ALBERT

Description: Background: T effector cells (Teff) within the stem cell graft in allogeneic hematopoietic stem cell transplantation (HSCT) can elicit disabling acute graft-versus-host disease (aGvHD) contributing to transplant-related mortality. Teff as donor lymphocyte infusion (DLI) are a therapeutic option to re-induce complete remission (CR) after leukemia relapse. Usually DLIs are given in dose escalating regimens until CR is achieved or first signs of aGvHD develop. To monitor the DLI induced allo-immune response and the efficacy of aGvHD treatment is a clinical challenge, since no established biomarkers are available. T regulatory cells (Tregs) are thought to play an important role in balancing immune responses and studies have shown effects of adoptive Treg transfer as a therapeutic option in GvHD. T cell receptor (TCR) sequencing of T cell subsets, such as Tregs, after DLI might allow the identification of clones inducing control of GvHD. Here we report data of 29 DLI patients with a median follow-up of 〉1 year allowing us to thoroughly analyze differences of the TCR repertoire in patients with or without aGvHD. Aims: We aimed to analyze Treg TCR diversity and clonality changes over time as a potential biomarker for development and treatment response of aGvHD following DLI. Patients and Methods: The study cohort consisted of 29 leukemia patients after HSCT who received DLI treatment for recurrence of disease, molecular relapse or high risk phenotype. Blood samples were taken before and after DLI. A median of 4 (range 2-6) blood samples per patient were available for TCR-sequencing. The last sample was taken at a median of 123 (27-530) days post DLI. After generation of PBMCs CD4+CD127-CD25+ Tregs were FACS-sorted for cDNA-based CDR3-region amplification of the TCR-β chain. CDR3 amplicons were sequenced on the Illumina MiSeq platform and annotated using the IMGT.org database. Further bioinformatic analyses were based on VDJtools and the tcR R-package. Results: In 18/29 patients we observed clinical symptoms of aGvHD, with blood samples available of the acute onset in 12/18 cases. Treg frequencies and absolute numbers did not differ between aGvHD and noGvHD samples. Treg TCR diversity, assessed via inverse Simpson's diversity index (1/D) increased on average by 322% at the first occurrence of aGvHD compared to the previous sample (Figure 1A, B). Stratifying for aGvHD severity (total grade 1-2 vs. 3-4) did not reveal any group differences. However, stratifying by organ involvement (skin vs. GI/liver) showed a more pronounced increase of 1/D in patients with aGvHD of the GI tract and/or the liver. Moreover, in 11 subjects blood samples were available a median of 7 (3-14) days prior to aGvHD diagnosis. Already at this preclinical time point we detected an increased 1/D of +361% (Figure 1A, B). Again, aGvHD organ involvement significantly affected the result, with GI/liver involvement mainly driving this effect (+567% vs. +1% 1/D). In contrast, patients who did not develop aGvHD at any time after DLI showed on average a slight decrease of -8% 1/D compared to the previous time point. Next, we analyzed all aGvHD patients with at least one sample available after initiation of treatment (local and/or systemic steroids). Control or remission of aGvHD symptoms was accompanied by decreased 1/D of on average -58% compared to the previous samples (Figure 2). In 9/11 patients we saw a focusing of the Treg TCR repertoire; in the other two (Figure 2, red lines) no or only partial clinical response to systemic steroids was reported. Conclusion: Our data describe detailed changes in the Treg compartment on the TCR level following development and treatment of aGvHD. Currently, aGvHD diagnosis following DLI treatment relies solely on clinical symptoms, deciding whether further dose increments of DLI can be administered. As our data suggest, Treg TCR sequencing may support transplant specialists with (1) detection of patients at risk for aGvHD, even prior to clinical symptoms, (2) identification of patients eligible for further dose increments (compare Figure 1B), and (3) assessment of aGvHD treatment with guidance whether higher dosage of steroids and/or alternative immunosuppressive treatment might be required (compare Figure 2, red lines). Future prospective studies are needed to replicate these findings in a large cohort, potentially enabling identification of specific Treg TCR clones controlling the allo-immune response in aGvHD. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees. Koenecke:BMS: Consultancy; abbvie: Consultancy; Amgen: Consultancy; Roche: Consultancy.

Description: Damage of endothelial cells (EC) is known to be involved in pathogenesis of microangiopathy, hepatic veno-occlusive disease, sepsis, capillary leak syndrome, and acute or chronic graft-versus-host disease (GvHD) as major causes of morbidity and mortality in patients after hematopoietic stem-cell transplantation (HSCT). We recently demonstrated in 39 patients undergoing allogeneic stem cell transplantation that numbers of circulating EC (CEC) increased significantly after conditioning regimens and that patients treated with reduced intensity conditioning (RIC) showed significantly lower cell numbers (Woywodt et al., Blood, 2004 May 1;103(9):3603-5). Here we report on the measurement of plasma levels of von Willebrand factor (vWF), thrombomodulin (TM), PAI-I and TAFI in the course of HSCT. Measurement of vWF, TM, PAI-I and TAFI was performed in the 39 patients (20 male, 19 female; n = 21: HLA-matched unrelated donor; n = 18: related donors). Blood samples were collected before starting conditioning regimen (day −7), the day before transplantation (day −1) and at day +7, +14 and +21 after transplantation. 28 patients received a conventional regimen with cyclophosphamide (120 mg/kg) and either total body irradiation with 12 Gy (n = 14) or busulfan (16 mg/kg, n = 14). 11 patients undergoing stem-cell transplantation were treated with reduced intensity conditioning with fludarabine (150 mg/m²) and busulfan (8 mg/kg body) or melphalan (120 mg/m²). Median baseline vWF was elevated (262%, range 68–612, normal range: 50–150). Median baseline PAI-I (11.3 U/l; range 1.8–34.4; normal range: 〈 20) and median baseline TAFI (90%; range 46–126; normal range: 70–120) were within normal limits. TM level was lower than normal values (median 3.95 ng/ml; range 2.69–9.36; normal range: 6.6–10.6). Levels of vWF increased after conditioning regimen and remained elevated until day +21 (day −1: median 262; day+7: 268; day +14: 327; day +21: 374; p 〈 0.01). Median TM remained low at all time points (day −1: median 4.26; day +7: 3.86; day +14: 3.97; day +21: 4.52). Levels of TAFI remained unchanged (day −1: median 82; day +7: median 91, day +14: median 88; day +21: median 89). There were also no differences in levels of PAI-I before or after conditioning regimen or after transplantation (day −1: median 11.4, day +7: median 10.8, day + 14: median 14, day + 21: median 11.8). There was no significant difference in vWF in patients undergoing reduced intensity conditioning compared to patients treated with conventional regimens. Interestingly, there was no correlation between the endothelial markers as vWF and TM and numbers of CEC. Our data indicate that significant alterations of the hemostatic system occur in patients undergoing HSCT. Further studies are warranted to define the clinical role of both, hemostatic alterations and CEC in the course of HSCT.

Description: In a phase I/II clinical trial investigating the prophylactic infusion of suicide gene-modified donor T cells in the context of haploidentical hemopoietic cell transplantation (haplo-HCT) for the treatment of high-risk leukemia, we observed a rapid and effective immune reconstitution. After activation with anti-CD3 antibodies, genetic modification of donor T cells was accomplished with a retroviral vector encoding for the Herpes Simplex thymidine kinase (TK). In vitro before infusion and in vivo at immune reconstitution, TK+ cells displayed an effector memory (EM) phenotype (CD45RA–CD62L−, CD28±CD27+, IL-2±IFN–γ+). The graft-versus-leukemia (GvL) effect was substantial in patients transplanted in remission, but failed to cure patients in relapse. Gene targeting with retroviral vectors is limited to memory T cells. Central memory (CM) T cells (CD45RA–CD62L+, CD28+CD27+, IL-2+IFN-γ±) share many characteristics with stem cells, namely the ability to self-renew and to differentiate into a progeny of effector cells. EM TK+ cells have a reduced alloreactivity. Recently, it has been proposed that alloreactivity may be confined to memory T cells with stem cell-features. Since alloantigens are the target not only of graft-versus-host disease (GvHD), but also of the GvL effect, crucial to the success of the strategy is the suicide gene-modification of this subset of memory T cells. We found that addition of CD28 costimulation on cell-sized beads and the use of homeostatic cytokines, such as IL-7 and IL-15, generates central memory (CM) TK+ cells. CM TK+ cells are highly alloreactive, both in vitro and in vivo in a humanized animal model of GvHD based on the grafting of human skin onto NOD/scid mice. Interestingly, CM TK+ cells express the IL7Rα, a marker associated with the stem cell-features of memory T cells. Moreover, IL7Rα expression is maintained after stimulation with alloantigens. Stimulation of CM, but not of EM TK+ cells with autologous dendritic cells pulsed with restricted peptides from the minor histocompatibility alloantigen (mHag) HA-1 or H-Y efficiently induces mHag-specific effector T cells that lyse natural ligand expressing HLA-A2+ targets. TK+ mHag-specific effector T cells also lysed mHag+HLA-A2+ leukemic cells and, when infused in conditioned NOD/scid mice harboring human leukemia, significantly delayed disease progression. Altogether, these data suggest that optimal T-cell receptor triggering and homeostatic cytokines are required for retroviral targeting of a suicide gene to alloreactive memory stem T cells and warrant their use for a safe and powerful GvL effect.

Description: Clinical responses of solid tumors after allogeneic human leukocyte antigen-matched stem cell transplantation (SCT) often coincide with severe graft-versus-host disease (GVHD). Targeting minor histocompatibility antigens (mHags) with hematopoiesis- and cancer-restricted expression, for example, HA-1, may allow boosting the antitumor effect of allogeneic SCT without risking severe GVHD. The mHag HA-1 is aberrantly expressed in cancers of most entities. However, an estimated 30% to 40% of solid tumors do not express HA-1 (ie, are HA-1neg) and cannot be targeted by HA-1–specific immunotherapy. Here, we investigated the transcriptional regulation of HA-1 gene expression in cancer. We found that DNA hypermethylation in the HA-1 promoter region is closely associated with the absence of HA-1 gene expression in solid tumor cell lines. Moreover, we detected HA-1 promoter hypermethylation in primary cancers. The hypomethylating agent 5-aza-2′-deoxycytidine induced HA-1 expression only in HA-1neg tumor cells and sensitized them for recognition by HA-1–specific cytotoxic T lymphocytes. Contrarily, the histone deacetylation inhibitor trichostatin A induced HA-1 expression both in some HA-1neg tumor cell lines and in normal nonhematopoietic cells. Our data suggest that promoter hypermethylation contributes to the HA-1 gene regulation in tumors. Hypomethylating drugs might extend the safe applicability of HA-1 as an immunotherapeutic target on solid tumors after allogeneic SCT.

Description: Background: Relapse of disease remains a frequent cause of mortality after allogeneic hematopoietic stem cell transplantation (HSCT). For patients with imminent relapse, therapeutic donor lymphocyte infusions (DLI) offer a treatment option for re-induction of complete remission of the underlying disease by employing the graft-versus-leukemia (GvL) effect. Moreover, DLI is used pre-emptively to prevent relapse in patients with high risk disease. DLI is given in increasing doses, adapted to clinical signs of graft-versus-host disease (GvHD) or molecular markers of disease, i.e. donor chimerism or minimal residual disease. There is a need for reliable monitoring of the immunologic response preceding the clinical outcome, enabling a precise administration of DLI dose escalation and thus possibly preventing overshooting immune reactions. With the advent of deep sequencing technology direct measure of high resolution T cell receptor (TCR) diversity can be used as a read-out of the immunologic response in the patient at the molecular level. Aims: In this study we aim to elucidate whether TCR-diversity can serve as a biomarker for clinical outcome of DLI treatment. Patients and Methods: We assessed 19 patients, who received DLI after HSCT. Therapeutic DLI was given in 14 patients and prophylactic DLI in 5 patients. The majority of patients (n=14) was treated for AML or MDS, 2 for ALL, and 3 for MPN. Peripheral blood samples for TCR-sequencing were obtained from the patient pre-DLI, at 2 and 4 weeks post DLI, and subsequently when available. Also, donor samples were collected. Donor's and patient's lymphocytes were FACS-sorted into CD8+ and CD4+ conventional (Tconv) and CD4+CD127-CD25+ regulatory T cells (Treg). Sorted subpopulations were subject to cDNA-based CDR3-region amplification by RACE-PCR allowing assessment of the entire TCR-β repertoire. Reliable generation of CDR3 amplicons was possible from as few as 4000 cells. These were then sequenced using the Illumina MiSeq platform. Reads were annotated by the IMGT.org database; further bioinformatics analyses included VDJtools and the tcR R-package. Results: GvL response (remission or stable disease) could be achieved in 14/19 patients; 8 of these patients developed acute GvHD ≥III°. Flow cytometric analysis showed that the ratio between CD8+ and CD4+ Tconv in the DLI is predictive of response to DLI: DLIs containing a majority of CD4+ Tconv were associated with development of GvHD (n=8, CD8:CD4 = 35.3%:54.9%) , whereas patients responding to DLI with GvL only had received a higher proportion of CD8+ T cells (n=6, CD8:CD4 = 59.1%:29.5%); comparison of ratios met statistical significance (Mann Whitney test, p=0.0027). TCR sequencing revealed that the diversity of the TCR-repertoire seems to be predictive of the clinical course of the patient after DLI. GvHD development within 2 weeks of blood sampling time (n=4) was preceded by an increase of Treg repertoire diversity (assessed with inverse Simpson's diversity index, 1/Ds). Compared to the pre-DLI repertoire 1/Ds, mean increase was 143.67% vs. a 36.04% decrease in patients not developing GvHD (n=7; Mann Whitney test, p=0.02). Steroid treatment of GvHD (n=2) led to a mean decrease of 49.71% of Treg TCR 1/Ds compared to the previous time point. Analysis of GvL response revealed an association of remission induction with a trend towards decreased 1/Ds of the CD8+ TCR-repertoire (mean decrease of 27.66% at d28 after DLI compared to pre-DLI vs. 0.31% decrease in patients showing no GvL effect). Assessment of TCR CDR3 region clonotype expansion over time is shown for a patient responding with GvHD (Fig. 1A) and GvL (Fig. 1C) to DLI and a patient progressing without response to DLI (no GvHD Fig. 1B, no GvL Fig. 1D). Conclusion: Our data indicate that TCR sequencing allows the assessment of TCR-diversity change as a surrogate parameter of DLI response. While an increase of Treg diversity seems to indicate the development of GvHD, repertoire compression of the CD8 compartment may be predictive of GvL response. Taken together, monitoring TCR repertoires may become a valuable predictive tool to improve DLI therapy and furthermore, analysis of expanding clonotypes after DLI enables identification of individual CDR3 sequences associated with DLI response. Disclosures Heuser: Pfizer: Research Funding; Novartis: Consultancy, Research Funding; BerGenBio: Research Funding; Karyopharm Therapeutics Inc: Research Funding; Tetralogic: Research Funding; Celgene: Honoraria; Bayer Pharma AG: Research Funding.

Description: Background: ADGRE2, CCR1, CD70, and LILRB2 expressed on the surface of myeloid blasts but not normal hematopoietic stem cells, T cells or other tissues have been recently suggested as candidate chimeric antigen receptor (CAR) targets for engineered T cells in acute myeloid leukemia (AML) patients. Aim: To validate the expression pattern of the recently identified candidate CAR targets ADGRE2, CCR1, CD70, and LILRB2 on leukemic blasts in a cohort of newly diagnosed AML patients. Methods: 109 patients with de novo (n=87) or secondary (n=22) AML were included in the analysis. Patients were classified according to the 2008 WHO classification and cytogenetically characterized by chromosome banding analysis. Molecular analyses were performed by Sanger and next-generation sequencing (NGS) with a panel of 46 genes. Bone marrow or peripheral blood samples from the time of first diagnosis were obtained to perform multi-color flow cytometry analysis evaluating the expression levels of ADGRE2 (also known as EMR2 or CD132), CCR1 (also known as CD191), CD70 and LILRB2 (also known as CD85d) antigens on the surface of myeloid blasts. Patients with expression of the marker on ≥20% of blast cells were defined positive. Informed consent was obtained from all patients in accordance to the declaration of Helsinki and institutional guidelines. Results: ADGRE2, CCR1, CD70 and LILRB2 were expressed in 100%, 70%, 27.5%, 27.5% of patients with a median expression on myeloid blasts of 87.8% (range 30.4-99.8%), 43.9% (range 21.1-86.2%), 29.0% (range 20.0-55.4%), and 35.6% (range 20.7-90.6%), respectively. A subset analysis was performed to determine expression levels of the candidate targets in CD3 positive cells. Of patients with positive marker expression on blast cells ADGRE2, CCR1, CD70 and LILRB2 were expressed on 14.5% (range 0.0-64.2%), 19.9% (0.0-73.0%), 15.3% (range 0.0-35.1%), and 2% (range 0.1-18.4%) of CD3+ T cells, respectively. The proportion of patients with ≥80% expression on blasts was 61.5% (n=67), 0.9% (n=1) and 3.7% (n=4) for ADGRE2, CCR1 and LILRB2, respectively. Of those 53.7%, 0%, and 100% had marker expression

Description: Introduction: The combination treatment of venetoclax (VEN) with both low-dose cytarabine (LDAC) and hypomethylating agents (HMA) in untreated primarily elderly AML patients yielded promising response rates leading to its approval for newly diagnosed AML patients who are 75 years or older, or who have comorbidities that preclude use of intensive induction chemotherapy. Prolonged cytopenias are of potential concern in venetoclax treated patients, especially in patients who underwent allogeneic hematopoietic cell transplantation (alloHCT) prior venetoclax treatment. Objective: To compare hematologic recovery in patients treated with VEN in combination with intensive and non-intensive chemotherapy regimens for the treatment of relapsed or refractory (R/R) acute myeloid leukemia (AML) depending on the pretreatment status for alloHCT. Methods: In this retrospective controlled study (www.clinicaltrials.gov NCT03662724), we included patients aged 18 years or older with R/R acute leukemia previously treated with VEN (days 1-7) combined with intensive salvage chemotherapy (fludarabine, cytarabine, idarubicin - FLAVIDA) or VEN combined with non-intensive regimens, namely HMA or LDAC. Eighty-one patients who were treated with FLA-IDA for R/R AML served as control for the intensively treated patients included in this analysis. Responses were evaluated per revised International Working Group criteria for AML. Main outcome measure was the rate of objective response (complete remission [CR] + CR with incomplete blood count recovery [CRi] + partial remission [PR] + morphologic leukemia-free state (MLFS; defined as less than 5% blasts in an aspirate sample). Safety and efficacy analyses included all patients who received at least one cycle of VEN combination treatment. This study was approved by the local Ethics Review Committee in accordance with the Declaration of Helsinki. Results: Between January 2017 and May 2019 49 patients with a median age of 59 years (range 18-80) received VEN with either FLA-IDA (n=14), HMA (n=31) or LDAC (n=4) and had safety and efficacy outcomes reported. The patient cohort was a high-risk cohort of relapsed (n=24) and refractory (n=25) patients. The analysis included 24 patients (49%) with secondary AML and two patients with biphenotypic acute leukemia (BAL). Twenty-two patients (45%) had received prior alloHCT and 7 (14%) had relapsed

Description: In allogeneic hematopoietic cell transplantation (HSCT), donor T lymphocytes mediate the graft-versus-leukemia (GVL) effect, but induce graft-versus-host disease (GVHD). Suicide gene therapy—that is, the genetic induction of a conditional suicide phenotype into donor T cells—allows dissociating the GVL effect from GVHD. Genetic modification with retroviral vectors after CD3 activation reduces T-cell alloreactivity. We recently found that alloreactivity is maintained when CD28 costimulation, IL-7, and IL-15 are added. Herein, we used the minor histocompatibility (mH) antigens HA-1 and H-Y as model alloantigens to directly explore the antileukemia efficacy of human T cells modified with the prototypic suicide gene herpes simplex virus thymidine kinase (tk) after activation with different stimuli. Only in the case of CD28 costimulation, IL-7, and IL-15, the repertoire of tk+ T cells contained HA-1– and H-Y–specific CD8+ cytotoxic T cells (CTL) precursors. Thymidine kinase–positive HA-1– and H-Y–specific CTLs were capable of self-renewal and differentiation into potent antileukemia effectors in vitro, and in vivo in a humanized mouse model. Self-renewal and differentiation coincided with IL-7 receptor expression. These results pave the way to the clinical investigation of T cells modified with a suicide gene after CD28 costimulation, IL-7, and IL-15 for a safe and effective GVL effect.

Description: Molecular measurable residual disease (MRD) assessment is not established in approximately 60% of acute myeloid leukemia (AML) patients because of the lack of suitable markers for quantitative real-time polymerase chain reaction. To overcome this limitation, we established an error-corrected next-generation sequencing (NGS) MRD approach that can be applied to any somatic gene mutation. The clinical significance of this approach was evaluated in 116 AML patients undergoing allogeneic hematopoietic cell transplantation (alloHCT) in complete morphologic remission (CR). Targeted resequencing at the time of diagnosis identified a suitable mutation in 93% of the patients, covering 24 different genes. MRD was measured in CR samples from peripheral blood or bone marrow before alloHCT and identified 12 patients with persistence of an ancestral clone (variant allele frequency [VAF] 〉5%). The remaining 96 patients formed the final cohort of which 45% were MRD+ (median VAF, 0.33%; range, 0.016%-4.91%). In competing risk analysis, cumulative incidence of relapse (CIR) was higher in MRD+ than in MRD− patients (hazard ratio [HR], 5.58; P 〈 .001; 5-year CIR, 66% vs 17%), whereas nonrelapse mortality was not significantly different (HR, 0.60; P = .47). In multivariate analysis, MRD positivity was an independent negative predictor of CIR (HR, 5.68; P 〈 .001), in addition to FLT3-ITD and NPM1 mutation status at the time of diagnosis, and of overall survival (HR, 3.0; P = .004), in addition to conditioning regimen and TP53 and KRAS mutation status. In conclusion, NGS-based MRD is widely applicable to AML patients, is highly predictive of relapse and survival, and may help refine transplantation and posttransplantation management in AML patients.

Description: Background: Relapse occurs in 30-40% of AML patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). Detecting molecular relapse before clinical relapse offers the opportunity of early interventions (e.g. donor lymphocyte infusions, reduction of immunosuppression etc.). Next-generation sequencing (NGS)-based error-corrected sequencing approaches have shown promising results in AML patients prior to alloHSCT, which identified MRD in 45% of patients and predicted a cumulative incidence of relapse of 66% versus 17% in MRD negative patients at 5 years. However, NGS-based MRD is not well studied in patients after alloHSCT. Aim: To evaluate the prognostic impact of MRD on day 90 and day 180 after alloHSCT in AML patients in morphologic complete remission (CR) using error-corrected NGS applicable to the majority of AML patients. Patients and Methods: We quantified MRD in 138 patients who underwent myeloablative (MA, n=47) or reduced-intensity conditioned (RIC, n=91) alloHSCT for AML on day 90 and 180 after alloHSCT. All patients had at least one mutation at the time of diagnosis that was identified by NGS with a myeloid panel on the Illumina platform. Amplicon-based error-corrected sequencing and bioinformatics analysis was applied to samples on day 90 (n=133) and day 180 (n=125) after alloHSCT as described previously (Thol et al. Blood 2018). In the first approach we analysed 1-2 diagnostic mutations (=limited marker approach). In the second approach an extended marker set with (2-4) markers was used (=extended marker approach). Genomic DNA from peripheral blood (PB) was used for the majority of analyses (PB n= 394; bone marrow n=17). Cumulative incidence of relapse (CIR) and non-relapse mortality (NRM) were evaluated by competing risk analysis. Results: The median follow up time of the cohort was 5.5 years. The mean limit of detection was a variant allele frequency (VAF) of 0.012% using error correction and 0.071 when using forward/reverse read error correction. MRD positivity on day 90 and/or day 180 was detected in 22 out of 138 patients (16%) with the limited marker approach, while MRD was found in 28 patients (20.3%) with the extended marker approach. Using the limited marker approach, the 5-year CIR was 52% for MRD positive and 30% for MRD negative patients (P=0.001), while NRM was similar between both groups (Figure 1A). Overall survival (OS) was shorter in MRD positive patients compared to MRD negative patients (P=.044, Figure 1B). In multivariate analysis using variables significant in univariate analysis (P