ALBERT

Description: Introduction: Acute leukemia is the most common malignancy in children and develops within the bone marrow. Consequently, bone marrow derived T cells of leukemia patients can be defined as tumor infiltrating lymphocytes (TILs). Dysfunctional TILs have been described in several other malignancies. However, in pediatric patients the interaction between leukemic blasts and TILs remains largely unknown. In order to understand the impact of leukemic blasts on bone marrow T cells we profiled T cells in the bone marrow of pediatric leukemia patients by surface marker and transcriptome wide analysis. Methods: First, artificial changes in marker expression due to cryopreservation and thawing were ruled out (n=5). Then, cryopreserved bone marrow samples from both pediatric patients with acute leukemia (n= 77; BCP-ALL: 18, TCP-ALL: 23, AML: 36) and age-matched healthy bone marrow donors (HD, n=23) were identified in a local biobank. Multicolor flow cytometry was performed to quantify co-inhibitory markers on CD4 and CD8 T cells in primary (n=49) and relapse leukemia samples (n=28). As we could not detect surface CTLA4 expression on T cells, CTLA4 was stained intracellularly. Additionally, RNA-Seq on sorted bone marrow derived CD8 T cells (n=48; TCP-ALL: 12, AML: 20, HD: 16) was performed. Analysis of RNA-Seq data was based on Reads Per Kilobase Million (RPKM) normalization and False Discovery Rate (FDR, Benjamini-Hochberg) statistics. 172 differentially expressed genes were found when comparing bone marrow derived CD8 T cells from healthy donors (n=16) and leukemia patients (n=32) using the following criteria: RPKM〉2 in both groups, fold change〉2 and FDR

Description: Introduction Pediatric acute lymphoblastic leukemia (ALL) is a cancer entity of minimal mutational load and low immunogenicity. The interaction of ALL cells with bone marrow (BM) T cells has not been investigated as a pathogenic driver or prognostic marker for pediatric ALL. We defined BM T cells of pediatric ALL patients as tumor-infiltrating lymphocytes (TILs) and investigated the prognostic relevance of co-stimulatory and co-inhibitory signals between ALL and BM T cells. Methods BM samples of 100 pediatric ALL patients were analyzed at time of initial diagnosis. T-cell subpopulations and expression of co-stimulatory and co-inhibitory molecules were defined by flow cytometry and correlated with clinical outcome of the patients. To investigate the role of TIM-3 for the interaction between T cells and leukemic cells, CRISPR/Cas9-mediated TIM-3 knockout (KO) was performed in primary T cells by ribonucleoprotein electroporation. T-cell activation and proliferation after contact with leukemic target cells were analyzed in TIM-3 KO cells and compared to wildtype T cells and T cells with retroviral TIM-3 overexpression. Interaction of T cells with leukemic target cells was induced by addition of anti-CD19/-CD3 bispecific T-cell engager (BiTE). Fold change (FC) of T-cell activation and proliferation was analyzed before and after co-culture. BM expression levels of known TIM-3 inducers were identified by RNA next generation sequencing of the bone marrow samples. Results Multivariate analyses identified high TIM-3 expression on CD4+ BM T cells at initial diagnosis as strong predictor for relapse of pediatric acute lymphoblastic leukemia (relapse free survival (RFS) 94.6% vs. 70.3%). The risk to develop ALL relapse was 7.1-fold higher in the group of TIM-3 high expressing patients (n=37) compared to TIM-3 low expressing patients (n=37). Expression levels of known TIM-3 ligands and inducers in the bone marrow of the patients were analyzed by RNA next generation sequencing and compared between patients with high TIM-3 expression (n=12) and low TIM-3 expression (n=15) on BM T cells. Presence of known TIM-3 ligands HMGB1 (High-Mobility-Group-Protein B1) and Galectin-9 was confirmed, but expression levels did not show significant differences. Known TIM-3 inducers IL-2, -7, -15 and -21 were not expressed on RNA level indicating that another mechanism must be responsible for TIM-3 overexpression. In vitro experiments showed that the interaction with leukemic cells induces TIM-3 expression on the surface of T cells (mean TIM-3 expression 51.1% vs. 29.7% on T cells with vs. without addition of leukemic cells, n=3). To investigate the functional relevance of TIM-3 expression in pediatric leukemia, TIM-3 KO and overexpression was performed on primary T cells. TIM-3 KO T cells showed higher activation levels after co-culture with leukemic cell lines plus CD3-/CD19-specific BiTE compared to wildtype (WT) T cells (FC of CD69 surface expression 5.0 vs. 3.2, n=3). FC of anti-leukemic proliferation was impaired in TIM-3 overexpressing T cells compared to WT T cells (FC 1.6 vs. 2.3, n=3) whereas TIM-3 KO T cells showed a higher proliferation FC compared to controls (FC 6.5 vs. 2.4, n=3). Conclusions Our study identifies TIM-3 expression on CD4+ bone marrow T cells at initial diagnosis as a strong predictor for pediatric ALL relapse. TIM-3 expression is induced by interaction of T cells with leukemic cells and results in impaired anti-leukemic T-cell activation and proliferation. TIM-3-mediated T-cell inhibition represents a new mechanism of impaired immune surveillance in pediatric ALL and blockade of this axis may be of importance for future immunotherapy in ALL. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Pearson syndrome (PS) was originally reported as a sideroblastic anemia in infancy with vacuolization of marrow precursors and exocrine pancreas dysfunction. It is now recognized as a fatal multisystem mitochondrial disorder caused by single mitochondrial DNA deletions (SLSMDs) presenting with anemia. PS, Kearns-Sayre Syndrome (KSS) and progressive external ophthalmoplegia (PEO) form a continuous spectrum of disease associated with SLSMDs. There have been only a few systematic studies on PS. Methods: We retrospectively reviewed hematological features and clinical course of 25 children with PS diagnosed between 1987 and 2019. Results: Patients presented with normo/macrocytic transfusion-dependent anemia (n=25), failure to thrive (n=3), diarrhea (n=1), acidosis (n=1) and/or omphalocele/esophageal atresia (n=1) at a median age of 5 (0-31) months. A median hemoglobin, platelet count and neutrophil count were 6.5 (1.9-9.8) g/dl, 104 (31-300) G/L, and 0.9 (0.1-2.4) G/L, respectively. Bone marrow (n=24) was normo- (n=15) or hypocellular (n=9). Vacuoles in erythroid and myeloid precursors were observed in all patients, but ring sideroblasts were present in only 16 of 23 patients examined. Morphology can resemble Diamond-Blackfan anemia (DBA) because of erythroid hypoplasia (15/21). Dysplastic features are often observed including micromegakaryocytes. Lactic acid was elevated in most patients examined (14/18). Exocrine pancreas insufficiency at diagnosis was documented in 5 patients only. The detection of SLSMDs confirmed the diagnosis of PS in all patients. The median age at the time of the last follow-up was 47 (7 - 183) months. Among 11 patients with hematological follow-up for more than 3 years after diagnosis, 8 had spontaneous resolution of anemia at a median age of 28 (12-67) months, and 3 died at the age of 3, 6 or 8 years without hematological recovery. Clinical course was highly heterogeneous and various organ dysfunctions appeared. Renal tubulopathy/Fanconi syndrome (n=7) and cardiomyopathy/arrhythmia (n=5) were often fatal complications which developed at the median age of 32 and 45 months, respectively. Failure to thrive/short stature (n=13) and muscle hypotonia (n=9) were commonly observed. Other complications included pancreas insufficiency (n=7), liver dysfunction (n=4), endocrine dysfunctions (n=7), hearing loss (n=1), ophthalmoplegia (n=1), retinitis pigmentosa (n=1), cataract (n=1), ataxia (n=2) and encephalopathy (n=1). Thirteen patients died of acute metabolic acidosis with/without other complications (n=7), arrhythmia (n=2), respiratory failure (n=3) and liver/renal failure (n=1) at the median age of 50 (14-183) months. Two patients developed KSS and PEO-like phenotypes at the age 92 months and 19 months, respectively. Summary: Anemia is generally the only presenting syndrome of PS. While the bone marrow morphology can resemble DBA or myelodysplastic syndrome, recognition of vacuolated myeloid/erythroid precursors lead to the correct diagnosis of PS in all cases. Other classical signs of PS, ring-sideroblasts and pancreas insufficiency, are often missing. Anemia spontaneously resolves in most patients surviving early childhood. However, PS is unexceptionally fatal (Figure), most patients succumb to metabolic acidosis and various forms of multi-organ failure. Since there is no effective therapy, the diagnosis of PS is one of the saddest news that pediatric hematologists have to break to parents of an anemic infant. Figure Disclosures Niemeyer: Celgene: Consultancy.

Description: Background: HLA haploidentical stem cell transplantation with T-replete grafts and post-transplant immunosuppression with high-dose cyclophosphamide after reduced intensity conditioning has evolved into an increasingly accepted method of alternative donor transplantations in adults. Reported transplantation related mortality, GVHD and relapse rates are at least comparable to those of HLA matched donor transplantations. Purpose: To assess the feasibility of this approach in children as a first or second allogeneic transplantation after myeloablative or reduced toxicity conditioning. Patients/methods: Ten consecutive transplantations in children with either malignant (n=6) or non-malignant diseases (n=4) at a single institution transplanted with unmanipulated bone marrow from parental HLA haploidentical donors were analyzed. In the malignant cohort (3 ALL, 2 AML, 1 MDS) four patients were in advanced disease status (2 NR, 2 CR3) and three had received a prior allogeneic transplantation (2 relapses, 1 rejection). Conditioning in these patients was either TBI-VP16 (n=3) or myeloablative treosulfan-based (n=3). Among the non-malignant patients (2 IL10R deficiency, 1 sickle cell disease, 1 XIAP), one had rejected a prior T-cell depleted HLA haploidentical transplantation. These patients were conditioned with alemtuzumab, treosulfan, fludarabine ± thiotepa. All patients in both groups received cyclophosphamide 14,5mg/kg on days -3 and -2. GVHD prophylaxis consisted of cyclophosphamide 50mg/kg on days +3 and +4, tacrolimus and MMF from day +5. In the absence of GvHD MMF was stopped on day +35, tacrolimus tapered from day +60 (malignant) or day+100 (non-malignant). Results: After a median follow-up of 6 months (1-25), 9 of 10 patients are fully engrafted, alive and free of disease. One patient with AML not in remission at transplantation died from pulmonary hemorrhage on day +24. He was also treated for CMV viremia pre-existing before transplantation. One ADV reactivation requiring pre-emptive treatment and no invasive fungal infections were noted in the other patients. Engraftment was recorded at a median of 17 days (neutrophils) and 26 days (platelets). Only in one patient transient acute skin GVHD (overall grade II) was observed, while no patient developed chronic GVHD. Mean CD3 counts of 310/µl, 937/µl and 1868/µl, CD4 115/µl, 291/µl and 1010/µl, CD8 176/µl, 589/µl and 802/µl and CD19 102/µl, 360/µl and 641/µl were measured on days +100, +180 and +365 respectively. Conclusion: HLA haploidentical transplantation with post-transplant cyclophosphamide appears to be a safe and promising approach with very low GVHD rates and excellent immune reconstitution in children with malignant as well as non-malignant diseases. This may be combined with myeloablative as well as reduced toxicity conditioning regimes and is a promising approach even as a second allogeneic procedure after relapse or rejection following a first allogeneic transplant. Disclosures No relevant conflicts of interest to declare.

Description: Emerging immunotherapies such as chimeric antigen receptor T cells have advanced the treatment of acute lymphoblastic leukemia. In contrast, long-term control of acute myeloid leukemia (AML) cannot be achieved by single lineage-specific targeting while sparing benign hematopoiesis. In addition, heterogeneity of AML warrants combinatorial targeting and several suitable immunotargets (HAVCR2/CD33 or HAVCR2/CLEC12A) were identified in adult AML. However, clinical and biologic characteristics differ between children and the elderly. Here, we analyzed 36 bone marrow (BM) samples of pediatric AML patients and 13 age-matched healthy donors using whole RNA-sequencing of sorted CD45dim and CD34+CD38-CD45dim BM populations and flow cytometry for surface expression of putative target antigens. Pediatric AML clusters apart from healthy myeloid BM precursors in principle component analysis. Immunotargets known from adult AML such as IL3RA were not overexpressed in pediatric AML compared to healthy precursors by RNA-sequencing. CD33 and CLEC12A were the most upregulated immunotargets on RNA level and showed the highest surface expression on AML detected by flow cytometry. KMT2A mutated infant AML cluster separately by RNA-sequencing, overexpress FLT3 and hence CD33/FLT3 co-targeting is an additional specific option for this subgroup. CLEC12A and CD33/CLEC12Adouble-positive expression was absent in CD34+CD38-CD45RA-CD90+ hematopoietic stem cells (HSC) and both are restricted to healthy hematopoietic tissue, while CD33 and FLT3 is expressed on HSC. In summary, we show that expression of immunotargets in pediatric AML differs from known expression profiles in adult AML. We identify CLEC12A/CD33 as preferential generic combinatorial immunotargets in pediatric AML and CD33/FLT3 as immunotargets specific for KMT2A mutated infant AML.

Description: The cure rate in pediatric acute lymphoblastic leukemia (ALL) is about 85%. Therapy failures are mainly due to relapse. Relapse rates could possibly be influenced by the anti-leukemic activity of the immune system, e.g. by the Th1/Th2 balance. It would be desirable to increase the cure rate if the immune system could be induced to participate in the elimination of leukemia cells. However, leukemia cells, especially ALL cells are poor stimulators for T cells and do not induce a Th1 response believed to be necessary for tumor cell elimination. Missing costimulatory molecules on the leukemia cells, especially CD80 and CD86, are commonly accepted as the main reason for their poor immunostimulatory activity. On the other hand, efficient costimulation does not guarantee improved cure rates. On the contrary, in a mouse model inoculation of CD86 transfected lymphoma cells led to tumor progression possibly by interaction with CD152 or induction of a Th2 response. Indeed, in an earlier study, we examined the mRNA expression of CD80 and CD86 in marrow samples of ALL patients and found an increased expression of CD86 and IL-4 in patients with a late leukemic relapse raising the possibility of a Th2 shift predisposing to relapse (Stachel et al, Eur J Med Res, 2006). It becomes increasingly clear that members of the costimulatory family other than CD80 and CD86 also influence the immune response. ICOS/ICOS ligand and PD-1/PD1 ligand both play an important role in the second step of Th2 induction. To determine whether increased or decreased expression of costimulatory molecules play a role in the pathogenesis of relapse in pediatric ALL we examined the expression of various costimulatory molecules in leukemic marrow samples in a prospective study. Samples from 49 consecutive pediatric patients with B cell precursor acute lymphoblastic leukemia (BCP ALL) were analyzed by semiquantitative RT-PCR for CD28, CD152 (CTLA-4), ICOS and ICOS ligand (B7RP-1). A total of 29 patients (60.4 %) remained relapse free and 19 (39.6 %) relapsed after a median follow up of 24.5 months (range: 6 to 50 months). Of those relapsing five patients suffered a very early relapse (VER, within 18 months from diagnosis), seven an early relapse (ER, between 18 and 30 months from diagnosis) and seven patients a late relapse (LR, later than 30 months from diagnosis). We found that in the subgroup of ALL patients experiencing a VER expression of mRNA ICOS ligand was significantly increased (2.0 +/− 0.7 (mean +/− SD relative intensity units) compared to non-relapsing patients (1.1 +/− 0.8, p=0.02). This trend was also visible in the late relapsing patient group (1.4 +/− 0.8 vs 0.9 +/− 0.6, ns). By comparative densitometry we estimated the copy number of ICOS ligand in the VER group to be 500,000 per 12,500 BM cells vs 350,000 per 12,500 BM cells in non-relapsing ALL patients. Since ICOS ligation on T-cells by ICOS ligand on B-cells appears to favor a Th2 polarization of the immune system our results could provide further evidence that a Th2 shift induced by leukemic blasts could confer an increased risk for very early ALL relapse. These data could provide a rationale for immunotherapeutic strategies aimed at strengthening a Th1 polarized immune response in the treatment for childhood BCP ALL.

Description: MicroRNAs (miRNAs) are conserved 21−23 nt non-coding RNA molecules that regulate gene expression either by mRNA cleavage or by repression of mRNA translation. miRNAs regulate many different processes, including apoptosis and cell proliferation and may therefore also play a critical role in oncogenic transformation. To date, most miRNAs have been discovered by cDNA cloning and sequencing, though other profiling methods, such as miRNA micro-arrays, have recently been applied. Profiling of miRNA expression by cloning has the advantage of identifying new miRNA genes, and if a large number of clones are sequenced, to also be quantitative. In addition the exact sequence is determined and polymorphisms and mutations in any miRNAs can readily be detected. To get an insight in the role of miRNAs in the differentiation and maturation of hematopoetic cells as well as their contribution to oncogenesis in ALL and lymphomas, we cloned and sequenced various cell lines and patient samples:five cell lines (B-ALL, AML, Burkitt Lymphoma); samples from sorted blood cells covering pluripotent stem cells, B-, T-, NK- cells, monocytes and granulocytes; eight patient samples with ALL (2 pro-B-ALL, 2 pre-B-ALL, 2 cALL, 2 T-ALL) at the time point of diagnosis; four additional samples of these patients with B-ALL and two samples of T-ALL patients each after 36 days of treatment according to the protocol of the German Cooperative Acute Lymphoblastic Leukemia study group (COALL-07-03). We also recorded the small RNA profiles of three patients with various forms of AML at diagnosis and after the first induction according to the protocol of the AML-BFM 2004 study; two Burkitt lymphoma samples and one B-Non Hodgkin Lymphoma (B-NHL) sample. We report here the identification of over 20 novel human miRNAs in these samples. To determine specific expression patterns, the miRNA profiles were compared to a reference set of 22 different human tissue types. Some miRNAs are expressed in a cell or tissue specific manner, others have a more general expression pattern between different cell types and tissues. For example human miRNA miR-142 is ubiquitously expressed in cells of the hematopoetic lineage, whereas human miR-150 is only expressed in differentiated hematopoetic cells, but not in hematopoetic stem cells. In hematopoetic stem cells human miR-126 is 3 to more than 10 times higher expressed than in differentiated hematopoetic cells. The existence of the latter two in humans are first described in this study. miR-16 on the other hand is expressed in all cell types examined including non-hematopoetic. Furthermore, miRNAs are up/down-regulated in ALL and NHL patient samples. In conclusion, this study identifies miRNAs that might be involved in hematopoetic cell differentiation and maturation and is important to identify miRNAs that might contribute to oncogenesis in leukemia and lymphomas.

Description: Although multi-agent chemotherapy is remarkably successful in the treatment of pediatric acute lymphoblastic leukemia (ALL), about 25% of patients experience relapse. A possible way to increase the cure rate for leukemia could be to modulate the immune response against leukemic cells. It is known that ALL blasts are poor stimulators of cellular immune responses. This is believed to be mainly the result of a reduced presentation of costimulatory molecules on the surface of the ALL blasts. For B cells, CD40 is an essential molecule which can be targeted therapeutically through its ligand, CD40 ligand or CD152. Normal B cells can induce cytotoxic T cell responses following stimulation with a soluble trimeric CD40 ligand. We therefore hypothesized that by coculture with CD40 ligand transfected HeLa cells for four days, pediatric ALL blasts could be induced to upregulate costimulatory molecules, apoptosis inducing ligands and cytokines. The expression of the surface antigens CD40, CD40 ligand, CD80, CD86, CD95/fas, HLA-ABC and HLA-DR was evaluated by flow cytometry and the RNA expression of CD40, CD80, CD86, Fas ligand (FasL), TRAIL, IL-4, IL-6, IL-10, TGF-β was assessed by semiquantitative RT-PCR. We found that coculture with CD40 ligand transfected HeLa cells significantly increased the expression of CD40 ligand, CD80, CD86 and CD95/fas while the expression of HLA-ABC, HLA-DR and CD40 remained unchanged. We found a significantly increased RNA expression of IL-6, TGF-β and CD80, a small but not significant increase of CD86, but no change in expression for IL-4, IL-10, FasL, TRAIL and CD40 after coculture. Addition of IL-4 to the culture did not influence the results. In summary CD40 ligand mediated stimulation lead to increased expression of costimulatory molecules CD80 and CD86 and the pro-apoptotic CD95/fas, while the anti-apoptotic cytokines IL-6 and TGF-β were also upregulated. Our results show that stimulation through CD40 ligation upregulates costimulatory molecules on pediatric ALL blasts, while it has differential effects on apoptosis inducers. This might have functional consequences which could be therapeutically exploited to modulate the immune system in an attempt to increase cure rates in pediatric ALL.