The major cause of death after allogeneic Hematopoietic Stem Cell Transplantation (HSCT) for acute myeloid leukemia (AML) is disease relapse. We investigated the expression of Inhibitory Receptors ...(IR; PD-1/CTLA-4/TIM-3/LAG-3/2B4/KLRG1/GITR) on T cells infiltrating the bone marrow (BM) of 32 AML patients relapsing (median 251 days) or maintaining complete remission (CR; median 1 year) after HSCT. A higher proportion of early-differentiated Memory Stem (T
) and Central Memory BM-T cells express multiple IR in relapsing patients than in CR patients. Exhausted BM-T cells at relapse display a restricted TCR repertoire, impaired effector functions and leukemia-reactive specificities. In 57 patients, early detection of severely exhausted (PD-1
Eomes
T-bet
) BM-T
predicts relapse. Accordingly, leukemia-specific T cells in patients prone to relapse display exhaustion markers, absent in patients maintaining long-term CR. These results highlight a wide, though reversible, immunological dysfunction in the BM of AML patients relapsing after HSCT and suggest new therapeutic opportunities for the disease.
The IL-6 family cytokine Oncostatin M (OSM) is involved in cell development, growth, hematopoiesis, inflammation, and cancer. Intriguingly, OSM has proliferative and antiproliferative effects ...depending on the target cell. The molecular mechanisms underlying these opposing effects are not fully understood. Previously, we found OSM upregulation in different myeloproliferative syndromes. However, OSM receptor (OSMR) expression was detected on stromal cells but not the malignant cells themselves. In the present study, we, therefore, investigated the effect of murine OSM (mOSM) on proliferation in stromal and fibroblast cell lines. We found that mOSM impairs the proliferation of bone marrow (BM) stromal cells, whereas fibroblasts responded to mOSM with increased proliferation. When we set out to reveal the mechanisms underlying these opposing effects, we detected increased expression of the OSM receptors OSMR and LIFR in stromal cells. Interestingly,
knockdown and
overexpression attenuated the OSM-mediated effect on proliferation in both cell lines indicating that mOSM affected the proliferation signaling mainly through the OSMR. Furthermore, mOSM induced activation of the JAK-STAT, PI3K-AKT, and MAPK-ERK pathways in OP9 and NIH/3T3 cells with differences in total protein levels between the two cell lines. Our findings offer new insights into the regulation of proliferation by mOSM.
The
JAK2 V617F
mutation can be detected with a high frequency in patients with myeloproliferative neoplasms (MPN). MPN treatment efficiency can be assessed by
JAK2 V617F
quantification. Real-time ...quantitative PCR (qPCR) is widely used for
JAK2 V617F
quantification. Emerging alternative technologies like digital droplet PCR (ddPCR) have been described to overcome inherent qPCR limitations. The purpose of this study was to evaluate the utility of ddPCR for
JAK2 V617F
quantification in patient samples with MPN. Sensitivity and specificity were established by using DNA artificial mixtures. In addition, 101 samples from 59 patients were evaluated for
JAK2 V617F
mutation. Limit of detection was 0.01 % for both qPCR and ddPCR. The
JAK2 V617F
mutation was detected in 43 out of 59 patients by both PCR platforms. However, in 14 % of the samples,
JAK2 V617F
mutation was detected only with ddPCR. This 14 % of discrepant samples were from patients shortly after allogeneic stem cell transplantation. Percentage of
JAK2 V617F
mutation measured by qPCR and ddPCR in clinical samples showed a high degree of correlation (Spearman
r
: 0.9637
p
< 0.001) and an excellent agreement assessed by Bland-Altman analysis. In conclusion, ddPCR is a suitable, precise, and sensitive method for quantification of the
JAK 2 V617F
mutation.
The multi-kinase inhibitor dasatinib has been implicated to be effective in pre-B-cell receptor (pre-BCR)-positive acute lymphoblastic leukemia (ALL) expressing the E2A-PBX1 fusion oncoprotein. The ...TGFβ signaling pathway is involved in a wide variety of cellular processes, including embryonic development and cell homeostasis, and it can have dual roles in cancer: suppressing tumor growth at early stages and mediating tumor progression at later stages. In this study, we identified the upregulation of the TGFβ signaling pathway in our previously generated human dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells using global transcriptomic analysis. We confirm the upregulation of the TGFβ pathway member SMAD3 at the transcriptional and translational levels in dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells. Hence, dasatinib blocks, at least partially, TGFβ-induced SMAD3 phosphorylation in several B-cell precursor (BCP) ALL cell lines as well as in dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells. Activation of the TGFβ signaling pathway by TGF-β1 leads to growth inhibition by cell cycle arrest at the G0/G1 stage, increase in apoptosis and transcriptional changes of SMAD-targeted genes, e.g. c-MYC downregulation, in pre-BCR+/E2A-PBX1+ ALL cells. These results provide a better understanding about the role that the TGFβ signaling pathway plays in leukemogenesis of BCP-ALL as well as in secondary drug resistance to dasatinib.
Relapse of the underlying disease is a frequent complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this study, we describe the clinical utility of measurable ...residual disease (MRD) and mixed chimerism (MC) assessment in circulating cell-free DNA (cfDNA) analysis to detect earlier relapse in patients with hematological malignancies after allo-HSCT. A total of 326 plasma and peripheral blood mononuclear cell (PBMCs) samples obtained from 62 patients with myeloid malignancies were analyzed by droplet-digital PCR (median follow-up: 827 days). Comparison of MC in patients at relapse and in complete remission identified an optimal discriminating threshold of 18% of recipient-derived cfDNA. After performing a targeted next-generation sequencing (NGS) panel, 136 mutations in 58 patients were detected. In a total of 119 paired samples, the putative mutations were detected in both cfDNA and PBMCs in 73 samples (61.3%). In 45 samples (37.8%) they were detected only in cfDNA, and in only one patient (0.9%) were they detected solely in DNA from PBMCs. Hence, in 6 out of 23 patients (26%) with relapse after allo-HSCT, MRD positivity was detected earlier in cfDNA (mean 397 days) than in DNA derived from PBMCs (mean 451 days). In summary, monitoring of MRD and MC in cfDNA might be useful for earlier relapse detection in patients with myeloid malignancies after allo-HSCT.
Introduction. Genomic loss of an HLA haplotype encoding incompatible alleles (“HLA loss”) has been described in previous single-center studies as a mechanism by which leukemic cells evade the ...graft-versus-leukemia effect mediated by alloreactive donor T cells and outgrow into a clinically evident relapse. HLA loss accounts for up to 30% of relapses after HLA-haploidentical transplants (Crucitti, Leukemia 2015), but the actual frequency and clinical relevance of this phenomenon in unrelated donor HSCTs, including cord blood transplants, are largely unknown. Here we present the first global collaborative study to investigate the incidence of HLA loss across different transplant settings.
Methods. Twenty transplant centers from Europe (n=16), North America (n=3) and Asia (n=1) joined to form the HLALOSS consortium. To date, we collected a total of 619 cases of hematologic relapse from adult patients with acute myeloid leukemia (78.5%), acute lymphoblastic leukemia (13.9%), myelodysplastic syndromes (4%) or myeloproliferative neoplasms (1.1%) after allogeneic HSCT from HLA-haploidentical relatives (31.7%), HLA-mismatched unrelated donors (MMUD, 21.3%), 10/10-matched unrelated donors (MUD, 37.2%), or unrelated cord blood units (UCB, 9.8%). Where available, the donor and patient germlines and the patient pre-transplant disease were collected in parallel. Until today, 476 cases were analyzed using conventional HLA typing of sorted leukemic blasts, the recently developed HLA-KMR assay (Ahci and Toffalori, Blood, 2017) or a novel Next-Generation Sequencing (NGS) method. The latter was developed adapting the HLA typing strategy in use at the DKMS (Lange, BMC Genomics 2013) to the study of chimeric samples, and allowing to cover all possible HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 alleles and to analyze at least 48 different cases in a single run.
Results. Out of the 476 relapses analyzed to date, 396 (83.2%) were informative for the study of HLA loss. Of these, 155 occurred after haploidentical HSCT, 101 after MMUD HSCT, 93 after 10/10-matched, HLA-DPB1 mismatched MUD, and 47 after UCB HSCTs. Three-hundred-two (76.2%) of cases were analyzed using the NGS platform. This method resulted particularly robust, reliable and sensitive in analyzing large sample series: the mean coverage across the 6 sequenced loci was over 8500x, up to 0.5% of the HLA allele of interest could be detected in artificial chimerism curves, and relapse samples tested in parallel via the sequencing platform and HLA-KMR (n=10) showed remarkable concordance between the two methods (R2=0.86, p<0.0001). In total, we detected 51 HLA loss post-transplantation relapses out of the 396 cases analyzed (12.8%). Of these, 35 occurred after haploidentical HSCT (22.6% of relapses in this setting), 12 after MMUD HSCT (11.9%), 4 after 10/10 MUD HSCT (4.3%) and, notably, none after UCB HSCT.
Conclusions. The present data, obtained from the largest collaborative study on the immunobiology of relapse to date, confirm the clinical relevance of HLA loss as a major mechanism of immune evasion and post-transplantation relapse after allogeneic HSCT, with an incidence which is proportional to the number of donor-recipient HLA mismatches. The only exception is represented by UCB HSCT which, despite being often performed across multiple major HLA incompatibilities, does not appear to be associated with this relapse modality. This finding might reflect the fact that in UCB HSCT, multiple HLA mismatches are often not encoded in cis on the same chromosome, thereby reducing the selective advantage for leukemic cells that undergo an HLA haplotype loss. This phenomenon might in turn contribute to the lower incidence of relapse reported for UCB HSCT compared to other stem cell sources.
Vago:Moderna TX: Research Funding; GENDX: Research Funding. Stoelzel:Neovii: Speakers Bureau. Gojo:Novartis: Membership on an entity's Board of Directors or advisory committees; Merck inc: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. Busca:Novartis: Speakers Bureau; Jazz Pharmaceuticals: Honoraria; Pfizer Pharmaceuticals: Honoraria, Speakers Bureau; Merk: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Luznik:WIndMIL Therapeutics: Equity Ownership, Patents & Royalties. Kobbe:Amgen: Honoraria, Research Funding; Celgene: Honoraria, Other: Travel Support, Research Funding; Roche: Honoraria, Research Funding. Kroeger:Novartis: Honoraria, Research Funding; Sanofi: Honoraria; Riemser: Honoraria, Research Funding; Neovii: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; JAZZ: Honoraria. Finke:Neovii: Consultancy, Honoraria, Other: travel grants, Research Funding; Medac: Consultancy, Honoraria, Other: travel grants, Research Funding; Riemser: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Other: travel grants, Research Funding. Mohty:Takeda: Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria; Servier: Consultancy; MaaT Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; Molmed: Consultancy; Jazz Pharmaceuticals: Honoraria, Research Funding, Speakers Bureau; Bristol Myers: Consultancy, Research Funding; Janssen: Honoraria, Research Funding, Speakers Bureau. Beelen:Medac: Consultancy, Other: Travel Support. Fleischhauer:GENDX: Research Funding.
Transplantation of hematopoietic cells from a healthy individual (allogeneic hematopoietic cell transplantation (allo-HCT)) demonstrates that adoptive immunotherapy can cure blood cancers: still, ...post-transplantation relapses remain frequent. To explain their drivers, we analyzed the genomic and gene expression profiles of acute myeloid leukemia (AML) blasts purified from patients at serial time-points during their disease history. We identified a transcriptional signature specific for post-transplantation relapses and highly enriched in immune-related processes, including T cell costimulation and antigen presentation. In two independent patient cohorts we confirmed the deregulation of multiple costimulatory ligands on AML blasts at post-transplantation relapse (PD-L1, B7-H3, CD80, PVRL2), mirrored by concomitant changes in circulating donor T cells. Likewise, we documented the frequent loss of surface expression of HLA-DR, -DQ and -DP on leukemia cells, due to downregulation of the HLA class II regulator CIITA. We show that loss of HLA class II expression and upregulation of inhibitory checkpoint molecules represent alternative modalities to abolish AML recognition from donor-derived T cells, and can be counteracted by interferon-γ or checkpoint blockade, respectively. Our results demonstrate that the deregulation of pathways involved in T cell-mediated allorecognition is a distinctive feature and driver of AML relapses after allo-HCT, which can be rapidly translated into personalized therapies.
Although typically found in cancers, frameshift mutations in microsatellites have also been detected in chronically inflamed tissues. Allogeneic hematopoietic cell transplantation (HCT) may ...potentially produce chronic tissue stress through graft-versus-host reactions. We examined non-neoplastic epithelial tissues (colon, buccal) obtained 1 to 5061 days after human allogeneic HCT for the presence of genomic alterations at 3 tetranucleotide and 3 mononucleotide microsatellite loci. Novel bands indicative of microsatellite instability (MSI) at tetranucleotide repeats were detected in laser-microdissected colonic crypts and in buccal smears of 75% and 42% of patients who received an allograft, respectively. In contrast, no MSI was found in similar tissues from control subjects and from patients after intensive chemotherapy or in buccal cells from patients after autologous HCT. The MSI found in colon, which was often affected by graft-versus-host disease, was not due to loss of expression or nitrosylation of DNA repair proteins. MSI in clinically intact oral mucosa was more frequently found at later time points after HCT. MSI was also found in 3 posttransplant squamous cell cancers examined. Our data show that genomic alterations in epithelium regularly occur after allogeneic HCT and may be implicated in the evolution of posttransplantation diseases, including secondary cancer.
Individuals with acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) in the gene encoding Fms-related tyrosine kinase 3 (FLT3) who relapse after allogeneic hematopoietic cell ...transplantation (allo-HCT) have a 1-year survival rate below 20%. We observed that sorafenib, a multitargeted tyrosine kinase inhibitor, increased IL-15 production by FLT3-ITD
leukemia cells. This synergized with the allogeneic CD8
T cell response, leading to long-term survival in six mouse models of FLT3-ITD
AML. Sorafenib-related IL-15 production caused an increase in CD8
CD107a
IFN-γ
T cells with features of longevity (high levels of Bcl-2 and reduced PD-1 levels), which eradicated leukemia in secondary recipients. Mechanistically, sorafenib reduced expression of the transcription factor ATF4, thereby blocking negative regulation of interferon regulatory factor 7 (IRF7) activation, which enhanced IL-15 transcription. Both IRF7 knockdown and ATF4 overexpression in leukemia cells antagonized sorafenib-induced IL-15 production in vitro. Human FLT3-ITD
AML cells obtained from sorafenib responders following sorafenib therapy showed increased levels of IL-15, phosphorylated IRF7, and a transcriptionally active IRF7 chromatin state. The mitochondrial spare respiratory capacity and glycolytic capacity of CD8
T cells increased upon sorafenib treatment in sorafenib responders but not in nonresponders. Our findings indicate that the synergism of T cells and sorafenib is mediated via reduced ATF4 expression, causing activation of the IRF7-IL-15 axis in leukemia cells and thereby leading to metabolic reprogramming of leukemia-reactive T cells in humans. Therefore, sorafenib treatment has the potential to contribute to an immune-mediated cure of FLT3-ITD-mutant AML relapse, an otherwise fatal complication after allo-HCT.