disease initiation that require further evaluation.

Relapse remains the most common cause of treatment failure for patients with acute myeloid leukemia (AML) who undergo allogeneic stem cell transplantation (alloSCT), and carries a grave prognosis. Multiple studies have identified the presence of measurable residual disease (MRD) assessed by flow cytometry before alloSCT as a strong predictor of relapse, but it is not clear how these findings apply to patients who test positive in molecular MRD assays, which have far greater sensitivity. We analyzed pretransplant blood and bone marrow samples by reverse-transcription polymerase chain reaction in 107 patients with NPM1-mutant AML enrolled in the UK National Cancer Research Institute AML17 study. After a median follow-up of 4.9 years, patients with negative, low (

Background: Kidney transplant recipients (KTRs) with “operational tolerance” (OT) maintain a functioning graft without immunosuppressive (IS) drugs, thus avoiding treatment complications. Nevertheless, IS drugs can influence gene-expression signatures aiming to identify OT among treated KTRs. Methods: We compared five published signatures of OT in peripheral blood samples from 18 tolerant, 183 stable, and 34 chronic rejector KTRs, using gene-expression levels with and without adjustment for IS drugs and regularised logistic regression. Findings: IS drugs explained up to 50% of the variability in gene-expression and 20–30% of the variability in the probability of OT predicted by signatures without drug adjustment. We present a parsimonious consensus gene-set to identify OT, derived from joint analysis of IS-drug-adjusted expression of five published signature gene-sets. This signature, including CD40, CTLA4, HSD11B1, IGKV4–1, MZB1, NR3C2, and RAB40C genes, showed an area under the curve 0⋅92 (95% confidence interval 0⋅88–0⋅94) in cross-validation and 0⋅97 (0⋅93–1⋅00) in six months follow-up samples. Interpretation: We advocate including adjustment for IS drug therapy in the development stage of gene-expression signatures of OT to reduce the risk of capturing features of treatment, which could be lost following IS drug minimisation or withdrawal. Our signature, however, would require further validation in an independent dataset and a biomarker-led trial. Funding: FP7-HEALTH-2012-INNOVATION-1 [305147:BIO-DrIM] (SC,IR-M,PM,DSt); MRC [G0801537/ID:88245] (MPH-F); MRC [MR/J006742/1] (IR-M); Guy's&StThomas’ Charity [R080530]&[R090782]; CONICYT-Bicentennial-Becas-Chile (EN-L); EU:FP7/2007–2013 [HEALTH-F5–2010–260687: The ONE Study] (MPH-F); Czech Ministry of Health [NV19–06–00031] (OV); NIHR-BRC Guy's&StThomas' NHS Foundation Trust and KCL (SC); UK Clinical Research Networks [portfolio:7521].

Introduction Allogeneic hematopoietic cell transplant (allo-HCT) in first or second complete remission (CR1 or CR2) plays a fundamental role in the management of FLT3-ITD mutant acute myeloid leukemia (AML). Measurable residual disease (MRD) detected prior to HCT by flow cytometry or NPM1 RT-qPCR indicates a high risk of post-HCT relapse (Dillon et al, 2020). FLT3-ITD has not routinely been used as an MRD marker due to concerns relating to clonal stability and assay sensitivity, however increasing use of FLT3 inhibitors post-HCT is likely to alter this recommendation, particularly in view of the routine use of midostaurin, which may increase clonal instability (Schmalbrock et al, 2021). To date evaluation of the prognostic utility of FLT3-ITD quantitation in the pre-HCT setting has mainly been limited to capillary electrophoresis (CE) (Gaballa et al, 2017, Helbig et al, 2020) which has low sensitivity (1-5%). Next-generation sequencing (NGS) enables FLT3-ITD MRD detection with higher sensitivity and specificity (lower limit of detection (LLD) 0.001%) (Blätte et al, 2019). Preliminary data indicate that pre-HCT detection of FLT3-ITD may provide powerful prognostic information (Hourigan et al, 2019). The current work evaluates the prognostic impact of FLT3-ITD MRD via deep NGS sequencing in the pre-HCT setting. Methods FLT3-ITD NGS was performed on stored DNA pre-HCT (69 bone marrow, 9 peripheral blood) from the UK NCRI AML17 trial (ISRCTN 55675535) and Alfred Hospital transplant database. Quantitation of FLT3-ITD was by amplicon-based sequencing of exons 14-15, detecting ITDs >6 base pairs (bp) length. Prepared libraries were sequenced (Illumina) using 150 bp paired-end reads with minimum coverage of 400,000 reads and bioinformatics analysis using getITD (Blatte et al, 2019). Sensitivity ~0.01% and LLD 0.001%. Kaplan-Meier method was used to determine relapse-free survival (RFS; HCT date to relapse or death from any cause) and overall survival (OS; HCT date to death or last follow-up). Cumulative incidence of relapse was estimated considering competing risk. Cox proportional hazards model was used to assess factors impacting RFS and OS. Categorical data evaluated using Fisher's exact and continuous variables with Mann-Whitney-U tests. The study was approved by Alfred Health Ethics Committee (181/21). Results The retrospective cohort included 78 patients with FLT3-ITD AML who received first allo-HCT in morphologic remission between 2010-2020. At diagnosis, median age was 48, 83% had NPM1 mutation, 45% FLT3-ITD allelic ratio (AR) ≥0.5, 43% received myeloablative conditioning, 82% transplanted in CR1, unrelated donor in 53% and 14% had received upfront FLT3-inhibitor. Pre-HCT FLT3-ITD MRD by NGS was not detected in 62% (MRD-neg) and detected in 38% (MRD-pos). Among FLT3-ITD MRD-pos pre-HCT, 90% had 1 ITD, median bp 54 (21-210) and median VAF 0.0632% (0.001-9.3519). Overall, 73% with MRD had FLT3-ITD VAF Conclusions Pre-HCT FLT3-ITD MRD detection at any level by deep NGS is associated with a higher risk of post-HCT relapse and poorer long-term survival. Among patients who were also NPM1 MRD-pos, FLT3-ITD MRD was more informative for outcome. Our findings support the use of FLT3-ITD NGS to assess pre-HCT risk and allows rational application of FLT3 directed targeted therapy in the pre- or post- HCT setting in this high-risk AML population. Figure 1 Figure 1. Disclosures Dillon: Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research Support, Educational Events; Amgen: Other: Research support (paid to institution); Astellas: Consultancy, Other: Educational Events , Speakers Bureau; Jazz: Other: Education events; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: educational events; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Session chair (paid to institution), Speakers Bureau; Menarini: Membership on an entity's Board of Directors or advisory committees; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees. Tiong: Amgen: Speakers Bureau; Servier: Consultancy, Speakers Bureau; Pfizer: Consultancy. Russell: Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees; Jazz: Research Funding, Speakers Bureau. Wei: Astellas: 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; Roche: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pfizer: 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; Astra Zeneca: 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; Gilead: Membership on an entity's Board of Directors or advisory committees; Macrogenics: 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.

BACKGROUND: An increased percentage of peripheral transitional B cells producing IL-10 has been observed in patients tolerant to kidney allografts. In healthy volunteers, the balance between the CD40 and B cell receptor (BCR) signalling modulated IL-10 production by B cells, with stimulation via the BCR decreasing CD40-mediated-IL-10 production. In this study, we evaluate whether in tolerant kidney transplant patients the increased IL-10 production by B cells was due to an altered CD40 and/or BCR signalling.METHODS: B cells obtained from a new cohort of tolerant renal transplant recipients and those from age- and gender-matched healthy volunteers, were activated via CD40 and BCR, either alone or in combination.RESULTS: In tolerant patients we observed higher percentages of B cells producing IL-10 after CD40 ligation and higher expression of CD40L on activated T cells, compared to healthy controls. Furthermore, B cells from tolerant recipients had reduced ERK signalling following BCR-mediated activation compared to healthy controls. In keeping with this, combining BCR signalling with CD40 ligation did not reduce IL-10 secretion as was observed in healthy control transitional B cells.CONCLUSION: Altogether our data suggests that the altered response of B cells in tolerant recipients may contribute to long-term stable graft acceptance.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial License 4.0 (CCBY-NC), where it is permissible to download, share, remix, transform, and buildup the work provided it is properly cited. The work cannot be used commercially.

Cytolytic proteins and peptide toxins are classical virulence factors of several bacterial pathogens which disrupt epithelial barrier function, damage cells and activate or modulate host immune responses. Such toxins have not been identified previously in human pathogenic fungi. Here we identify the first, to our knowledge, fungal cytolytic peptide toxin in the opportunistic pathogen Candida albicans. This secreted toxin directly damages epithelial membranes, triggers a danger response signalling pathway and activates epithelial immunity. Membrane permeabilization is enhanced by a positive charge at the carboxy terminus of the peptide, which triggers an inward current concomitant with calcium influx. C. albicans strains lacking this toxin do not activate or damage epithelial cells and are avirulent in animal models of mucosal infection. We propose the name 'Candidalysin' for this cytolytic peptide toxin; a newly identified, critical molecular determinant of epithelial damage and host recognition of the clinically important fungus, C. albicans.

Steroid conversion (HSD11B1, HSD11B2, H6PD) and receptor genes (NR3C1, NR3C2) were examined in kidney-transplant recipients with "operational tolerance" and chronic rejection (CR), independently and within the context of 88 tolerance-associated genes. Associations with cellular types were explored. Peripheral whole-blood gene-expression levels (RT-qPCR-based) and cell counts were adjusted for immunosuppressant drug intake. Tolerant (n = 17), stable (n = 190) and CR patients (n = 37) were compared. Healthy controls (n = 14) were used as reference. The anti-inflammatory glucocorticoid receptor (NR3C1) and the cortisol-activating HSD11B1 and H6PD genes were up-regulated in CR and were lowest in tolerant patients. The pro-inflammatory mineralocorticoid gene (NR3C2) was downregulated in stable and CR patients. NR3C1 was associated with neutrophils and NR3C2 with T-cells. Steroid conversion and receptor genes, alone, enabled classification of tolerant patients and were major contributors to gene-expression signatures of both, tolerance and CR, alongside known tolerance-associated genes, revealing a key role of steroid regulation and response in kidney transplantation.

BACKGROUND: The development of spontaneous kidney transplant tolerance has been associated with numerous B cell-related immune alterations. We have previously shown that tolerant recipients exhibit reduced B cell receptor (BCR) signalling and higher IL-10 production than healthy volunteers. However, it is unclear whether CD4T cells from tolerant recipients also display an antiinflammatory profile that could contribute to graft maintenance.METHODS: CD4T cells were isolated from kidney transplant recipients who were identified as being tolerant recipients, patients with chronic-rejection or healthy volunteers. CD4T cells from the 3 groups were compared in terms of their gene expression profile, phenotype and functionally upon activation.RESULTS: Gene expression analysis of transcription factors and signalling proteins, in addition to surface proteins expression and cytokine production, revealed that tolerant recipients possessed fewer Th17 cells and exhibited reduced Th17 responses, relative to patients with chronic rejection or healthy volunteers. Furthermore, impaired TCR signalling and altered cytokine cooperation by monocytes contributed to the development of Th17 cells in tolerant recipients.CONCLUSIONS: These data suggest that defective proinflammatory Th17 responses may contribute to the prolonged graft survival and stable graft function, which is observed in tolerant recipients in the absence of immunosuppressive agents.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial License 4.0 (CCBY-NC), where it is permissible to download, share, remix, transform, and buildup the work provided it is properly cited. The work cannot be used commercially without permission from the journal.

Introduction Most children with acute myeloid leukaemia (AML) harbour fusion genes which are ideal targets for molecular minimal residual disease (MRD) monitoring. However, evidence of prognostic significance is currently lacking and consequently most current paediatric AML treatment protocols rely on flow cytometric (FCM) evaluation to allocate treatment. Molecular MRD techniques provide significantly greater sensitivity and specificity and could allow more accurate outcome prediction, and consequently more personalised therapy, which is highly relevant in a disease where treatment related mortality, morbidity and relapse remain significant. Methods Between June 2016 and February 2019, MyeChild01 enrolled 170 children aged 0-18y with newly diagnosed AML who were randomly assigned to induction therapy with liposomal daunorubicin or mitoxantrone with cytarabine with or without gemtuzumab ozogamicin. Consolidation treatment was determined by karyotype, mutational profile and MRD status. Comprehensive centralised diagnostic assessment consisted of: Karyotype and fluorescence in-situ hybridisation (FISH) using a custom panel of probes to detect paediatric AML-associated gene fusions.PCR based screening for mutations in FLT3, NPM1 and CEPBA.Targeted capture of known fusion loci and paired end sequencing.RNA-seq using the Illumina TruSight fusion panel. Where a fusion gene was identified, RT-qPCR assays were designed and optimised for each patient. NPM1 mutation was also used as an MRD target if present. Paired PB and BM samples were requested after each cycle of treatment. Patients could have sequential monitoring after completion of therapy although this was not mandated. For this analysis, patients with core-binding factor (CBF) leukaemias i.e. inv(16)(p13q22) or t(8;21)(q22q22) with transcript levels above previously defined thresholds (Yin et al, 2012) were considered MRD positive. For all other targets, amplification in at least 2/3 replicates at For patients with CBF or NPM1 mutation, both molecular and FCM MRD status contributed to treatment allocation. Otherwise, FCM MRD was used unless there was no FCM target. Allogeneic stem cell transplantation (HSCT) was recommended for all patients with poor risk cytogenetics, those with intermediate risk cytogenetics who were MRD positive after cycle 2, and those with favourable risk cytogenetics who were MRD positive at the end of cycle 3. After completion of treatment, no specific advice was given regarding management of positive MRD results. Results We identified fusion genes in 126/170 patients (74%). We designed 55 unique RT-qPCR assays to specifically amplify 27 fusions with calculated sensitivity between 1:104 and 1:106. 62/126 children provided a complete set of samples: 31 (60%) with favourable, 18 (29%) with intermediate and 13 (21%) with high risk cytogenetic / molecular profiles. We analysed the effect of molecular MRD status at the end of protocol specified treatment, which included SCT in 17/62 (27%). One year event-free survival from diagnosis was 70% (95% confidence interval 52-82%) in patients who tested MRD negative compared to 11% (1-39%) in patients who tested MRD positive at the end of treatment (p Four patients received non-protocol specified SCT due to physician / family preference based on persistently positive or serially rising transcript levels after completion of treatment. At last follow up 4/4 were alive in complete remission. Rising transcript levels were observed in a further 8 patients to whom no pre-emptive treatment was given due to individual preference or lack of appropriate therapy. Haematological relapse subsequently occurred in 8/8. Conclusion Molecular MRD monitoring is feasible in the majority of children with AML, permitting refinement of response-adapted therapy. Molecular MRD status at the end of treatment is highly predictive of outcome, identifying molecular complete remission as a treatment goal for these children. Serially rising MRD levels reliably predict relapse in the absence of therapeutic intervention. Disclosures Dillon: Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; TEVA: Consultancy, Honoraria. Vyas:Astellas: Speakers Bureau; Abbvie: Speakers Bureau; Forty Seven, Inc.: Research Funding; Celgene: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Pfizer: Speakers Bureau; Daiichi Sankyo: Speakers Bureau. Amrolia:UCLB: Patents & Royalties. Baruchel:Bellicum: Consultancy; Servier: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Introduction: Molecular MRD monitoring in NPM1 mutant (mut) AML is now standard of care in many countries as prospective clinical trials have demonstrated that suboptimal reduction and/or rising levels of NPM1mut transcript are associated with disease relapse (Ivey, NEJM 2016; Balsat, JCO 2017). Higher levels of NPM1mut MRD positivity are associated with poorer outcomes after allogeneic hematopoietic stem transplantation (allo-HSCT) (Dillon, EHA 2019). Data regarding the effectiveness of pre-emptive MRD intervention is scant. A reduction in NPM1mut MRD was reported in 17/33 (52%) receiving azacitidine (Platzbecker, Lancet Oncol 2018). Among patients with NPM1mut molecular relapse in the NCRI AML17 trial, 16/27 (59%) achieved MRD negativity with 1-2 cycles of FLAG-Ida salvage chemotherapy (manuscript in preparation). As the BCL-2 inhibitor venetoclax in combination with low-dose cytarabine (LDAC) or hypomethylating agents (HMA) are known to be highly active in NPM1mut AML (Wei, JCO 2019; Strickland, EHA 2018), we hypothesized that venetoclax-based regimens could represent an effective low intensity option for patients with persistent or rising NPM1mut MRD. Methods: Consecutive patients with NPM1mut AML from 4 sites treated with venetoclax-LDAC/HMA for MRD eradication were included if previously received at least 1 cycle of intensive chemotherapy and achieved complete remission. cDNA was analyzed on bone marrow aspirate ± peripheral blood samples using a NPM1 mutant specific RT-qPCR (Ivey, NEJM 2016) according to Europe Against Cancer guidelines (Gabert, Leukemia 2003). Results are expressed as MRD level relative to the diagnostic sample (%) with both samples normalized to a control gene (ABL). Results: A total of 10 patients were treated with venetoclax (600 mg PO daily D1-14) in combination with LDAC (20 mg/m2 SC twice daily D1-10; n=8), or venetoclax (400 mg PO daily D1-14) azacitidine (75 mg/m2 SC daily D1-7; n=2) as summarized in Table 1. If concurrent posaconazole was used, the dose of venetoclax was adjusted to 100 mg PO daily. The median age of the cohort was 61.2 years (range 31-81). All patients had intermediate risk cytogenetics, including 8 with normal karyotype at diagnosis. Concurrent mutations in addition to NPM1 are shown in Table 1. The number of consolidation cycles received prior to venetoclax ranged from 0-4 and typically included high-dose cytarabine. Three patients had failed other MRD-directed therapies prior to treatment with venetoclax. Six patients (Cases #1-6) received venetoclax-LDAC/HMA for NPM1mut molecular relapse after complete molecular remission (CRMRD-), or progression after molecular persistence at low copy number (CRMRD+). Five of six (83%) achieved CRMRD- after only 1-2 cycles, with ongoing CRMRD- after a median of 134 days (range 59-447). One patient had failed to respond to FLAG-Ida but achieved CRMRD- after just 1 cycle of venetoclax-LDAC (Figure 1A). Three patients proceeded to subsequent allo-HSCT, including 2 in CRMRD- (Figures 1B and C). Follow up post-HSCT is ongoing ( Four patients (Cases #7-10) were treated for molecular persistence after completion of induction and 0-3 cycles of consolidation chemotherapy. All 4 patients responded, including 3 (75%) who achieved CRMRD-. Case #7 (Figure 1D) had received prior gemtuzumab ozogamicin and azacitidine without molecular response but then achieved CRMRD- after receiving venetoclax-azacitidine, which was sustained for >23 months without an allo-HSCT. Venetoclax in combination with LDAC/HMA was well tolerated. Febrile neutropenia occurred in 2 patients (20%) and one patient had grade 4 sepsis. Grade 4 neutropenia occurred in 70% (median duration 10 days) and grade 4 thrombocytopenia in 40% (median duration 16 days). Conclusion: In the setting of molecular NPM1mut MRD relapse or persistence, venetoclax in combination with LDAC/HMA was highly effective, with MRD reductions in 100% and complete molecular remission in 80%. Future clinical exploration is warranted to validate the role of venetoclax as a pre-emptive approach to suppress persistent or rising NPM1mut MRD. Disclosures Dillon: Pfizer: Consultancy, Honoraria; TEVA: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Ivey:Novartis: Honoraria. Vassili:Amgen: Honoraria. Taussig:Celgene: Research Funding. Russell:Jazz: Consultancy, Honoraria, Speakers Bureau; Astellas: Consultancy, Honoraria, Speakers Bureau; DSI: Consultancy, Honoraria, Speakers Bureau; Pfizer Inc: Consultancy, Honoraria, Speakers Bureau. Raj:Jazz: Honoraria, Speakers Bureau; Daiichi Sankyo: Honoraria; Pfizer: Honoraria; MSD: Honoraria; Celgene: Honoraria; Novartis: Honoraria, Speakers Bureau; Mallinckrodt: Honoraria, Speakers Bureau. Fong:Pfizer: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Astellas: Consultancy. Grigg:Janssen: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees; Roche: Other: Travel. Knapper:Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Honoraria; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Tolero: Membership on an entity's Board of Directors or advisory committees. Wei:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: AHW is a former employee of the Walter and Eliza Hall Institute and receives a fraction of its royalty stream related to venetoclax, Research Funding, Speakers Bureau; Astellas: 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, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Macrogenics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, 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; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Honoraria, Research Funding; Janssen: Honoraria. OffLabel Disclosure: Venetoclax is a BCL-2 inhibitor that is FDA-approved in some indications. This presentation will focus on venetoclax as a MRD-directed therapy in AML, which is not an approved indication.

Oral epithelial cells discriminate between the yeast and hyphal forms of Candida albicans via the mitogen-activated protein kinase (MAPK) signaling pathway. This occurs through phosphorylation of the MAPK phosphatase MKP1 and activation of the c-Fos transcription factor by the hyphal form. Given that fungal cell wall polysaccharides are critical in host recognition and immune activation in myeloid cells, we sought to determine whether β-glucan and N - or O -glycosylation was important in activating the MAPK/MKP1/c-Fos hypha-mediated response mechanism and proinflammatory cytokines in oral epithelial cells. Using a series of β-glucan and N - and O -mannan mutants, we found that N -mannosylation (via Δ och1 and Δ pmr1 mutants) and O -mannosylation (via Δ pmt1 and Δ mnt1 Δ mnt2 mutants), but not phosphomannan (via a Δ mnn4 mutant) or β-1,2 mannosylation (via Δ bmt1 to Δ bmt6 mutants), were required for MKP1/c-Fos activation, proinflammatory cytokine production, and cell damage induction. However, the N - and O -mannan mutants showed reduced adhesion or lack of initial hypha formation at 2 h, resulting in little MKP1/c-Fos activation, or restricted hypha formation/pseudohyphal formation at 24 h, resulting in minimal proinflammatory cytokine production and cell damage. Further, the α-1,6-mannose backbone of the N -linked outer chain (corresponding to a Δ mnn9 mutant) may be required for epithelial adhesion, while the α-1,2-mannose component of phospholipomannan (corresponding to a Δ mit1 mutant) may contribute to epithelial cell damage. β-Glucan appeared to play no role in adhesion, epithelial activation, or cell damage. In summary, N - and O -mannosylation defects affect the ability of C. albicans to induce proinflammatory cytokines and damage in oral epithelial cells, but this may be due to indirect effects on fungal pathogenicity rather than mannose residues being direct activators of the MAPK/MKP1/c-Fos hypha-mediated immune response.

Introduction Relapse remains the most common cause of treatment failure for patients with acute myeloid leukaemia (AML) who undergo allogeneic stem cell transplantation (SCT) and carries a grave prognosis. Multiple studies have identified the presence of minimal residual disease (MRD) prior to SCT assessed by flow cytometry (FCM) as a strong predictor of relapse: however, little is known about the impact of molecular MRD pre-SCT. Molecular techniques can provide 100-1000 fold greater sensitivity than FCM in NPM1 mutated AML allowing identification of patients with very low level MRD prior to SCT. Peri-transplant management decisions for patients with positive molecular MRD are highly challenging due to the absence of robust outcome data. Methods Between 2009-14 the NCRI AML17 study enrolled 3215 adult non-APML patients aged 16-77 eligible for intensive chemotherapy. Central screening for NPM1 mutations was positive in 861/2949 (29%); 530 patients provided serial samples for MRD monitoring. Paired blood (PB) and bone marrow aspirates (BM) were requested after each chemotherapy cycle and then quarterly; additional samples were requested pre- and post-SCT. Samples were analysed by RT-qPCR using a suite of 27 mutation-specific reverse primers. Results were only fed back to clinicians after June 2012 when patients could be treated for confirmed re-emergent or persistent molecular positivity. Post-remission treatment was determined according to the validated NCRI risk score, with poor-risk patients recommended for SCT during first complete remission (CR1). Survival analysis was performed using the logrank test. Results In total 108/530 patients received SCT (CR1 57 (52%), after molecular relapse or progression (MR) 30 (28%), CR2 21 (19%)). Five-year survival post-SCT (5y-OS) was 65% in CR1, and 54% in MR/CR2 (p=0.3). After MR 26/30 patients received chemotherapy prior to SCT. Evaluable pre-SCT PB and BM samples taken within 60 days of SCT were available for 104 and 78 patients (both available n=74). Considering PB samples, 5y-OS was 73% (median OS (mOS) not reached (NR)) for MRD-ve patients (n=74) vs. 30% (mOS 8.1 m) for any PB positivity (n=30) (p Of the 47 patients who received additional chemotherapy for morphologic or molecular relapse or progression, 26 (55%) converted to MRD negativity accounting for 44% of 59 patients who were MRD negative pre-SCT. Ninety-three per cent of 59 pre SCT-MRD negative patients had been MRD negative in the PB after the second cycle of induction (a previously identified marker of favourable outcome). A threshold of 200 mutant NPM1 transcripts/105 ABL copies in the pre-SCT PB sample split patients into 3 groups with 2y-OS of 81% (negative, n=74), 54% (low, n=13) and 9% (high, n=17; p Thirty four patients were positive for FLT3-ITD at diagnosis (5y-OS 55%) and 74 were negative (5y-OS 62%; p=0.3). For patients without FLT3 ITD, negative, low and high levels of MRD in the pre-SCT PB sample were associated with 2y-OS of 79% (n=53), 88% (n=8) and 0% (n=9) (p We assigned 80 patients to high and low risk groups based on FLT3 ITD status and pre-SCT MRD level in PB and BM. High-risk patients (FLT3 ITD and any detectable level of MRD in the PB or BM, or FLT3 ITD negative with > 200 copies in the PB or > 1000 copies in the BM) had 3y-OS of 14% (n=27, mOS 6.5 months) compared with 80% for low-risk patients (n=53, p Conclusions Patients who test negative for NPM1 mutant transcripts immediately before SCT have a favourable outcome which is also observed in FLT3 ITD negative patients who are MRD positive at levels which do not exceed 200 copies in the PB or 1000 copies in the BM. FLT3 ITD mutated patients with any level of MRD prior to SCT, and FLT3 ITD negative patients with transcript levels above these thresholds, have a very high risk of relapse and may benefit from further chemotherapy or FLT3 inhibition prior to or after SCT; studies to investigate this are urgently needed. Disclosures Hills: Daiichi Sankyo: Consultancy, Honoraria. Cavenagh:Celgene: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Russell:Daiichi Sankyo: Consultancy; Pfizer: Consultancy, Honoraria, Speakers Bureau; Jazz Pharma: Speakers Bureau.

SummaryDiscriminating between commensal and pathogenic states of opportunistic pathogens is critical for host mucosal defense and homeostasis. The opportunistic human fungal pathogen Candida albicans is also a constituent of the normal oral flora and grows either as yeasts or hyphae. We demonstrate that oral epithelial cells orchestrate an innate response to C. albicans via NF-κB and a biphasic MAPK response. Activation of NF-κB and the first MAPK phase, constituting c-Jun activation, is independent of morphology and due to fungal cell wall recognition. Activation of the second MAPK phase, constituting MKP1 and c-Fos activation, is dependent upon hypha formation and fungal burdens and correlates with proinflammatory responses. Such biphasic response may allow epithelial tissues to remain quiescent under low fungal burdens while responding specifically and strongly to damage-inducing hyphae when burdens increase. MAPK/MKP1/c-Fos activation may represent a “danger response” pathway that is critical for identifying and responding to the pathogenic switch of commensal microbes.

Background. The ability of epithelial cells (ECs) to discriminate between commensal and pathogenic microbes is essential for healthy living. Key to these interactions are mucosal epithelial responses to pathogen-induced damage.Methods. Using reconstituted oral epithelium, we assessed epithelial gene transcriptional responses to Candida albicans infection by microarray. Signal pathway activation was monitored by Western blotting and transcription factor enzyme-linked immunosorbent assay, and the role of these pathways in C. albicans-induced damage protection was determined using chemical inhibitors.Results. Transcript profiling demonstrated early upregulation of epithelial genes involved in immune responses. Many of these genes constituted components of signaling pathways, but only NF-κB, MAPK, and PI3K/Akt pathways were functionally activated. We demonstrate that PI3K/Akt signaling is independent of NF-κB and MAPK signaling and plays a key role in epithelial immune activation and damage protection via mammalian target of rapamycin (mTOR) activation.Conclusions. PI3K/Akt/mTOR signaling may play a critical role in protecting epithelial cells from damage during mucosal fungal infections independent of NF-κB or MAPK signaling.

The fungus C. albicans uses adhesins to interact with human epithelial surfaces in the processes of colonization and pathogenesis. The C. albicans ALS (agglutinin-like sequence) gene family encodes eight large cell-surface glycoproteins (Als1-Als7 and Als9) that have adhesive function. This study utilized C. albicans Δals mutant strains to investigate the role of the Als family in oral epithelial cell adhesion and damage, cytokine induction and activation of a MAPK-based (MKP1/c-Fos) signaling pathway that discriminates between yeast and hyphae. Of the eight Δals mutants tested, only the Δals3 strain showed significant reductions in oral epithelial cell adhesion and damage, and cytokine production. High fungal:epithelial cell multiplicities of infection were able to rescue the cell damage and cytokine production phenotypes, demonstrating the importance of fungal burden in mucosal infections. Despite its adhesion, damage and cytokine induction phenotypes, the Δals3 strain induced MKP1 phosphorylation and c-Fos production to a similar extent as control cells. Our data demonstrate that Als3 is involved directly in epithelial adhesion but indirectly in cell damage and cytokine induction, and is not the factor targeted by oral epithelial cells to discriminate between the yeast and hyphal form of C. albicans.

We previously reported that a bi-phasic innate immune MAPK response, constituting activation of the mitogen-activated protein kinase (MAPK) phosphatase MKP1 and c-Fos transcription factor, discriminates between the yeast and hyphal forms of Candida albicans in oral epithelial cells (ECs). Since the vast majority of mucosal Candida infections are vaginal, we sought to determine whether a similar bi-phasic MAPK-based immune response was activated by C. albicans in vaginal ECs. Here, we demonstrate that vaginal ECs orchestrate an innate response to C. albicans via NF-κB and MAPK signaling pathways. However, unlike in oral ECs, the first MAPK response, defined by c-Jun transcription factor activation, is delayed until 2 h in vaginal ECs but is still independent of hypha formation. The ‘second’ or ‘late’ MAPK response, constituting MKP1 and c-Fos transcription factor activation, is identical to oral ECs and is dependent upon both hypha formation and fungal burdens. NF-κB activation is immediate but independent of morphology. Furthermore, the proinflammatory response in vaginal ECs is different to oral ECs, with an absence of G-CSF and CCL20 and low level IL-6 production. Therefore, differences exist in how C. albicans activates signaling mechanisms in oral and vaginal ECs; however, the activation of MAPK-based pathways that discriminate between yeast and hyphal forms is retained between these mucosal sites. We conclude that this MAPK-based signaling pathway is a common mechanism enabling different human epithelial tissues to orchestrate innate immune responses specifically against C. albicans hyphae.

Oral epithelial cells detect the human pathogenic fungus Candida albicans via NF-κB and a bi-phasic mitogen-activated protein kinase (MAPK) signaling response. However, discrimination between C. albicans yeast and hyphal forms is mediated only by the MAPK pathway, which constitutes activation of the MAPK phosphatase MKP1 and the c-Fos transcription factor and is targeted against the hyphal form. Given that C. albicans is not the only Candida species capable of filamentation or causing mucosal infections, we sought to determine whether this MAPK/MKP1/c-Fos mediated response mechanism was activated by other pathogenic Candida species, including C. dubliniensis, C. tropicalis, C. parapsilosis, C. glabrata and C. krusei. Although all Candida species activated the NF-κB signaling pathway, only C. albicans and C. dubliniensis were capable of inducing MKP1 and c-Fos activation, which directly correlated with hypha formation. However, only C. albicans strongly induced cytokine production (G-CSF, GM-CSF, IL-6 and IL-1α) and cell damage. Candida dubliniensis, C. tropicalis and C. parapsilosis were also capable of inducing IL-1α and this correlated with mild cell damage and was dependent upon fungal burdens. Our data demonstrate that activation of the MAPK/MKP1/c-Fos pathway in oral epithelial cells is specific to C. dubliniensis and C. albicans hyphae.

Parasite-derived glycosylphosphatidylinositol (GPI) is believed to be a major inducer of the pathways leading to pathology and morbidity during Plasmodium falciparum infection and has been termed a malaria “toxin.” The generation of neutralizing anti-GPI (“antitoxic”) antibodies has therefore been hypothesized to be an important step in the acquisition of antidisease immunity to malaria; however, to date the GPI-neutralizing capacity of antibodies induced during natural Plasmodium falciparum infection has not been evaluated. Here we describe the development of an in vitro macrophage-based assay to assess the neutralizing capacity of malarial GPI-specific IgG. We demonstrate that IgG from Plasmodium falciparum- exposed individuals can significantly inhibit the GPI-induced activation of macrophages in vitro , as shown by reduced levels of tumor necrosis factor production and attenuation of CD40 expression. The GPI-neutralizing capacity of individual IgG samples was directly correlated with the anti-GPI antibody titer. IgG from malaria-exposed individuals also neutralized the macrophage-activating effects of P. falciparum schizont extract (PfSE), but there was only a poor correlation between PfSE-neutralizing activity and the anti-GPI antibody titer, suggesting that PfSE contains other macrophage-activating moieties, in addition to GPI. In conclusion, we have established an in vitro assay to test the toxin-neutralizing activities of antimalarial antibodies and have shown that anti-GPI antibodies from malaria-immune individuals are able to neutralize GPI-induced macrophage activation; however, the clinical relevance of anti-GPI antibodies remains to be proven, given that malarial schizonts contain other proinflammatory moieties, in addition to GPI.