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The search for novel targets in chronic myeloid leukaemia (CML) is ongoing, to improve treatment efficacy in refractory disease and increase eligibility for tyrosine kinase inhibitor (TKI) discontinuation. Increased frequency of Tregs and effector Tregs was evident at diagnosis, together with increased expression of T-cell exhaustion markers, including in regulatory T cells at diagnosis and in patients with refractory disease. Plasma analysis revealed significantly increased levels of cytokines including tumour necrosis factor (TNF)-a and interleukin (IL)-6 at diagnosis, in keeping with a pro-inflammatory state prior to treatment. We hence demonstrate T-cell exhaustion and a pro-inflammatory state at diagnosis in CML, likely secondary to leukaemia-associated antigenic overload associated with increased disease burden.

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Introduction: The search for potential new targets to improve outcomes in patients with CML is ongoing, with a view to improve treatment efficacy for those with refractory disease and increase the proportion of those eligible to attempt TKI discontinuation. CML is recognised as a particularly immune sensitive tumour and as such immune checkpoint inhibitors, which can enhance inherent immune surveillance mechanisms are an attractive proposition. Methods: We performed flow cytometric analysis of peripheral blood mononuclear cells for expression of PD1, CTLA-4, TIM-3 and LAG-3 on T effectors and regulatory T cells. T effectors included CD4+ and CD8+ subsets and a gating strategy of CD4+/CD25+/CD127 lo/FOXP3+ cells for Tregs was employed, whilst FOXP3 hi/CD45RA-ve cells denoted effector Tregs. FMO controls were used to determine positive populations for each immune checkpoint molecule under investigation. Results: Samples from 22 patients were analysed, including samples from two different time points in 4 patients. This included patients at diagnosis (n=8), those with refractory disease defined as Patients at diagnosis had higher Tregs as proportion of total CD4+ cells compared to those with low disease burden with a mean of 6.3 vs 4.6 (p=0.048). Similarly, effector Tregs, the most functionally suppressive Treg subset, were also higher at diagnosis at 10.3 vs 5.4 (p=0.05). No differences were observed in frequency of Tregs between refractory and low disease burden groups. PD1 expression was higher at diagnosis in CD4+, CD8+ cells and Tregs when compared to those with low disease burden (4.75 vs 2.75, 5.85 vs 2.43 and 5.2 vs 2.78, p=0.034/p=0.003/p=0.002). Similarly, TIM3 expression was significantly higher at diagnosis compared to at low disease burden; in CD4+ at 6 vs 1.9, CD8+ at 15.71 vs 4.41 and Tregs at 5.15 vs 1.88 (p=0.027/p= Despite low sample size, PD1 expression was also significantly higher in refractory patients compared to those with low disease burden, in CD4+ cells at 5.34, and Tregs at 5.6 (p=0.031/p=0.026). TIM3 expression was higher in CD8+ subset only at 11.74 (p=0.028). LAG3 showed higher expression in CD4+ cells at 1.56 vs 0.45 and in CD8+ cells at 8.81 (p=0.004/P= One patient was analysed at diagnosis and then again after achieving MMR following dasatinib treatment for 11 months. A significant trend of downregulation of immune checkpoint expression with successful TKI treatment was observed. For example, Treg expression of PD1 decreased from 7.03 to 4.86, TIM3 decreased from 2.57 to 0.83 and LAG3 decreased from 2.23 to 0.16 (Figure 1a), with CTLA4 remaining at a similar level. Similarly, another patient was analysed whilst in MMR and then again following haematologic relapse, one week after regaining CHR with ponatinib. In contrast, this patient showed evidence of upregulation of checkpoint molecules, particularly in TIM3 expression increasing from 9.2 to 22.2 in CD4+ cells and from 12.6 to 20.1 in CD8+ cells (Figure 1b). Discussion: We have shown through an extended analysis of immune checkpoint expression on lymphocytes from blood samples of patients with CML, that expression correlates strongly with leukaemia disease burden. We provide the first report, to our knowledge, to describe the increased expression of TIM3 and LAG3 in peripheral blood lymphocytes, which is of significance given the recent development of inhibitors of these molecules. These data support the potential future use of immune checkpoint inhibitors in certain patients with high-risk disease at diagnosis as well as addition in those with inadequate response, alongside conventional TKI therapy. Moreover, these data provide a potential basis for the use of combination immune checkpoint blockade which has proven highly efficacious in other settings. Further laboratory and clinical studies evaluating these agents in CML are warranted. Figure 1 Figure 1. Disclosures Harrington: Bristol Myers Squibb: Research Funding; Incyte: Honoraria. Dillon: Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Menarini: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Other: Educational Events , Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Session chair (paid to institution), Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: educational events; Jazz: Other: Education events; Amgen: Other: Research support (paid to institution); Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research Support, Educational Events. Radia: Cogent Biosciences Incorporated: Other: Study Steering Committee; Blueprint Medicines Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Study steering group member, Research Funding; EXPLORER and PATHFINDER studies: Other: Member of the Response Adjudication Committee; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Education events. Kordasti: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Alexion: Honoraria; Beckman Coulter: Honoraria. Harrison: Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Keros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Galacteo: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Geron: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI BioPharma: 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, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Constellation Pharmaceuticals: Research Funding; Incyte Corporation: Speakers Bureau; Sierra Oncology: Honoraria; AOP Orphan Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Promedior: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. de Lavallade: Incyte: Honoraria, Research Funding; Novartis: Speakers Bureau; Bristol Myers Squibb: Research Funding.

Background: Systemic mastocytosis (SM) is a disorder of neoplastic mast cells ranging from indolent to aggressive multi-system disease. We previously reported our large single centre experience managing SM. Since 2019 patients have had access to new treatments within trials. Prognostic scoring systems developed in SM to improve outcome predictions and guide treatment have been a focus of recent studies, although are yet to be validated in real-world setting. We sought to apply these scoring systems within our UK cohort, with a particular focus on the international prognostic score (IPSM) and the mutation-adjusted risk score (MARS). Methods: We performed a retrospective study of 192 adult patients diagnosed with SM between 2009 and 2021 including demographics, clinical data, as well as next generation sequencing (NGS) based myeloid gene panels carried out in advanced SM patients (AdvSM) where available. Prognosis for all patients was calculated based on IPSM (Sperr et al. [2019]) in all our SM patients and MARS scores in those with AdvSM (Jawhar et al. [2019]). Results: There was no gender bias in our cohort with 87 (46%) males and 105 (54%) females. Median age at diagnosis was 52 years (range 5-84) and majority of patients had indolent SM (ISM; 129/192 [67%]), 8/192 (4%) smouldering SM (SSM) and 55/192 (29%) advanced SM (AdvSM). In those with AdvSM, 43/55 (78%) had an associated haematological neoplasm (SM-AHN); 9/55 (17%) aggressive SM (ASM) and 3/55 (5%) mast cell leukaemia. As expected, AHN sub-types were myeloid with majority being CMML (16/55; 29%) [Table 1]. Median tryptase at diagnosis in ISM patients was 41ug/L (range 3-351ug/L), while in SSM it was 494ug/L (range 174-682ug/L) and in AdvSM it was 155ug/L (range 10-1551). Most patients were positive for the KIT D816V mutation (131/192; 68%). As expected, ISM and SSM (non-AdvSM) patients had better prognosis compared to those with AdvSM [Figure 1a], with median overall survival (OS) not reached in the former and 12 months in the latter with 11-year follow-up. We next applied the IPSM score to the AdvSM cohort, with the largest proportion categorised as AdvSM-3 (25/55; 45%) while 10/55 (18%) were AdvSM-4 [Table 1]. Survival outcome was notably higher in the AdvSM-1 group (4/55; 7%) with no deaths recorded, while 6/25 (24%) and 4/10 (40%) deaths were reported in AdvSM-3 and AdvSM-4 groups respectively [Figure 1b]. SM patients are known to carry somatic mutations in addition to KIT, in particular SRSF2, ASXL1 and RUNX1 (S/A/R) which are associated with adverse outcome. In our AdvSM cohort, 10/55 (18%) of patients were positive for one of these mutations on NGS-based myeloid gene panel and 8/55 (15%) carried ≥2. Additional clinically significant mutations are summarised in Table 1. We next examined the MARS score; most of our AdvSM patients categorised as low-risk (28/55, 51%), 21/55 (38%) were intermediate-risk and 6/55 (13%) high-risk [Table 1]. Survival outcome as expected in the low-risk group was higher compared to the intermediate- and high-risk groups, with 10-year OS of 87% versus 35% and 30% respectively [Figure 1c]. On reviewing our AdvSM cohort, 6/55 (11%) had progressive disease: 5 transforming to acute myeloid leukaemia (AML) and 1 to mast cell leukaemia (MCL). Interestingly, when reviewing cause of death, the highest proportion were due to AHN progression (6/16; 38%) while 2/16 (13%) were from transformation to AML [Table 1]. As expected within our cohort, there was a discrepancy between the IPSM and MARS scores as the latter includes the presence of S/A/R mutations. 2 patients diagnosed with MCL in 2016 and 2019 respectively, were categorised high risk based on both scores. Both are alive to date having received the KIT inhibitor avapritinib, with one continuing on this treatment. The other progressed to AML and underwent allogeneic stem cell transplant, with ongoing complete remission. Other treatments received by our AdvSM patients are summarised in Table 1. Conclusion: Application of the IPSM and MARS prognostic scores to our data reflects findings of other groups with better outcomes seen in non-AdvSM compared to AdvSM patients. Furthermore, although our AdvSM cohort is small, the presence of adverse risk factors could be overcome through recent advances in treatments and consolidation with stem cell transplant. Hence, identification of high-risk patients using these prognostic scores can direct targeted first-line therapy and improve outcomes. Figure 1 Figure 1. Disclosures Sriskandarajah: Celgene: Speakers Bureau; Novartis: Other: Education. Green: Novartis: Other: Education. Ong: Novartis: Other: Education. Harrison: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte Corporation: Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Constellation Pharmaceuticals: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Geron: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Keros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Galacteo: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sierra Oncology: Honoraria; AOP Orphan Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Promedior: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Grattan: Novartis: Speakers Bureau. Radia: Blueprint Medicines Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Study steering group member, Research Funding; Cogent Biosciences Incorporated: Other: Study Steering Committee; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Education events; EXPLORER and PATHFINDER studies: Other: Member of the Response Adjudication Committee.