Immunotherapy Of Acute Lymphoblastic Leukemia Research Articles

Newly Developed Treatments for Acute Lymphoblastic and Acute Myeloid Leukemia

Chemotherapy has been dominating the field of cancer treatment for a long time, however, its limitations have been revealed over time. Therefore, several antigen proteins and chimeric antigen receptorT cells (CAR-T) involved in the immunotherapy of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) have been introduced. This paper provides details on the mechanisms, implemented investigations, and drawbacks of the immunotherapy for ALL and AML. Current studies have shown that CAR-T cell therapy can eliminate pediatric ALL relapse along with treating B cell ALL. With the appearance of CAR-T cell therapy, especially CD19-, CD20-, and CD22-directed CAR-T cells, aggressive acute lymphomas involving ALL become treatable. Studies have also shown that AML can be treated with FLT3 inhibitors and immunotherapy including monoclonal antibodies (mbA) and CD33-, CD123-directed CAR-T cells. Anti-CD33 monoclonal antibodies can combine with calicheamicin, a cytotoxic agent in DNA strand cleavage, and monotherapy of gemtuzumab ozogamicin (GO), an antibody-drug conjugate, and this combination has been proved to extend the overall survival of both newly treated patients and R/R AML patients who are unable to tolerate standard chemotherapy.

Preclinical characterization and comparison between CD3/CD19 bispecific and novel CD3/CD19/CD20 trispecific antibodies against B-cell acute lymphoblastic leukemia: targeted immunotherapy for acute lymphoblastic leukemia.

The CD19-targeting bispecific T-cell engager blinatumomab has shown remarkable efficacy in patients with relapsed/refractory B-cell precursor acute lymphoblastic leukemia. However, several studies showed that blinatumomab has a short plasma half-life due to its low molecular weight, and thus its clinical use is limited. Furthermore, multiple trials have shown that approximately 30% of blinatumomab-relapsed cases are characterized by CD19 negative leukemic cells. Here, we design and characterize two novel antibodies, A-319 and A-2019. Blinatumomab and A-319 are CD3/CD19 bispecific antibodies with different molecular sizes and structures, and A-2019 is a novel CD3/CD19/CD20 trispecific antibody with an additional anti-CD20 function. Our in vitro, ex vivo, and in vivo experiments demonstrated that A-319 and A-2019 are potent antitumor agents and capable of recruiting CD3 positive T cells, enhancing T-cell function, mediating B-cell depletion, and eventually inhibiting tumor growth in Raji xenograft models. The two molecules are complementary in terms of efficacy and specificity profile. The activity of A-319 demonstrated superior to that of A-2019, whereas A-2019 has an additional capability to target CD20 in cells missing CD19, suggesting its potential function against CD19 weak or negative CD20 positive leukemic cells.

Acute lymphoblastic leukemia-derived exosome inhibits cytotoxicity of natural killer cells by TGF-β signaling pathway.

This study was conducted to explore whether acute lymphoblastic leukemia (ALL)-derived exosomes affect natural killer (NK) cells. Exosomes were isolated and identified from Jurkat cells and co-cultured with NK cells. Then, the cytotoxicity, viability, and release of perforin and granzyme B in NK92-MI cells were measured. PCR arrays were used to detect gene expression alterations in the transforming growth factor (TGF)-β pathway of NK92-MI cells treated or not treated with exosomes. The morphology and size of the exosomes isolated from Jurkat cells showed typical characteristics of exosomes, and the expression of cluster of differentiation 63 was detected. Jurkat-derived exosomes were internalized by NK92-MI cells, further inhibiting the proliferation and cytotoxicity of NK92-MI cells. An enzyme-linked immunosorbent assay revealed that the release of perforin and granzyme B from NK92-MI cells decreased after co-culture with exosomes. Similarly, western blot and immunofluorescence staining verified that Jurkat-derived exosomes inhibited the expression of granzyme B and perforin. Furthermore, Jurkat-derived exosomes enhanced the signaling of the TGF-β pathway in NK92-MI cells via the MDS1 and EVI1 complex loci and homeodomain interacting protein kinase 2. In conclusion, we found that ALL-derived exosomes inhibit the biological function of NK cells and provide support for the immunotherapy of ALL.

Immunotherapy of Acute Lymphoblastic Leukemia and Lymphoma With T Cell-Redirected Bispecific Antibodies.

Immunotherapy of Acute Lymphoblastic Leukemia and Lymphoma With T Cell-Redirected Bispecific Antibodies.

CSPG4-Specific CAR T Cells for High-Risk Childhood B Cell Precursor Leukemia.

The advent of CD19-specific chimeric antigen receptor (CAR) T cells has proven to be a powerful asset in the arsenal of cancer immunotherapy of acute lymphoblastic leukemia and certain B cell lymphomas. However, a sizable portion of patients treated with CD19-CAR T cells relapse with CD19-negative cancer cells, necessitating the quest for back-up antigens. Chondroitin sulfate proteoglycan 4 (CSPG4) expression has been reported on leukemic blasts bearing the ill-fated MLL 11q23 rearrangement. We aimed at exploring the use of CSPG4-specific CAR T cells against mixed-lineage leukemia (MLL)-rearranged leukemic blasts, using the precursor B cell leukemia cell line KOPN8 (MLL–MLLT1 translocation) as a model. First, we confirmed CSPG4 expression on KOPN8 cells. Bulk T cells electroporated with mRNA encoding a CSPG4-specific CAR upregulated activation markers and secreted the Th1 cytokines TNF and IFNγ in an antigen-specific manner upon co-culture with KOPN8 cells. More importantly, CSPG4-specific CAR T cells evinced specific degranulation towards KOPN8 cells and specifically lysed KOPN8 target cells in chromium lysis experiments. CSPG4 is a well-established CAR target in cutaneous melanoma. Here, we provide proof-of-principle data for the use of CSPG4-specific CAR T cells against MLL-translocated leukemias.

Advances in hematopoietic cell transplant for the treatment of hematologic malignancies.

Incorporation of minimal residual disease (MRD) testing in acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML) has transformed the landscape of hematopoietic cell transplantation (HCT). Pre-HCT MRD has allowed prognostication of HCT outcomes for high-risk leukemia patients, whereas the detection of post-HCT MRD has allowed for interventions to decrease relapse. In this review, we emphasize studies from the past two decades that highlight the critical role of MRD in HCT in pediatric ALL and AML. Advances in MRD detection methodology, using next-generation sequencing, have improved the sensitivity of MRD testing allowing for more accurate predictions of HCT outcomes for patients with relapsed and refractory ALL and AML. In addition, novel pre-HCT therapies, especially immunotherapy in ALL, have dramatically increased the number of patients who achieve MRD-negative remissions pre-HCT, resulting in improved HCT outcomes. Post-HCT MRD remains a challenge and new therapeutic interventions are needed to reduce post-HCT relapse. As immunotherapy increases pre-HCT MRD-negative remissions, and next-generation sequencing-MRD is incorporated to improve the sensitivity of MRD detection, future clinical studies will investigate less toxic HCT approaches to reduce long-term sequelae and to identify which patients may benefit most from early post-HCT intervention to reduce relapse.

TIM-3 Expression on CD4+ Bone Marrow T Cells Predicts Relapse of Pediatric B-Precursor Acute Lymphoblastic Leukemia

IntroductionPediatric 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.MethodsBM 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.ResultsMultivariate 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).ConclusionsOur 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. DisclosuresNo relevant conflicts of interest to declare.

The Bold World of Immunotherapy for Acute Lymphoblastic Leukemia

The Bold World of Immunotherapy for Acute Lymphoblastic Leukemia

Reducing Ex Vivo Culture Improves the Antileukemic Activity of Chimeric Antigen Receptor (CAR) T Cells

The success of chimeric antigen receptor (CAR)-mediated immunotherapy in acute lymphoblastic leukemia (ALL) highlights the potential of T-cell therapies with directed cytotoxicity against specific tumor antigens. The efficacy of CAR T-cell therapy depends on the engraftment and persistence of T cells following adoptive transfer. Most protocols for T-cell engineering routinely expand T cells ex vivo for 9 to 14 days. Because the potential for engraftment and persistence is related to the state of T-cell differentiation, we hypothesized that reducing the duration of ex vivo culture would limit differentiation and enhance the efficacy of CAR T-cell therapy. We demonstrated that T cells with a CAR-targeting CD19 (CART19) exhibited less differentiation and enhanced effector function in vitro when harvested from cultures at earlier (day 3 or 5) compared with later (day 9) timepoints. We then compared the therapeutic potential of early versus late harvested CART19 in a murine xenograft model of ALL and showed that the antileukemic activity inversely correlated with ex vivo culture time: day 3 harvested cells showed robust tumor control despite using a 6-fold lower dose of CART19, whereas day 9 cells failed to control leukemia at limited cell doses. We also demonstrated the feasibility of an abbreviated culture in a large-scale current good manufacturing practice-compliant process. Limiting the interval between T-cell isolation and CAR treatment is critical for patients with rapidly progressing disease. Generating CAR T cells in less time also improves potency, which is central to the effectiveness of these therapies. Cancer Immunol Res; 6(9); 1100-9. ©2018 AACR.

Current Status and Challenges of CAR-T Immunotherapy in Hematologic Malignancies -Review

The chimeric antigen receptor (CAR) T cell therapy has gradually became a new trend in the treatment of refractory and relapsed hematologic malignancies by developing for 30 years. With the exciting development of genetic engineering, CAR-T technology has subjected to 4 generations of innovation. Structure of CAR-T started from a single signal molecule to 2 or more than 2 co-stimulatory molecules, and then coding the CAR gene or promoter. CAR-T can specifically recognize tumor antigens, and does not be restricted by major histocompatibility complex (MHC), thus making a breakthrough in clinical treatment. In this review, the history, structure and mechanism of action of CAR-T, as well as the current status and challenges of CAR-T immunotherapy in acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia and multiple myeloma are summarized.

Resistance to anti-CD19/CD3 BiTE in acute lymphoblastic leukemia may be mediated by disrupted CD19 membrane trafficking

AbstractThe CD19 antigen is a promising target for immunotherapy of acute lymphoblastic leukemia (ALL), but CD19− relapses remain a major challenge in about 10% to 20% of patients. Here, we analyzed 4 CD19− ALL relapses after treatment with the CD19/CD3 bispecific T-cell engager (BiTE) blinatumomab. Three were on-drug relapses, with the CD19− escape variant first detected after only 2 treatment courses. In 1 patient, the CD19− clone appeared as a late relapse 19 months after completion of blinatumomab treatment. All 4 cases showed a cellular phenotype identical to the primary diagnosis except for CD19 negativity. This argued strongly in favor of an isolated molecular event and against a common lymphoid CD19− progenitor cell or myeloid lineage shift driving resistance. A thorough molecular workup of 1 of the cases with early relapse confirmed this hypothesis by revealing a disrupted CD19 membrane export in the post–endoplasmic reticulum compartment as molecular basis for blinatumomab resistance.

Immunotherapy for acute lymphoblastic leukemia: from famine to feast

Publisher's Note: This article has a companion Point by Jabbour and Kantarjian. Publisher's Note: Join in the discussion of these articles at Blood Advances Community Conversations.

EphA3 as a target for antibody immunotherapy in acute lymphoblastic leukemia

The human EphA3 gene was discovered in a pre-B acute lymphoblastic leukemia (pre-B-ALL) using the EphA3-specific monoclonal antibody (mAb), IIIA4, which binds and activates both human and mouse EphA3. We use two models of human pre-B-ALL to examine EphA3 function, demonstrating effects on pre-B-cell receptor signaling. In therapeutic targeting studies, we demonstrated antitumor effects of the IIIA4 mAb in EphA3-expressing leukemic xenografts and no antitumor effect in the xenografts with no EphA3 expression providing evidence that EphA3 is a functional therapeutic target in pre-B-ALL. Here we show that the therapeutic effect of the anti-EphA3 antibody was greatly enhanced by adding an α-particle-emitting 213Bismuth payload.

FHIT Gene Expression in Acute Lymphoblastic Leukemia and its Clinical Significance

To investigate the expression of the fragile histidine triad (FHIT) gene in acute lymphoblastic leukemia and its clinical significance. The level of expressed FHIT mRNA in peripheral blood from 50 patients with acute lymphoblastic leukemia (ALL) and in 50 peripheral blood samples from healthy volunteers was measured via RT-PCR. Correlation analyses between FHIT gene expression and clinical characteristics (gender, age, white blood count, immunophenotype of acute lymphoblastic leukemia and percentage of blast cells) of the patients were performed. The FHIT gene was expressed at 2.49±7.37 of ALL patients against 14.4±17.9 in the healthy volunteers. The difference in the expression levels between ALL patients and healthy volunteers was statistically significant. The rate of gene expression did not significantly vary with immunophenotype subtypes. Gene expression was also found to be correlated with increase of total leukocyte and decrease in platelets, but not with age, gender, immunophenotyping or percentage of blast cells. FHIT gene expression is low in acute lymphoblastic leukemia and could be a useful marker to monitor minimal residual disease. This gene is also a candidate target for the immunotherapy of acute lymphoblastic leukemia.

Humoral and cellular immunotherapy in ALL in children, adolescents, and young adults.

Although the event-free survival for children and adolescents with acute lymphoblastic leukemia (ALL) has dramatically improved over the past half century, it has plateaued over the past decade. Children and adolescents with refractory/relapsed ALL continue to have a dismal prognosis with hematopoietic stem cell transplant being their most viable option for cure. There is an obvious need for the development of novel agents to further enhance overall outcomes. In this review we focus on the development of humoral and cellular immunotherapeutic agents in the treatment of childhood, adolescent, and young adult ALL. Immunotherapy in various forms has shown immense promise. To date we have seen numerous safety studies using monoclonal antibody therapy, antibody conjugates, bispecific T cell and bispecific natural killer (NK) cell antibodies and genetically reengineered T and NK cells expressing targeted chimeric antigen receptors. Initial success has been found with the anti-CD20 monoclonal antibodies followed by promising results using anti-CD22 and anti-CD19 therapies alone or in combination. Genetic modification of T and NK cells to express targeted chimeric antigen receptors offers a novel immunotherapy option that demonstrates enhanced cytotoxicity in otherwise resistant tumor cells. There is great potential to combine immunotherapies to further improve overall cure rates in children, adolescents, and young adults with poor-risk ALL. A number of humoral and cellular immunotherapy strategies have been investigated and found to be effective, safe, and well tolerated. Ideally, the targeted approach of immunotherapy will result in an overall decrease in toxicities experienced by patients. Future studies are required to determine when in the course of treatment with humoral and cellular therapy will have the safest and optimal effect in ALL.

T-cell adoptive immunotherapy for acute lymphoblastic leukemia

Substantial progress has been made in the treatment of precursor B-cell acute lymphoblastic leukemia (B-ALL), but recurrent disease remains a leading cause of death in children due to cancer and outcomes for adults with B-ALL remain poor. Recently, complete clinical responses have been observed in small numbers of patients with B-ALL treated with adoptive immunotherapy using T cells genetically engineered to express chimeric antigen receptors (CARs) targeting CD19, a cell surface molecule present in essentially all cases of B-ALL. Preclinical data suggest that CARs targeting CD22, another antigen present in the majority of B-ALL cases, are similarly potent. Several clinical studies already under way will soon more clearly define the rate of response to this novel therapy in B-ALL. Further work is needed to identify optimal platforms for CAR-based adoptive immunotherapy for leukemia, to establish guidelines for managing toxicity, and to determine whether the remissions induced by this approach can be rendered durable.

ALL Progression Induces PD1 on T Cells and Blockade of PD1 Enhances Adoptive Immunotherapy

Abstract 2587 Background:Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and, despite tremendous success in therapy over the past 3 decades, remains a primary cause of cancer-related mortality in children. Enthusiasm for the use cellular immunotherapy for ALL has been tempered by the poor response to donor lymphocyte infusions following allogeneic hematopoietic stem cell transplantation. However, ALL blasts are susceptible to T cell and NK cell mediated lysis in vitro suggesting that poor response to in vivo immune interventions may be due to events occurring during the priming of the immune response. Using a murine model of precursor B cell ALL we examined the impact of leukemia progression on T cells in vivo. Methods: We developed a transplantable syngeneic model of pediatric ALL derived from transgeneic mice expressing human E2aPBX1, a recurring translocation present in 5% of pediatric leukemia (Bijl et al, Genes and Development, 2005). This murine line displays a precursor B cell phenotype and results in 100% lethality following injection of 100,000 cells (Qin et al, ASH, 2010). Using congenic (CD45.1) B6 recipients, we tracked the early progression of ALL in vivo and examined the T cells in the leukemia-containing compartments by flow cytometry and PCR. Results: Using congenic markers, ALL cells can be detected in bone marrow as early as 3 days following intravenous injection of 1,000,000 cells with a sensitivity of 0.01%. Spleen and lymph node involvement was seen later (10 days) followed by the detection of circulating blasts by 2 weeks. E2aPBX1 cells express variable levels of costimulatory molecules in vitro with no change in expression during in vivo progression. Notably, PDL1 and PDL2 are expressed both in vitro and in vivo at higher levels than on non-malignant precursor B cells in leukemia-bearing mice. Remarkably, although PD1+ T cells are not seen in the bone marrow of non-leukemia-bearing mice, PD1 expression on bone marrow T cells was markedly increased during progression such that 60–80% of all bone marrow CD4 and CD8 T cells were positive by 2 weeks following leukemia injection (figure). In addition to expression of PD1, these T cells also co-expressed Tim3, a phenotype associated with T cell exhaustion. Blockade of PD1 or PDL1 starting 3 days following leukemia injection had no impact on leukemia progression. However, combining PD1 blockade with the adoptive transfer of T cells from leukemia-primed donors resulted in improved survival compared to primed T cells alone (p=0.0004). Conclusions: Early progression of ALL results in the induction of PD1 and Tim3 on T cells in vivo. Combination of PD1 blockade plus adoptive T cell therapy results in therapeutic benefit suggesting that this axis may be an attractive target in ALL. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Flow cytometric study of potential target antigens (CD19, CD20, CD22, CD33) for antibody-based immunotherapy in acute lymphoblastic leukemia: analysis of 552 cases

Monoclonal antibody (MoAb)-based therapies have opened innovative treatment avenues that have impacted on the management of patients with both neoplastic and non-neoplastic hematological diseases. The aim of our study was to evaluate in a large series of cases of acute lymphoblastic leukemia (ALL) the expression of specific antigens, CD19, CD20, CD22, and CD33, for which MoAbs are available for clinical use. For each antigen, evaluation was based on the percentage of positive leukemic cells and the degree of antigen expression by mean fluorescence intensity (MFI) and antibody binding capacity (ABC) that were correlated with age, immunophenotype, and presence/absence of particular molecular markers. We can document that some of the analyzed antigens showed a degree of expression related to the B-cell maturation profile, and that the antigen expression intensity appeared to vary according to the presence of specific genetic markers. These findings suggest that the possible clinical use of a given MoAb in patients with ALL should take into account both the maturation profile of the leukemic cells and the presence of a given molecular transcript. Only clinical studies will conclusively demonstrate whether the differences in antigenic expression truly correlate with the different therapeutic efficacies of the various clinical grade MoAbs.

Selective Reprogramming of Cd19-Specific T Cells With IL-21 and CD28 Signaling for Adoptive Immunotherapy of Acute Lymphoblastic Leukemia

Selective Reprogramming of Cd19-Specific T Cells With IL-21 and CD28 Signaling for Adoptive Immunotherapy of Acute Lymphoblastic Leukemia

WT1-Specific CD8+ T Lymphocytes May Participate in the Elimination of Acute Lymphoblastic Leukemia Following Allogeneic Stem Cell Transplantation.

There is clinical evidence that a graft-versus-leukemia (GVL) effect occurs following allogeneic stem cell transplantation for acute lymphoblastic leukemia (ALL). However, the potency of this GVL effect is often associated with unwanted graft-versus-host-disease (GVHD) and disease relapse remains a major contributor to treatment failure. Wilms' tumor gene 1 (WT1) is overexpressed in 70–90% of cases of ALL and has been identified as a convenient minimal residual disease (MRD) marker. WT1 is an attractive immunotherapeutic target in ALL because peptides derived from WT1 can induce CD8+ T-cell responses, and being non-allelic, WT1 would be unlikely to provoke GVHD. We investigated whether CD8+ T-cells directed against an HLA-A*0201 restricted epitope of WT1 (WT126) occur in ALL patients during the early phase of immune reconstitution post-SCT (days 30–180). We analyzed CD8+ T-cell responses against WT1 in 10 HLA-A*0201+ ALL SCT recipients and their respective donors using WT1/HLA-A*0201 tetrameric complexes and flow cytometry for intracellular IFN-gamma. We studied the kinetics WT1-specific CD8+ T-cell responses in consecutive samples obtained post-SCT. CD8+ T-cells recognizing WT1 were detected ex vivo in samples from 5 of 10 ALL patients post-SCT but not in patients pre-SCT. WT1-tetramer+ CD8+ T cells had a predominantly effector memory phenotype (CD45RO+CD27−CD57+). WT1 gene expression in pre-SCT and donor samples was assayed by quantitative real-time PCR (RQ-PCR). WT1 expression in PBMC from healthy donors was significantly lower than in patients (median 0, range 0–66 ×10−4 WT1/ABL compared to patients, median 12, range 0–2275 ×10−4 WT1/ABL) (P < 0.01). There was a strong correlation between the emergence of WT1-specific CD8+ T cells and a reduction in WT1 gene expression (P < 0.001) (as depicted below) suggesting direct anti-ALL activity post-SCT. Disappearance of WT1-specific CD8+ T-cells from the blood coincided with reappearance of WT1 gene transcripts, consistent with a molecular relapse, further supporting the direct involvement of WT1-specific CD8+ T-cells in the GVL response. These results provide evidence for the first time of spontaneous T-cell reactivity against a leukemia antigen in ALL patients. Our results support the immunogenicity of WT1 in ALL patients post-SCT and a potential application for WT1 peptides in post-transplant immunotherapy of ALL. [Display omitted]