In myelodysplastic syndromes (MDS), cytogenetic characteristics of the malignant bone marrow cells influence the clinical course. The aim of this study was to evaluate whether cytogenetics is useful to predict outcome and response in patients with del(5q) under azacitidine (AZA) ± lenalidomide (LEN) therapy. We therefore performed comprehensive cytogenetic analyses in MDS patients with del(5q) treated within the randomized phase II trial NMDSG10B. Seventy-two patients were enrolled in the study and 46 patients (64%) had sufficient cytogenetics at inclusion and response evaluation. Karyotyping was significantly more sensitive during follow-up to detect del(5q) compared to FISH, 34 patients (97%) versus 27 patients (77%) (p = 0.027). The overall response rate (ORR) did not differ between the 11 patients with < 3 aberrations (median 1 aberration) and the 59 patients with ≥ 3 aberrations (median 7 aberrations, range 3-16), while ≥ 3 aberrations were associated with shorter overall survival (OS), 9.9 months versus 25.2 months (p = 0.004). OS was significantly shorter in patients with unbalanced translocation of 5q than patients with del (5)(q14q34), 8.4 months versus 21.1 months (p = 0.004). Both complex karyotype and multi-hit TP53 alterations were more frequent in patients with unbalanced translocations of 5q versus del (5)(q14q34), 98% and 88% versus 67% and 47% (each p = < 0.001). Most patients with cytogenetic progression had multi-hit TP53 alterations at inclusion. Cytogenetic progression occurred at a similar frequency in the AZA arm and in the AZA + LEN arm. In summary, this study in homogenously treated MDS patients with different abnormalities of 5q demonstrates the influence of cytogenetics on treatment results. Trial Registration: EudraCT number: 2011-001639-21; ClinicalTrials.gov identifier: NCT01556477.

Immune aplastic anemia (AA) is a life-threatening bone marrow failure disorder driven by an autoimmune T cell attack against hematopoietic stem and progenitor cells (HSPCs). However, the exact autoantigen targets and role of other immune cells in the pathogenesis of AA are unknown. Here, we analyzed a cohort of 218 patients with AA using single-cell RNA and T cell receptor (TCR) αβ sequencing, TCRβ sequencing, flow cytometry, and plasma cytokine profiling. We identified natural killer (NK) cells and CD8+ terminally differentiated effector T (TEMRA) cells expressing NK receptors with AA-associated TCRβ motifs as the most dysregulated immune cell populations in AA bone marrow. Functional coculture experiments using primary HSPCs and immune cells showed that NK cells cannot kill HSPCs alone but may sensitize HSPCs to CD8+ T cell-mediated killing through production of interferons. Furthermore, HSPCs induced activation of T cell clones with CD8+ TEMRA NK-like phenotype in coculture. Our results reveal a convergent phenotype of innate and adaptive immune cells that may drive AA.

Lenalidomide (LEN) can induce RBC transfusion independence (RBC-TI) in 60–70% of del(5q) myelodysplastic syndrome (MDS) patients. Current recommendation is to continue LEN in responding patients until failure or progression, with likelihood of toxicity and a high cost for healthcare systems. This HARMONY Alliance study investigated the outcome of MDS del(5q) patients who discontinued LEN in RBC-TI. We enrolled 118 patients with an IPSS-R low-intermediate risk. Seventy patients (59%) discontinued LEN for intolerance, 38 (32%) per their physician decision, nine (8%) per their own decision and one (1%) for unknown reasons. After a median follow-up of 49 months from discontinuation, 50/118 patients lost RBC-TI and 22/30 who underwent cytogenetic re-evaluation lost complete cytogenetic response. The median RBC-TI duration was 56 months. In multivariate analysis RBC-TI duration after LEN discontinuation correlated with low transfusion burden before LEN therapy, treatment ≥ 12 LEN cycles, younger age and higher Hb level at LEN withdrawal. Forty-eight patients were re-treated with LEN for loss of response and 28 achieved RBC-TI. These data show that stopping LEN therapy in MDS del(5q) patients in RBC-TI allows prolonged maintenance of TI in a large subset of patients.

PURPOSE Clinical relapse is the major threat for patients with myelodysplastic syndrome (MDS) undergoing hematopoietic stem-cell transplantation (HSCT). Early detection of measurable residual disease (MRD) would enable preemptive treatment and potentially reduced relapse risk. METHODS Patients with MDS planned for HSCT were enrolled in a prospective, observational study evaluating the association between MRD and clinical outcome. We collected bone marrow (BM) and peripheral blood samples until relapse, death, or end of study 24 months after HSCT. Patient-specific mutations were identified with targeted next-generation sequencing (NGS) panel and traced using droplet digital polymerase chain reaction (ddPCR). RESULTS Of 266 included patients, estimated relapse-free survival (RFS) and overall survival (OS) rates 3 years after HSCT were 59% and 64%, respectively. MRD results were available for 221 patients. Relapse was preceded by positive BM MRD in 42/44 relapses with complete MRD data, by a median of 71 (23-283) days. Of 137 patients in continuous complete remission, 93 were consistently MRD-negative, 39 reverted from MRD+ to MRD–, and five were MRD+ at last sampling. Estimated 1 year-RFS after first positive MRD was 49%, 39%, and 30%, using cutoff levels of 0.1%, 0.3%, and 0.5%, respectively. In a multivariate Cox model, MRD (hazard ratio [HR], 7.99), WHO subgroup AML (HR, 4.87), TP53 multi-hit (HR, 2.38), NRAS (HR, 3.55), and acute GVHD grade III-IV (HR, 4.13) were associated with shorter RFS. MRD+ was also independently associated with shorter OS (HR, 2.65). In a subgroup analysis of 100 MRD+ patients, presence of chronic GVHD was associated with longer RFS (HR, 0.32). CONCLUSION Assessment of individualized MRD using NGS + ddPCR is feasible and can be used for early detection of relapse. Positive MRD is associated with shorter RFS and OS (ClinicalTrials.gov identifier: NCT02872662 ).

Background: Lenalidomide can lead to red blood cell-transfusion independence (RBC-TI) in 60-70% of del(5q) transfusion dependent MDS patients, and to complete cytogenetic response (CCyR) in 30-40% of them. Currently, patients achieving RBC-TI and CCyR are suggested to continue treatment indefinitely. However, the optimal duration of lenalidomide therapy has never been formally addressed. Besides, lenalidomide is associated with possibly serious side effects and high treatment costs and could potentially trigger disease progression. Some small retrospective experiences showed that the majority of RBC-TI patients who stopped lenalidomide maintained the response. Aims: This study aimed to investigate the outcome of patients with del(5q) MDS RBC-TI who discontinue lenalidomide in RBC-TI response. The primary end point was event free survival (EFS) (events included RBC-TI loss, CCyR loss, disease progression, lenalidomide re-start or death). Secondary end points were RBC-TI duration, overall survival (OS), progression free survival. Methods: Within the MDS working group of the Harmony project, we retrospectively included patients from different European Countries (Italy, Germany, Spain, France, Finland, Switzerland, Sweden) with a del(5q) MDS diagnosis according to 2008 or 2016 WHO classification, at IPSS-r very low, low or intermediate at lenalidomide treatment start, who discontinued therapy after achieving RBC-TI. Patients who stopped lenalidomide for lack of response or disease progression were excluded. Results: We included 114 del(5q) MDS patients who started lenalidomide between December 2003 and January 2021 and discontinued treatment between July 2004 and March 2022. Median age was 77 years (range 42-93) and 81.6% were female. After a median of 12 lenalidomide cycles (range 1-70) before discontinuation, all the patients achieved RBC-TI, 12% also a partial CyR and 41% a CCyR. The reason for lenalidomide stop was intolerance for 71 patients (62%), optimal response for 32 (28%), patient decision for 9 (8%) and clonal evolution for 2 (2%).After a median follow-up of 44 months 40% of patients (n 46) lost RBC-TI, 55% lost CCyR (25 out of 45 who had a cytogenetic re-evaluation), 24% (n 27) progressed to high risk MDS or acute myeloid leukemia and 38% (n 44) died. Median EFS was 36 months (range 1-129) and was affected by IPSSr (p=0.035) and by treatment duration (> o ≤ 12 cycles) before discontinuation (median 53 vs 26 months, p=0.007). Median duration of RBC-TI was 60 months (range 1-119) and was longer for patients who achieved a CCyR before discontinuation (median 85 vs 35 months, p=0.028 Fig A) and received more than 12 lenalidomide cycles (median 86 vs 35 months, p <0.001 Fig B). However, patients who lost RBC-TI had a shorter survival (median 71 vs 101 months, p=0.046). Forty-six patients were re-treated with lenalidomide for response loss and 56% of them achieved at least a hematological response (3 also a CCyR). Summary/Conclusion: This is the largest series to date exploring the outcome of MDS del(5q) patients who discontinue lenalidomide while in RBC-TI response. Lacking prospective studies on the optimal duration of lenalidomide treatment, our retrospective data suggest that stopping lenalidomide in responding patients, particularly after at least 12 months of treatment and achievement of CCyR, is possible and may spare toxicities, reduce the economic impact on national healthcare systems and likely improve patients’ quality of life. Prospective studies could confirm this promising data.Keywords: Therapy, Health care, MDS

Background: Immune aplastic anemia (AA) is a bone marrow failure (BMF) disorder driven by an autoimmune response against the hematopoietic stem and progenitor cells (HSPCs), yet the mechanisms of breaking self-tolerance and sustaining inflammation are incompletely understood. Aims: Characterize and functionally validate the immune cell phenotypes and T cell receptor (TCR) targets in AA and associate them with clinical variables. Methods: With scRNA+TCRαβ-seq, we profiled AA (n=13, N=17) from bone marrow (BM) and peripheral blood (PB) and compared these to healthy controls (HC) (n=35) and hematological malignancies (n=36). With TCRβ-seq, we profiled AA (n=144, N=248) from BM and PB and compared these to other BMFs (n=137), other autoimmune disorders (n=45), hematological malignancies (n=39), and HC (n=826, N=866). With multiplexed cytokine platform, we profiled AA (n=78, N=88) and HC (n=20). Findings were validated in co-cultures of HPSCs with immune cells with scRNA+TCRαβ-seq readout (AA n=7, HC n=4; N=57). The presence of clonal hematopoiesis, somatic loss of HLA alleles, and somatic mutations in the T cells were assessed in a subset of patients. Results: Compared to HC, the most statistically significant finding in the AA BM was the expansion of NK cell cluster. The increased proportion of NK cells was validated in a larger flow cytometry cohort (AA n=49, HC n=26) and was associated with more severe disease. NK cells had mostly terminally differentiated or adaptive NK cell phenotype with upregulation of cytotoxic markers and lineage-defining KLRC2/NKG2C and LAG3. Patients with the highest NK cell infiltration had also distinct cytokine profiles, with upregulated IL2/4/13/20 and CCL28. Compared to HC, patients with AA had also significantly more cytotoxic CD8+ T cells. The most expanded CD8+ T cell populations were terminally differentiated, highly cytotoxic CD8+ TEMRA cells, with upregulation of exhaustion markers (LAG3, TIGIT), but with retained stimulation capacity in vitro. Interestingly, these clonally expanded T cells also upregulated NK cell receptors, such as FCGR3A/CD16 and KLRC1/2/3. The expansion of these NK-like CD8+ TEMRA cells was validated in an additional scRNA-seq cohort (AA n=8, HC n=2) and was more pronounced in severe AA than in non-severe AA. The TCR-seq analysis showed oligoclonal expansions in patients with AA. With a sophisticated in-house TCR-seq analysis pipeline, we found 200 TCR-motifs that were associated with AA, and not found in HC, MDS, or other associated disorders or viral infections. These motifs were successfully validated in two large AA and HC TCR-seq cohorts and were associated with clinical variables such as loss of HLA class I alleles. T cells with AA-associated TCR motifs had NK-like CD8+ TEMRA phenotype, implying NK receptor expression in T cells could be a result of chronic antigen stimulation. Finally, the function of NK cells, NK-like CD8+ TEMRA cells, and clones with AA-associated TCR-motifs was assessed with autologous immune cell and HPSC co-cultures with scRNA+TCRαβ-seq readout and results will be presented at the conference. Summary/Conclusion: Our results show convergent shift of both innate and adaptive immune cells, where NK cells gain T cell-like memory properties and T cells gain NK-like TCR independent killing abilities to maintain autoinflammation in AA. Keywords: Autoimmunity, Aplastic anemia, T cell, Natural killer

Background: One third of patients with myelodysplastic syndrome (MDS) relapse after allogeneic stem cell transplantation (HCT). Early detection of impending relapse would enable pre-emptive treatment and potentially reduce relapse risk but is limited by the lack of sensitive markers for measurable residual disease (MRD). We developed a pipeline where patient-specific mutations, as determined by a myeloid next generation sequencing (NGS) panel are tracked using digital droplet PCR (ddPCR). Aims: To evaluate if personalized MRD detection by ddPCR can predict clinical relapse earlier than conventional methods. Methods: The prospective study (NCT02872662) enrolled patients with MDS, MDS/MPN or MDS-AML with < 30% marrow blasts undergoing HCT. Patients were included before HCT, and serial bone marrow (BM) samples were collected every third month post-HCT for 2 years. Peripheral blood (PB) samples were collected monthly. MRD results were not available for the treating physician. Results: We screened 286 pts between 2016 and 2020, whereof 20 were excluded mainly due to lack of genetic aberration or no HCT performed. 266 pts were included from 12 HCT centers. Median age was 64 (18-78) years and 59% were male. Myeloid panel NGS screening identified a median of 2 (0-9) mutations. The most common mutations were TET2 (n=85), ASXL1 (n=73) and SRSF2 (n=59). Median time of follow up was 886 (4-1934) days. Sixty pts relapsed after a median of 189 (53-1281) days and 46 died due to non-relapse mortality after a median of 121 (4-1036) days. Remaining pts (n=160) were in continuous complete remission (CCR) after a median follow-up of 1053 (479-1934) days. Estimated 1 and 2y overall survival was 79%, and 71%, respectively, while estimated 1 and 2y relapse-free survival (RFS) was 75% and 66%, respectively. MRD data was missing in 46 pts; no post-HCT samples available (n=15), no mutation detected (n=14) and difficulties to design ddPCR primers (n=11). 221 pts were available for MRD analysis with a median number of 4 (0-13) and 5 (0-23) samples from BM and PB, respectively. Of 53 clinical relapses with MRD results available, 42 were preceded by pos MRD (>0.1%) with a median of 70 (range 20-425) days between first pos MRD and clinical relapse. For the 11 remaining pts, 8 were inadequately sampled with a median time of 189 (82-397) days between last sampling and clinical relapse. One patient had an extramedullary relapse only. Of 31 pts who died without relapse, 19 were consistently MRD neg, while 5 were borderline positive (MRD > 0.1% and <0.5%) during the first 100 days but negative thereafter. Four MRD+ patients died without clinical relapse. Three pts were initially MRD+ but turned negative, all of which had chronic GVHD (cGVHD). Of 136 CCR patients, 94 were consistently MRD neg; 26 were borderline pos (MRD > 0.1% and <0.5%) during the first 100 days followed by neg samples; 16 were MRD positive (either > 0.5% during the first 100d or > 0.1% after 100d) of which 10 had a transition from pos to neg samples (all had cGVHD); one patient was treated for a molecular relapse detected by clinical routine method (FISH) and five patients were MRD positive at time of last follow-up. MRD used as a time-dependent co-variate was negatively associated with RFS (HR 7.1, p<0.01). Estimated cumulative incidence of relapse and non-relapse mortality 2y after pos MRD was 60% and 7% respectively (see figure). Image:Summary/Conclusion: We report the development of a highly functional personalized MRD pipeline based on patient-specific mutations showing a high sensitivity to predict relapse and relapse-free survival.

The prevalence and functional impact of somatic mutations in nonleukemic T cells is not well characterized, although clonal T-cell expansions are common. In immune-mediated aplastic anemia (AA), cytotoxic T-cell expansions are shown to participate in disease pathogenesis. We investigated the mutation profiles of T cells in AA by a custom panel of 2533 genes. We sequenced CD4+ and CD8+ T cells of 24 AA patients and compared the results to 20 healthy controls and whole-exome sequencing of 37 patients with AA. Somatic variants were common both in patients and healthy controls but enriched to AA patients’ CD8+ T cells, which accumulated most mutations on JAK-STAT and MAPK pathways. Mutation burden was associated with CD8+ T-cell clonality, assessed by T-cell receptor beta sequencing. To understand the effect of mutations, we performed single-cell sequencing of AA patients carrying STAT3 or other mutations in CD8+ T cells. STAT3 mutated clone was cytotoxic, clearly distinguishable from other CD8+ T cells, and attenuated by successful immunosuppressive treatment. Our results suggest that somatic mutations in T cells are common, associate with clonality, and can alter T-cell phenotype, warranting further investigation of their role in the pathogenesis of AA.

1 Hematology Research Unit Helsinki, University of Helsinki and Helsinki University 8 Hospital Comprehensive Cancer Center, Helsinki, Finland 9 2 Translational Immunology Research Program, University of Helsinki, Helsinki, 10 Finland 11 3 iCAN Digital Precision Cancer Medicine Flagship, Helsinki, Finland 12 4 Helsinki University Hospital Comprehensive Cancer Center, Department of 13 Hematology, Helsinki, Finland 14 5 Institute for Molecular Medicine Finland, HiLIFE, University of Helsinki, Helsinki, 15 Finland 16 6 Department of Pathology, HUSLAB, Helsinki University Hospital and University of 17 Helsinki, Helsinki, Finland 18 7 HUS Diagnostic Center, HUSLAB, Helsinki University Hospital, Helsinki, Finland 19 8 Department of Cancer Genetics, Institute for Cancer Research, Oslo University 20 Hospital, and Oslo Centre for Biostatistics and Epidemiology, University of Oslo, 21 Oslo, Norway 22 9 Novartis Pharmaceuticals, Basel, Switzerland 23 10 Department of Clinical Chemistry and Hematology, University of Helsinki, 24 Helsinki, Finland 25 26 27 * Corresponding authors 28 † These authors contributed equally to this work. 29 30 31 Address for correspondence: 32 Drs S Mustjoki and O Brück, Hematology Research Unit Helsinki, University of 33 Helsinki and Helsinki University Hospital Comprehensive Cancer Center, 34 Haartmaninkatu 8, FIN-00290 Helsinki, Finland. E-mail: satu.mustjoki@helsinki.fi, 35 oscar.bruck@hus.fi 36 Running title: Histopathological Landscape of MDS 37 Word count: Abstract 150, Manuscript 4490 38 Number of Figures and tables: 4 figures; 1 table, 10 supplemental figures, 3 39 supplemental tables 40 41 Disclosure of Conflicts of Interest 42 K.P. received honoraria and research funding from Celgene, Incyte, Novartis and 43 Bristol-Myers Squibb. S.M. received honoraria and research funding from Pfizer, 44

Abstract In myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN), bone marrow (BM) histopathology is assessed to identify dysplastic cellular morphology, cellularity, and blast excess. Yet, other morphologic findings may elude the human eye. We used convolutional neural networks to extract morphologic features from 236 MDS, 87 MDS/MPN, and 11 control BM biopsies. These features predicted genetic and cytogenetic aberrations, prognosis, age, and gender in multivariate regression models. Highest prediction accuracy was found for TET2 [area under the receiver operating curve (AUROC) = 0.94] and spliceosome mutations (0.89) and chromosome 7 monosomy (0.89). Mutation prediction probability correlated with variant allele frequency and number of affected genes per pathway, demonstrating the algorithms' ability to identify relevant morphologic patterns. By converting regression models to texture and cellular composition, we reproduced the classical del(5q) MDS morphology consisting of hypolobulated megakaryocytes. In summary, this study highlights the potential of linking deep BM histopathology with genetics and clinical variables. Significance: Histopathology is elementary in the diagnostics of patients with MDS, but its high-dimensional data are underused. By elucidating the association of morphologic features with clinical variables and molecular genetics, this study highlights the vast potential of convolutional neural networks in understanding MDS pathology and how genetics is reflected in BM morphology. See related commentary by Elemento, p. 195.

In myelodysplastic syndrome (MDS), bone marrow (BM) histopathology is visually assessed to identify dysplastic cellular morphology, cellularity, and blast excess. Yet, many morphological findings elude the human eye. Here, we extracted visual features of 236 MDS, 87 MDS/MPN, and 10 control BM biopsies with convolutional neural networks. Unsupervised analysis distinguished underlying correlations between tissue composition, leukocyte metrics, and clinical characteristics. We applied morphological features in elastic net-regularized regression models to predict genetic and cytogenetic aberrations, prognosis, and clinical variables. By parallelizing tile, pixel, and leukocyte-level image analysis, we deconvoluted each model to texture and cellular composition to dissect their pathobiological context. Model-based mutation predictions correlated with variant allele frequency and number of affected genes per pathway, demonstrating the models’ ability to identify relevant visual patterns. In summary, this study highlights the potential of deep histopathology in hematology by unveiling the fundamental association of BM morphology with genetic and clinical determinants.

Introduction One third of patients with myelodysplastic syndrome (MDS) will relapse after allogeneic stem cell transplantation (SCT), with a dismal prognosis. Early detection of relapse enables pre-emptive treatment and may potentially reduce relapse risk, but is limited by the lack of sensitive markers for minimal residual disease (MRD). We developed a pipeline where patient-specific mutations, as determined by a myeloid next generation sequencing (NGS) panel are tracked using sensitive digital droplet PCR (ddPCR). Method We designed a prospective Nordic study (NMDSG14B; NCT02872662) enrolling all patients with MDS, mixed MDS / MPN or AML with myelodysplasia related disease and < 30% marrow blasts undergoing SCT in the Nordic region. We hypothesized that personalized MRD detection by ddPCR can predict clinical relapse earlier than conventional methods. Patients were included before SCT and serial bone marrow samples were collected before, and 1 and 3 months post SCT, and thereafter every third month for 2 years or until relapse or death. Blood samples were collected monthly. The MRD results were not available for the treating physicians. MRD positivity was defined based on the background noise of the specific ddPCR-assays and varied between 0.05-0.1% VAF. Results Three-hundred and sixteen patients were screened between 2016 and 2020, of which 19 were excluded due to lack of mutation or disease progression preventing SCT. We here present data of 254 patients followed ≥ 6 months after SCT. Median age was 64 (18-78) years and 59% were male. Most WHO subgroups of MDS (n=166), MDS/MPN (n=39), AML (n=8) and therapy-related disease (n=41), were represented. Risk profile according to IPSS-R was very low (n=13), low (n=32), intermediate (n=46), high (n=60) and very high (n=32). The majority of patients received pre-SCT treatment consisting of HMA (n=159) and / or intensive chemotherapy (n=59) while 60 patients did not receive disease-modifying treatment prior to SCT. The most common mutations were ASXL1 (n=69), TET2 (n=58), SRSF2 (n=57) and TP53 (n=44). No mutation was identified in 10 pts, and NGS data is still pending for 11 patients. After a median follow-up of 436 days, estimated 2 years overall survival and relapse free survival were 72% and 63%, respectively. Cumulative incidence of NRM and relapse at 2 years was 16% and 20%, respectively. Forty-six patients relapsed after a median of 170 (53-733) days, and the estimated median survival following relapse was 197 days. The most common pre-SCT mutations in the relapsed cohort were TP53 (n=19), DNMT3A (n=11) and RUNX1 (n=9). Thirty-seven patients died due to non-relapse mortality (NRM) after a median of 83 (4-754) days. To date, MRD results are available for 64 patients. Relapse was preceded by positive MRD in 14 out of 15 patients a median of 79 (21-173) days before clinical relapse. The 15th patient had an extra-medullary relapse only. Borderline positive MRD samples < 0.2% VAF within 100 days after SCT followed by negative samples were seen in 11 non-relapse patients. Twenty-four of 37 patients in continuous complete remission (CCR) were consistently MRD neg. Six CCR patients had positive MRD after 100 days; two with transient borderline peaks (<0.1%) at 6 months; two with transient peaks > 0.1%, which turned negative when the patients developed GVHD; one patient with slowly decreasing MRD which turned negative first after 1 y, and finally one patient with prevailing KIT mutation (>700 days post-SCT) despite negative BCOR and STAG2. Two MRD+ patients died from NRM without showing signs of clinical relapse. Discussion In summary, we show that our pipeline of personalized MRD-assessment, based on patient-specific mutations is feasible with a high sensitivity to predict relapse. An update of study progression will be presented at the meeting. Figure 1 Disclosures Illman: Sanofi-Genzyme: Other: Travel Support; Celgene: Other: Travel Support. Mielke:DNA Prime: Honoraria, Other: received via my institution , Speakers Bureau; KIADIS Pharma: Honoraria, Other: received via my institution , Speakers Bureau; Miltenyi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: received via my institution , Speakers Bureau; Kite/Gilead: Honoraria, Other: received via my institution , Speakers Bureau; Bellicum: Honoraria, Other: received via my institution, Speakers Bureau; Novartis: Honoraria, Other: received via my institution, Speakers Bureau; Celgene/BMS: Honoraria, Other: received via my institution , Speakers Bureau. Ebeling:Accord Healthcare: Other: Travel Support; Amgen: Other: Travel Support; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel Support; Otsuka Pharma Scandanavia AB: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Background: MDS del(5q) is a distinct MDS subgroup with an isolated interstitial 5q deletion involving 5q31, characterized by bone marrow erythroid hyperplasia, hypolobated megakaryocytes and refractory anemia. The phenotype is due to haplo-insufficiency of multiple genes. Heterozygous loss of the RPS14 gene on 5q leads to activation of p53 in the erythroid lineage and the macrocytic anemia characteristic of the 5q- syndrome. The megakaryocytic and platelet phenotype of the 5q- syndrome has been attributed to heterozygous deletion of miR145 and miR146a. The haploinsufficient gene expression is known to sensitize cells to lenalidomide therapy. Lenalidomide also co-stimulates T cells through degradation of interleukin 2 expression repressors (Gandhi et al., Br J Haematol 2014) and has been found to activate cytotoxic T and NK cells (Kerdivel et al., Leukemia 2018). The current treatment standard in MDS del(5q), where severe anemia is usually the main clinical problem, is after erythropoietin refractoriness or failure, lenalidomide taken 10 mg per day in 21/28-day cycles. An erythroid response is reached in 76% after a median of 4,6 weeks. A complete cytogenetic remission (CCyR) has been reported in 45%. Lenalidomide is continued until relapse of red blood cell (RBC) transfusion dependence or progression of disease, which may mean several years of treatment. Stem cells harboring del(5q) have, however, been detected even in patients in CCyR. Rare MDS del(5q) cases have been reported with persistent RBC transfusion independence (RBC-TI) even after discontinuing lenalidomide. RBC- TI lasting several years has been described in eight patients in CCyR for at least 6 months during lenalidomide before stopping (Giagounidis et al., Leukemia 2012). Lenalidomide has been associated with long-term adverse effects such as development of secondary malignancies and possible selection of resistant clones, especially clones with TP53 mutation, predisposing to AML progression. Therefore, possible beneficial effects of temporary or sequential lenalidomide treatment have been suggested. Aims: We describe five MDS del(5q) patients with prolonged RBC-TI after interruption of lenalidomide use (Table). Methods The clinical data was retrospectively collected from records of available patients in Finland. Results: All the still living patients (1,2, 4 and 5) had normal hemoglobin levels at the time of analysis. Some of the patients had an inflammatory reaction temporally closely associated with lenalidomide initiation (erythrodermia, myocarditis, pruritic eczema after the second lenalidomide initiation in patient 3). In addition, patient 2 had pre-existing rheumatoid arthritis. In patient 5, the interleukin 2 receptor concentration measured during the acute erythrodermic phase was as high as 6080 kU/l, and also a temporary plasma cell clone in the bone marrow of the patient was detected. The clinical course and various immunological features/reaction models will be analyzed in blood and bone marrow samples collected from the most recent patient 5.Summary/Conclusion: A limited lenalidomide exposure may result in prolonged transfusion free time in MDS del(5q) patients. This beneficial response might be associated with an early immune reaction inducing long-term suppression of the disease clone. Acute inflammatory events in patients could represent development of a host-versus-malignancy effect. Prospective clinical trials in MDS del(5q) with lenalidomide interruption for instance after hemoglobin level normalization or at CCyR, and close follow-up, are warranted.

Immune aplastic anemia (AA) is a life-threatening bone marrow failure syndrome in which the hematopoietic stem cells are destroyed, leading to pancytopenia. Although the exact biological process leading to AA remains largely unknown, bone marrow destruction is thought to be mediated by an autologous T cell response. We hypothesized that the autoimmune process in AA would create a T cell fingerprint unique to aplastic anemia. To decipher this signature, we collected an international, multi-centre cohort of 245 AA-samples from bone marrow and peripheral blood profiled with T-cell receptor beta (TCRβ) -sequencing. CD8+ T cell- and MNC-sorted samples from 153 clinically annotated AA patients were obtained from diagnosis, during remission and at relapse. To compare AA to similar diseases, we gathered 116 samples from other bone marrow failure syndromes, including MDS, PNH and hypoplastic LGL, and 45 samples from other autoimmune disorders. As healthy controls, we profiled 60 CD4+ and CD8+ T cells and utilized 786 MNC samples from public data repositories. To gain insight into T cell phenotypes, we also profiled 6 longitudinal samples with scRNA+TCRαβ-sequencing. As there are 1x1012-16 different TCRs and most of them are exclusive to individuals (private), we reasoned that by studying the most biologically interesting clonotypes from each individual, we could explain differences in disease severities, variation in treatment responses and pathogenesis. From all subjects, we selected private response clonotypes: highly expanded clonotypes (at least 1% of the total repertoire), convergent clonotypes (in which multiple nucleotide sequences converge to encode the same amino acid sequence) and from patients with AA, treatment-responding clonotypes (clonotypes that were suppressed/expanded after immune therapy). To analyse epitope-specificities of these clonotypes we leveraged TCRGP, our recently described Gaussian process method that can predict if TCRs recognize previously known epitopes. Clonotypes recognising viral epitopes (CMV, EBV and Influenza A) were enriched among private response clonotypes in comparison to the total repertoire (Fisher's exact test, p=2e-16), indicating that our filtering strategy indeed enriched for epitope-specific clonotypes. Of interest, the healthy donors' private response clonotypes showed more anti-viral clonotypes than did AA-patients (p=0.003), suggesting that in AA the epitope-specifities of private response clonotypes are not driven by common viral antigens. To identify specifities against unknown epitopes of the private response AA clones, we used an unsupervised learning strategy, GLIPH,that groups TCRs recognising the same epitope based on amino acid level similarities. Clonotypes in AA showed high convergence in their epitope-targets, as 1709 of 5744 (29.75%) clonotypes formed a single, potentially epitope-specific cluster that was not viral-specific. Similar analysis of control samples resulted in fewer clones clustering to the most prominent cluster (23.20%, p=1.63e-10), suggesting for a more homogenous target population within AA patients' clones. After showing sequence-level similarity of the private response clonotypes in AA, we aimed to link these pathological clonotypes to transcriptomes at the single-cell level using scRNA+TCRαβ-sequenced samples. The cells of the private response clonotypes showed multiple T cell phenotypes, but most cells (47.13%) in the bone marrow environment were recently activated CD8+ effector phenotype, marked by expression of GZMH, GNLY and PRF1. In comparison, the anti-viral clonotypes were mostly (37.3%) central memory phenotype (CCR7, TCF7). In serially sampled patients, anti-thymocyte globulin treatment suppressed private response clonotypes in a responding patient (55.03% of T cells to 12.79%), while the amount of these clonotypes increased in a non-responding patient (18.65% to 37.86%), where treatment mostly affected the viral-specific clonotypes. In summary, our data suggest that the private response clonotypes in immune AA patients may recognise a common antigen, which was not predicted to be viral. Further, at the single-cell level AA signature clonotypes are of effector phenotype and fluctuate following immunosuppressive treatment. Monitoring of these clonotypes throughout treatment may provide insight into disease biology and variation in treatment responses. Figure Disclosures Blombery: Janssen: Honoraria; Novartis: Consultancy; Invivoscribe: Honoraria. Maciejewski:Novartis: Consultancy; Alexion: Consultancy. Mustjoki:BMS: Honoraria, Research Funding; Novartis: Research Funding; Pfizer: Research Funding.