T-ALL Blasts Research Articles

Anti-CD21 Chimeric Antigen Receptor (CAR)-T Cells for T Cell Acute Lymphoblastic Leukaemia (T-ALL)

CAR-T cell therapy against CD19 has changed the treatment landscape in relapsed/refractory (r/r) B-ALL. R/r T-ALL has a dismal prognosis, with an unmet need for effective targeted therapies. Several unique challenges mean that CAR-T cell therapy has yet to be successfully translated to T-ALL. Most strategies have targeted pan-T cell antigens (CD7, CD5) but these are limited by T cell aplasia and fratricide, requiring elimination of CAR-T antigen surface expression during manufacture. An ideal target would be exclusively or largely confined to the malignant T cell component but published examples of these (CD1a and TRBC1) are expressed in only minor T-ALL subsets.We previously showed that CD21 is expressed in a NOTCH-dependent manner in T-ALL (Leukemia. 2013, 27:650) and have developed it as a potential immunotherapy target, being primarily expressed on normal B cells, with minimal expression on mature T cells.70% of human T-ALL cell lines (9/16) expressed surface CD21 by flow cytometry (FACS), with a median antigen density in positive lines of 2545/cell. In primary T-ALL, 57% of presentation samples (n=58) expressed CD21 (median antigen density 1168/cell). 45% of relapse (n=11) and 20% of primary refractory cases (n=30) expressed CD21, with a similar antigen density to presentation samples. CD21 positivity varied by maturational stage, with highest expression in cortical T-ALL (80% of cases) followed by pre-T (72%), mature (67%), ETP (25%) and pro-T (17%).Healthy donor blood (n=14) showed CD21 expression limited to B cells and a low proportion (11%) of T cells (10-fold lower intensity v B cells, 316 antigens/cell). T cell CD21 expression was not up-regulated upon activation with CD3/CD28 antibodies (n=6) and was not associated with markers of differentiation/exhaustion.To target CD21, DNA gene-gun vaccination of rats with a plasmid encoding full-length CD21, followed by phage display was performed and multiple anti-CD21 scFvs isolated. These were cloned into 4-1BBζ CARs and expressed in primary T cells but failed to kill or secrete cytokines in response to CD21+ SupT1 cells.CD21 is a bulky molecule, with 15/16 sushi repeats in the extracellular domain. All isolated scFvs were found to bind membrane-distal domains. We hypothesized that ineffective signalling due to inadequate synapse formation was responsible for poor performance of anti-CD21 CAR-T, and that binders to membrane-proximal epitopes would signal more efficiently. We re-vaccinated rats with the first 5 sushi repeats of CD21 and generated a library of binders which bound CD21 at this membrane-proximal region.Multiple candidate binders expressed as CARs were functional, with cytotoxicity and interferon-γ secretion in response to CD21+ target cells. However, non-specific background cytokine secretion was seen against CD21 negative cells, and no IL-2 secretion was seen. Re-cloning binders into a fragment antigen binding (Fab)-CAR architecture yielded constructs capable of specific cytotoxicity, IFN-γ and IL2 secretion against a CD21+ cell line but not its CD21 negative counterpart (n=6). Our lead anti-CD21 candidate CAR specifically proliferated in vitro, without fratricide or premature exhaustion/ differentiation, and was active against low-density CD21-positive cell lines (n=3) and primary cells from 2 T-ALL patients. Improved functionality of Fab v scFv-based CAR was not driven by higher affinity binding or CAR surface expression.We tested anti-CD21 CAR in murine models of T-ALL. NSG mice were injected with SupT1-luciferase cells and treated with aCD19 or aCD21 CAR-T on day +5. At 2 weeks post treatment, markedly lower disease burden was seen in CD21 CAR-T v CD19 recipients by bioluminescence imaging (median radiance 71700 v 790000 p=0.0079). Further, we injected primary T-ALL blasts in another cohort, treating with aCD19 or aCD21 CAR-T on D+20. Serial bleeds from day 27 post CAR-T showed tumour control in aCD21 CAR treated mice (p=0.024) with an overall survival advantage (median OS 44 days vs undefined, HR = 19.8, p = 0.0069, n=4/group).In summary, we propose CD21 as a novel target for CAR-T cell therapy in T-ALL. Its expression is largely restricted to the malignant T cell compartment, overcoming issues with fratricide and on-target off-tumour effects seen in many T-ALL CAR-T strategies to date. Despite the complexity of the target, we have successfully generated an aCD21 CAR that is functional both in vitro and in vivo. DisclosuresMaciocia: Autolus: Current equity holder in publicly-traded company. Onuoha: Autolus: Ended employment in the past 24 months. Khwaja: Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Maciocia: Autolus: Current equity holder in publicly-traded company, Research Funding. Pule: Autolus: Current Employment, Current equity holder in publicly-traded company.

Assessment of Comprehensive Mutational Profiling in T-lymphoblastic leukemia/lymphoma (T-ALL/LBL): A Single Center Experience

Abstract Introduction/Objective T-lymphoblastic leukemia/lymphoma (T-ALL/LBL) is a malignancy arising from immature precursor T cells with T-ALL involving bone marrow/blood and T-LBL occurring in the thymus and nodal/extranodal sites. Studies have now revealed >100 recurrently altered genes that are not necessarily disease initiating but can provide diagnostic, prognostic, and predictive information which can then be utilized in personalized therapy. Methods/Case Report Next-generation sequencing was performed on DNA and/or RNA extracted from blood/marrow aspirates or tissue at an external CLIA-certified, CAP-accredited laboratory. The hematology panel sequenced DNA of 406 genes, introns of 31 gene rearrangements, and RNA of 265 genes. This retrospective single-center study highlights salient findings noted in genomic profiles of 15 T-ALL/LBL cases out of 83 total patients with ALL from 2018-2021. While the majority were B-ALL cases, T-ALL accounted for 18%, and all but 1 case were pediatric patients (ages 9-21 years). Results (if a Case Study enter NA) In our pediatric cohort (14 patients; 9 males, 5 females), as in literature, NOTCH signaling was most frequently involved with NOTCH1 (50%) and FBXW7 (36%) mutations, followed by those in cell cycle process CDKN2A/2B (36%) and PTEN (28%) mutations. Other mutations: PHF6 (21%), BCOR and TAL1 (14%) each. The prognostic effect of mutations: NOTCH1 favorable, FBXW7 no effect but trend toward favorable when FBXW7 co-occurs with NOTCH1 while PTEN is unfavorable (3 patients had relapses). Some unusual or useful findings: a patient diagnosed initially as AML with aberrant CD3 was re-classified as early T-cell precursor ALL, supported by RELN mutation (occurs in 4% ETP-ALL). The adult with NOTCH1 and BCOR mutations in addition to BCR-ABL1 fusion was diagnosed as having T-ALL blasts with CML. We could not study detailed nuances in mutational profiles of T-ALL vs T-LBL with only 1 case of T-LBL showing FBXW7, PTEN, NF1, RB1, BCOR and NRAS mutations (latter is typically noted in pediatric T-LBL cases). Conclusion Clinical molecular testing in our pediatric T-ALL patients revealed gene alterations that provide refinement of diagnosis, prognosis, and risk stratification. It also contributes a useful data set for further analysis and potential use of clinically actionable therapeutic targets in some cases. Longer term follow-up incorporating therapy and outcomes information would be valuable.

Abstract 3185: Integrin signaling is critical for myeloid-mediated support of T-ALL

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is a malignant transformation of immature T cells. Although T-ALL harbors multiple pro-leukemic genetic alterations, T-ALL blasts require exogenous signals to survive. We recently reported that tumor-associated myeloid cells provide critical support for T-ALL in mouse models and in pediatric patient samples, at least partially through activation of IGF1R signaling. However, IGF1 alone is not sufficient to support T-ALL survival, implicating additional mechanisms in myeloid-mediated T-ALL support. We previously found that T-ALL cells make prolonged and frequent contacts with tumor-associated myeloid cells in live tissue sections; thus, we hypothesized that cell-cell interactions are essential for myeloid-mediated support of T-ALL. Here, we confirm that cell contact is required for myeloid cells to support T-ALL cell survival in vitro. Analysis of transcriptional profiling data revealed enrichment of gene signatures associated with integrin pathways in T-ALL cells relative to healthy T cells, implicating integrin-mediated cell adhesion as a potential mechanism underlying contact-dependent T-ALL support. Blockade of integrin αL ± integrin β1 or their ligands ICAM-1 ± VCAM-1 via antibodies or pharmacologic inhibitors significantly decreased T-ALL survival and reduced activation of IGF1R in T-ALL cells co-cultured with tumor-associated myeloid cells. Consistent with these findings, inhibition of integrins or their ligands in vivo via antibody blockade or genetic manipulation resulted in a significant reduction of T-ALL burden at multiple leukemic sites and prolonged survival. Consistent with mouse models, blockade of integrin-mediated cell adhesion significantly reduced survival of primary pediatric T-ALL cells co-cultured with myeloid cells. Furthermore, analyses of published transcriptomic datasets from pediatric T-ALL patients showed that enrichment of integrin pathways correlates with elevated myeloid gene signatures and poor patient prognosis. Together, our studies demonstrate that tumor-associated myeloid cells promote survival of T-ALL cells by activating integrin signaling, implicating the associated integrins and downstream signals as targets for therapeutic intervention. Citation Format: Aram Lyu, Seo Hee Nam, Zicheng Hu, Dhivya Arasappan, Terzah M. Horton, Lauren I. Ehrlich. Integrin signaling is critical for myeloid-mediated support of T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3185.

Issue Highlights - March 2019.

This issue features, among others, two reviews and four original manuscripts in the field of clinical cytometry, all of them in the area of leukemia/lymphoma immunophenotyping. These may be summarized as follows: Jevremovic and colleagues reviewed the position of flow cytometry in the diagnosis, prognosis and follow-up of T- and NK-cell lymphoproliferations 1. The diagnosis of these entities is generally based on the demonstration of immunophenotypic abnormalities and demonstration of clonality. The latter studies involve V-beta T-cell receptor repertoire analysis and killer immunoglobulin like receptors (KIR). In this context it is important to realize that a T-cell clone does not need to be synonymous with “malignancy” but could also reflect the transient clonal proliferation of “reactive” T cells. Among the mature T-cell lymphoproliferative disorders, T-cell large granular lymphocytic leukemia (T-LGLL), anaplastic large T-cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), mycosis fungoides or Sezary syndrome (MF/SS), T-cell prolymphocytic leukemia (T-PLL), hepatosplenic T-cell lymphoma (HSTCL) and peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) are distinguished. Within the NK-cell disorders, chronic lymphoproliferative disorder of NK cells (CLPD-NKs), extranodal NL/T-cell lymphoma, nasal type, aggressive NL-cell leukemia/lymphoma and NK-cell enteropathy are defined. Nowadays, flow cytometry is a sensitive and specific approach to detect T and NK-cell neoplasms but requires a thorough knowledge of the normal T-cell and NK-cell immune spectrum. Jan W. Gratama, Deputy Editor [Color figure can be viewed at wileyonlinelibrary.com] CD123, the alpha chain of IL3 receptor, is expressed in variable intensities on human leukocytes; the strongest expression is seen on plasmacytoid dendritic cells and basophils, whilst the marker is absent on nucleated red blood cells; a range of intermediate expression levels are seen on T cells, mature B cells, granulocytes and eosinophils. CD123 is expressed on AML blasts and most B-cell precursor ALL blasts whilst it is negative on most T-ALL blasts. The teams of the Laboratory for Medical Immunology at Erasmus MC (Rotterdam, Netherlands) in collaboration with the Dutch Childhood Oncology Group (The Hague, Netherlands) set out to study CD123 expression levels in 846 patients with acute leukemia in comparison to normal cell populations obtained in 1,252 unique bone marrow and peripheral blood samples 6. Immunophenotyping was performed using standardized procedures as developed by the EuroFlow collaborative project 7. Whilst published information on this topic was relatively scarce and difficult to compare due to different methodologies, Bras and colleagues confirmed most observations as they extensively document in this report 6. Importantly, they observed that in paired B-cell precursor ALL, CD123 expression was considerably higher at relapse than at initial diagnosis, whilst in AML, the leukemic stem cells (CD34pos, CD38neg fraction of tumor cells) expressed similar CD123 levels as the remainder of AML blasts. These observations make CD123 an interesting target for antibody-targeted therapy 8. Early positive minimal residual disease (MRD) is a strong predictor of response to treatment in ALL and AML. In an observational study of 125 children with B-cell precursor ALL, Loosveld and colleagues 9 investigated the prognostic value of flow cytometric assessment of MRD in peripheral blood on days 8 and 15 after start of cytoreductive therapy, compared to day 35 MRD on bone marrow using molecular techniques. Children with detectable PB MRD on day 15 and negative day 35 BM results had lower disease-free survival at 4 years follow-up, indicating the prognostic potential of this less invasive, blood-based procedure early after start of therapy as compared to conventional bone marrow studies. Mora and colleagues 10 studied 120 consecutive patients with chronic B-lymphoproliferations (81 within the CLL spectrum and 39 with other monoclonal B-cell disorders) to establish whether CD200, a member of the Ig receptors membrane proteins superfamily, would have added value to the immunological scoring system already developed by Matutes et al. 11. CD200 had already been suggested to be rather specific for B-CLL in earlier studies. This study confirmed the value of CD200 as diagnostic marker and revealed that it's addition to the Matutes score increased the accuracy of that ranking algorithm. Beke Debreceni et al. 12 analyzed the regulation of expression of the adhesion receptor L-selectin (CD62L) in a cohort of untreated patients with B-CLL. Expression of surface-bound CD62L and tumor necrosis factor alpha-converting enzyme (TACE), as well as total phosphatase and protein phosphatase-2A activities, were lower on B-CLL than on normal B cells whilst the reverse was true for soluble CD62L and phosphorylated mitogen-activated protein kinase (pp38MAPK). In separate in vitro experiments confirmed that a phosphatase inhibitor (calyculin A) could reduce the effect of pp38MAPK inhibitor on CD62L shedding. The authors concluded that the reduction of phosphatase activity in B-CLL has resulted in a downstream signaling cascade with subsequent reduction of surface-bound CD62L which is mediated by enhanced phosphorylation of p38MAPK and reduced TACE expression. Jan W. Gratama, Deputy Editor Email: j.w.gratama@erasmusmc.nl

Abstract SY42-01: Targeting T cell malignancies with gene edited CAR-T

Abstract T-cell malignancies represent a class of devastating hematologic cancers with high rates of relapse and mortality in both children and adults for which there are currently no effective or targeted therapies. Despite intensive multiagent chemotherapy regimens, fewer than 50% of adults and 75% of children with T-ALL survive beyond five years. For those who relapse after initial therapy, salvage chemotherapy regimens induce remissions in 20-30% of cases. Allogeneic stem cell transplant, with its associated risks and toxicities, is the only curative therapy. Targeted therapy against T-cell malignancies represents a significant unmet medical need. Such targeted therapies have shown great potential for inducing both remissions and even long-term relapse-free survival in patients with B-cell leukemia and lymphoma. Engineered T cells that express a chimeric antigen receptor (CAR) directed against T-cell malignancies are limited by several significant obstacles, but are a promising cancer immunotherapy. First, the shared expression of target antigens between T effector cells and T-cell malignancies results in fratricide, or self-killing, of CAR-T cells. Second, harvesting adequate numbers of autologous T cells without contamination by malignant cells is, at best, technically challenging and prohibitively expensive. Third, the use of genetically modified CAR-T cells from allogeneic donors may result in life-threatening graft-vs.-host disease (GvHD) when infused into immune-compromised HLA-matched or mismatched recipients. We hypothesized that deletion of CD7 and the T-cell receptor alpha chain (TRAC) using CRISPR/Cas9 in CAR-T targeting CD7 (UCART7) would result in the efficient targeting and killing of malignant T cells without significant effector T-cell fratricide or induction of GvHD. We chose to target CD7 on malignant T cells because it is overexpressed on the vast majority of T-cell and NK-cell malignancies. Second, germline biallelic deletion of CD7 resulted in mice with normal T-cell numbers, T-cell subsets, and T-cell function. We generated a CD7 CAR using a anti-CD7 single chain variable fragment (scFv) created using commercial gene synthesis and cloned into the backbone of a 3rd-generation CAR with CD28 and 4-1BB internal signaling domains. The construct was modified to express CD34 via a P2A peptide to enable detection of CAR following viral transduction. Human primary T cells were activated using anti-CD3/CD28 beads for 48 hours prior to bead removal and electroporation with CD7 gRNA, TRAC gRNA, and Cas9 mRNA. On day three, T cells were transduced with lentivirus particles encoding either CD7 CAR or CAR CD19 control and allowed to expand for a further 6 days. Transduction efficiency and ablation of CD7 and TRAC was confirmed by flow cytometry. Multiplex CRISPR/Cas9 gene-editing resulted in the simultaneous deletion of both CD7 and TRAC in 72.8%±1.92 of cells, as determined by FACS analysis. To prevent alloreactivity, CD3+ CAR-T were removed from the product by magnetic depletion. UCART7 effectively killed T-ALL cell lines (CCRF-CEM, MOLT3, and HSB2) and human primary T-ALL blasts in vitro. Next, we tested the capacity of UCART7 to kill primary T-ALL in vivo without xenogeneic GvHD. Considerable expansion of alloreactive T cells, severe GvHD (mean clinical GvHD score = 5.66), and a robust graft vs. leukemia effect were observed in recipients of WT T cells. In contrast, GvHD was completely absent, T cells were undetectable, and considerable tumor burden was observed in mice receiving TRACΔ T cells. Mice receiving UCART7 had no GvHD and unlike UCART19 controls, effectively cleared T-ALL blasts. Fratricide-resistant and allo-tolerant "off-the-shelf" UCART7 signifies a novel strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin's T-cell lymphoma. Citation Format: John F. Dipersio, Matthew Cooper. Targeting T cell malignancies with gene edited CAR-T [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY42-01.

An Off-the-Shelf™ Fratricide-Resistant CAR-T for the Treatment of T Cell Hematologic Malignancies

T cell malignancies represent a class of devastating hematologic cancers with high rates of relapse and mortality in both children and adults for which there are currently no effective or targeted therapies. Despite intensive multi-agent chemotherapy regimens, fewer than 50% of adults and 75% of children with T-ALL survive beyond five years. For those who relapse after initial therapy, salvage chemotherapy regimens induce remissions in 20-40% of cases. Allogeneic stem cell transplant, with its associated risks and toxicities, is the only curative therapy.T cells engineered to express a chimeric antigen receptor (CAR) are a promising cancer immunotherapy. Such targeted therapies have shown great potential for inducing both remissions and even long-term relapse-free survival in patients with B cell leukemia and lymphoma7-9. Thus, a targeted therapy against T cell malignancies represents a significant unmet medical need. However, several challenges have limited the clinical development of CAR-T cells against T cell malignancies. First, the shared expression of target antigens between T effector cells and T cell malignancies results in fratricide, or self-killing, of CAR-T cells. Second, harvesting adequate numbers of autologous T cells, without contamination by malignant cells is, at best, technically challenging and prohibitively expensive. Third, the use of genetically modified CAR-T cells from allogeneic donors may result in life-threatening graft-vs.-host disease (GvHD) when infused into immune-compromised HLA-matched or mismatched recipients.We hypothesized that deletion of CD7 and the T cell receptor alpha chain (TRAC) using CRISPR/Cas9 in CAR-T targeting CD7 (UCART7) would result in the efficient targeting and killing of malignant T cells without significant effector T cell fratricide or induction of GvHD.To generate the CD7 CAR, the anti-CD7 single chain variable fragment (scFv) was created using commercial gene synthesis and cloned into the backbone of a 3rd generation CAR with CD28 and 4-1BB internal signaling domains. The construct was modified to express CD34 via a P2A peptide to enable detection of CAR following viral transduction. Human primary T cells were activated using anti-CD3/CD28 beads for 48 hours prior to bead removal and electroporation with CD7 gRNA, TRAC gRNA, and Cas9 mRNA. On day three, T cells were transduced with lentivirus particles encoding either CD7 CAR or CAR CD19 control and allowed to expand for a further 6 days. Transduction efficiency and ablation of CD7 and TRAC were confirmed by flow cytometry. Multiplex CRISPR/Cas9 gene-editing resulted in the simultaneous bi-allelic deletion of both CD7 and TRAC in 72.8%±1.92 of cells, as determined by both non-homologous end joining (NHEJ) and FACS analyses. To prevent alloreactivity, CD3+ CAR-T were removed from the product by magnetic depletion. Of particular importance is that by using two distinct methods for assessing “off-target” nuclease activity across the entire human T cell genome (Guide-seq and probe capture), we could only detect one gene, an intronic modification of RMB33, that was inappropriately targeted using this approach. No obvious genomic rearrangements were detected by these analyses.UCART7 effectively expanded and killed T-ALL cell lines (CCRF-CEM, MOLT3, and HSB2) and human primary T-ALL blasts in vitro. Next, we tested the capacity of UCART7 to kill primary T-ALL in vivo without xenogeneic GvHD. Considerable expansion of alloreactive T cells, severe GvHD (mean clinical GvHD score = 5.66), and a robust graft vs. leukemia effect were observed in recipients of WT T cells. In contrast, GvHD was completely absent, T cells were undetectable, and considerable tumor burden was observed in mice receiving TRACΔ T cells. Mice receiving UCART7, however, had no GvHD and, unlike UCART19 controls, effectively cleared primary human T-ALL in NSG mice.Fratricide-resistant and allo-tolerant ‘off-the-shelf’ UCART7 signifies a novel strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin's T cell lymphomas without a requirement for autologous T cells and represents the first clinically feasible adoptive T cell therapy for T cell malignancies. DisclosuresNo relevant conflicts of interest to declare.

Exonal switch down-regulates the expression of CD5 on blasts of acute T cell leukaemia.

To date, CD5 expression and its role in acute T cell lymphoblastic leukaemia (T-ALL) have not been studied closely. We observed a significant reduction in surface expression of CD5 (sCD5) on leukaemic T cells compared to autologous non-leukaemic T cells. In this study, we have shown the molecular mechanism regulating the expression and function of CD5 on leukaemic T cells. A total of 250 patients suffering from leukaemia and lymphoma were immunophenotyped. Final diagnosis was based on their clinical presentation, morphological data and flow cytometry-based immunophenotyping. Thirty-nine patients were found to be of ALL-T origin. Amplification of early region of E1A and E1B transcripts of CD5 was correlated with the levels of surface and intracellular expression of CD5 protein. Functional studies were performed to show the effect of CD5 blocking on interleukin IL-2 production and survival of leukaemic and non-leukaemic cells. Lack of expression of sCD5 on T-ALL blasts was correlated closely with predominant transcription of exon E1B and significant loss of exon E1A of the CD5 gene, which is associated with surface expression of CD5 on lymphocytes. High expression of E1B also correlates with increased expression of cytoplasmic CD5 (cCD5) among leukaemic T cells. Interestingly, we observed a significant increase in the production of IL-2 by non-leukaemic T cells upon CD5 blocking, leading possibly to their increased survival at 48 h. Our study provides understanding of the regulation of CD5 expression on leukaemic T cells, and may help in understanding the molecular mechanism of CD5 down-regulation.

Sphingosine-1-Phosphate Induces Dose-Dependent Chemotaxis or Fugetaxis of T-ALL Blasts through S1P1 Activation.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in several physiological processes including cell migration and differentiation. S1P signaling is mediated through five G protein-coupled receptors (S1P1-S1P5). S1P1 is crucial to the exit of T-lymphocytes from the thymus and peripheral lymphoid organs through a gradient of S1P. We have previously observed that T-ALL and T-LBL blasts express S1P1. Herein we analyzed the role of S1P receptors in the migratory pattern of human T-cell neoplastic blasts. S1P-triggered cell migration was directly related to S1P1 expression. T-ALL blasts expressing low levels of S1P1 mRNA (HPB-ALL) did not migrate toward S1P, whereas those expressing higher levels of S1P1 (MOLT-4, JURKAT and CEM) did migrate. The S1P ligand induced T-ALL cells chemotaxis in concentrations up to 500 nM and induced fugetaxis in higher concentrations (1000–10000 nM) through interactions with S1P1. When S1P1 was specifically blocked by the W146 compound, S1P-induced migration at lower concentrations was reduced, whereas higher concentrations induced cell migration. Furthermore, we observed that S1P/S1P1 interactions induced ERK and AKT phosphorylation, and modulation of Rac1 activity. Responding T-ALL blasts also expressed S1P3 mRNA but blockage of this receptor did not modify migratory responses. Our results indicate that S1P is involved in the migration of T-ALL/LBL blasts, which is dependent on S1P1 expression. Moreover, S1P concentrations in the given microenvironment might induce dose-dependent chemotaxis or fugetaxis of T-ALL blasts.

Targeting Rac-Gtpases Reverses Stroma-Induced Resistance to Notch and mTOR-Inhibition in Acute T-Cell Leukemia

Molecular hallmarks of T-ALL are the aberrant activation of NOTCH signaling and high activity of the PI3K-AKT-mTOR pathway. Upregulated mTOR and NOTCH have been linked to the resistance of T-ALL to chemotherapy as well as to high frequencies of leukemia-initiating cells. Hence, the mTOR and the NOTCH pathways are promising therapeutic targets in T-ALL. However, clinical success of the mTOR inhibitor Rapamycin in acute leukemia has been disappointing. Similar results have been observed in mouse models of T-ALL treated with Notch inhibitors. To investigate the impact of mTOR and Notch inhibition in a genetically complex T-ALL, we developed an aggressive murine T-ALL model, driven by tyrosine kinase signaling, loss of Pten, Cux1-haploinsufficiency and constitutive Notch signaling. In vitro, T-ALL blasts were highly sensitive to inhibition of AKT, mTOR and Notch signaling. We transplanted the leukemias into secondary recipients and initiated treatment with Rapamycin after the onset of leukemia. Rapamycin significantly prolonged survival of the animals (placebo: 27 days, Rapamycin 49 days, p<0.001). Eventually, all Rapamycin treated animals succumbed to the T-ALL that extensively infiltrated the bone marrow and solid organs despite continuous drug administration. When Rapamycin-resistant blasts were explanted and cultured in petri dishes they again became susceptible to Rapamycin, demonstrating a context-dependent resistance rather than outgrowth of intrinsically resistant clones. Gene set enrichment analysis revealed that Rapamycin-resistant T-ALL in vivo upregulated genetic networks associated with cell-cell interactions. Stromal cell support from OP9-cells as well as from mesenchymal stem cells recapitulated the in vivo effect and induced resistance to mTOR and Notch-inhibition in T-ALL blasts. Coating the tissue culture wells with Collagen, Fibronectin, Retronectin or Matrigel, did not elicit resistance. By using trans-well assays we show that the stroma-induced resistance was dependent on direct cell-cell interactions. Immunoblots and PhosFlow probing the mTORC1/C2 and Notch pathway demonstrated an identical drug effect on their intracellular targets in resistant T-ALL blasts cultured on stroma cells and susceptible cells in suspension. Since the number of molecules potentially involved in cell-to-cell contacts is vast, we focused on central nodes that organize this process in order to find a potentially druggable target that is critically involved in stroma-induced resistance. Transcriptome profiling pointed towards upregulation of Rac-associated pathways. We determined the activation of Rac1 by PAK-pull down assays in T-ALL blasts grown in suspension or on stromal cells. We observed an increase (FC=1.96 ± 0.58, p=0.04) in activated Rac1 in the T-ALL blasts in contact with a stromal layer. To determine whether Rac activation plays a role in stroma-induced resistance, we devised a strategy to abrogate Rac signaling in T-ALL blasts, but not in the stromal cells, since inhibition of Rac in stromal cells by the Rac-inhibitor NSC23766 led to the their detachment. Furthermore, Rac1,2 and 3 can be functionally redundant, making knock down experiments using shRNAs challenging. The Clostridium difficile serotype F strain 1470 produces toxin B isoform (TcdBF), that selectively glucosylates and inactivates Rac(1,2,3). We pretreated T-ALL blasts with TcdBF and observed a dose-dependent functional inhibition of Rac GTPases monitored by dephosphorylation of the Rac effector kinase pS144/141-PAK-1/2. T-ALL blasts were then incubated for 5 hours with increasing toxin doses, washed 3 times and incubated in toxin-free medium. Eighteen hours after the end of the exposure to the toxins, Rac was still inhibited. Strikingly, in the TcdBF-pretreated T-ALL, the stroma-induced resistance effect was abrogated and clusters of apoptotic cells were clearly visible (>2 fold reduction of the input, p=0.002). In contrast, the carrier-treated T-ALL exhibited resistance to the inhibitors on stroma (>10 fold expansion of the input, p<0.0003). Altogether, we identify the Rac-GTPases as a nexus of stroma-induced drug resistance and show that inhibition of Rac and mTOR is synthetically lethal to T-ALL blasts T-ALL blasts that are in contact with stromal cells, paving the way to augment the effectiveness of small molecule inhibitors in acute leukemia. DisclosuresNo relevant conflicts of interest to declare.

411. CD5 CAR for the Treatment of T-Cell Malignancies

Chimeric antigen receptors (CARs) have emerged as a powerful therapeutic tool by redirecting patients’ own T cells to tackle hematologic malignancies – in particular, those of B cell origin. However, T cell malignancies remain a more challenging task for CAR T cells owing to the shared expression of targetable tumor-associated antigens between normal and cancerous T cells, potentially invoking fratricide of CAR T cells and/or profound immunodeficiency.We designed a novel CAR targeting CD5, a common marker expressed in ~80% of T-cell leukemia/lymphoma blasts as well as in normal T cells. Upon transduction with CD5 CAR, more than 95% of T cells expressed the CAR while producing limited and transient fratricide. Fratricide of CD5 CAR T cells was mainly directed against naive phenotype and central memory cells, eliminating more than 50% of cells in these subsets and resulting in preferential expansion of effector memory and effector T cells. CD5 CAR T cells acquired resistance to self-killing by 3 days post-transduction and could then expand normally, up to 100,000-fold. Expansion of CD5 CAR T cells coincided with downregulation of CD5 protein from the cell surface while preserving CAR expression.CD5 CAR T cells mounted an effective anti-tumor response, selectively killing five CD5-positive T-ALL and T lymphoma cell lines in 4-hour chromium release assays while displaying no activity against CD5-negative cell lines. Upon longer-term coculture, CD5 CAR T cells eliminated >95% of leukemia cells from 3 T-ALL lines within 48h and 100% by day 7. We also observed the ability of CD5 CAR T cells to eliminate leukemia cells in sequential killing assays where we recurrently replenished fresh target cells for at least 4 iterations. Lack of functional exhaustion in sequential killing assays supports the fitness of CD5 CAR T cells for eradicating large numbers of tumor cells in vivo. CD5 CAR T cells dramatically suppressed systemic in vivo disease progression in 3 different xenograft mouse models, doubling median survival. Importantly, CD5 CAR T cells demonstrated significant cytokine production and cytotoxicity against primary T-ALL blasts (n=6), highlighting the therapeutic potential of CD5 CAR for patients with T cell malignancies. Overall, we demonstrated for the first time that CD5 CAR redirects T cells to eliminate CD5-positive malignant T cells in vitro and in vivo while producing only limited fratricide of the normal T cell population.

Abstract 997: Targeting the Jak/Stat signaling pathway is highly effective in xenograft models of early T cell precursor (ETP) acute lymphoblastic leukemia (ALL)

Abstract ETP ALL is a recently described subtype of T-ALL with poor prognosis and is defined by a unique immunophenotype, expressing T-lineage and myeloid/early progenitor markers. ETP ALL blasts have a diverse array of genetic alterations without a unifying mutated gene or pathway. A fraction have mutations in genes involved in Jak/Stat signaling, and we hypothesized targeting Jak/Stat may be effective for some ETP ALL patients. We evaluated Jak/Stat signaling by phosphoflow cytometry and immunoblotting of blasts collected from patients with ETP ALL with and without Jak/Stat pathway mutations and compared them to non-ETP T-ALL blasts. We xenografted blasts from 6 different ETP ALL patient samples with heterogeneous mutations (Table) into immunodeficient NSG mice and tested the in vivo efficacy of the Jak1/2 inhibitor ruxolitinib (rux). Mice were randomized to rux or vehicle after they developed &amp;gt;1% peripheral blood (pb) blasts (cytoCD3+/CD45+) by flow cytometry (FACS). Disease burden was assessed by FACS enumeration of pb blasts weekly and splenic blasts at sacrifice. We found that the Jak/Stat signaling pathway was dysregulated in ETP ALL samples, including in samples without identifiable mutations in Jak/Stat. ETP ALL blasts had marked increased expression of pSTAT1 (p = 0.01), pSTAT3 (p = 0.01) and pSTAT5 (p = 0.02) when compared to non ETP T-ALL by immunoblot, results confirmed by phosphoflow. All 6 ETP samples had a profound in vivo response to rux; circulating and splenic blasts were markedly reduced in all treated animals compared to controls (Table). The degree of response was comparable to that seen with TKIs in BCR-ABL ALL, using the same xenograft model. These results strongly suggest that ETP ALL blasts may be addicted to Jak/Stat signaling, irrespective of the presence of identifiable Jak/Stat mutations and that clinical trials testing Jak/Stat inhibitors are needed. Ruxolitinib is active in ETP ALLGenetic alterationAvg splenic blasts in millions. Control vs Rux Treated (p value by 2-tailed t-test)DNM2 m, JAK3 m157 vs 5 (p &amp;lt;0.01)PTPN11 SV124 vs 31 (p &amp;lt;0.01)GATA3 del, JAK1m, SH2B3 SV192 vs 18 (p &amp;lt;0.01)EZH2 m, RUNX1 m, NOTCH1 m, IKZF1m, SUZ12 m, SH2B3 SV452 vs 25 (p &amp;lt;0.01)EED m256 vs 1 (p &amp;lt;0.01)GATA3 m, JAK1 m, PTEN del, RB1 del, CDKN2A/B del332 vs 4 (p &amp;lt;0.01)m = mutation; del = deletion; SV = structural variationPK obtained from mice demonstrated serum levels that correlate with humans treated with drug and that would be expected to target JAK1 and JAK2 but not JAK3. Citation Format: Shannon L. Maude, Sibasish Dolai, Cristina Delagdo-Martin, Tiffaney Vincent, Alissa Robbins, Arthavan Selvanathan, Theresa Ryan, Stephen P. Hunger, Mignon L. Loh, Charles G. Mullighan, Brent L. Wood, Michelle L. Hermiston, Stephan A. Grupp, Richard B. Lock, David T. Teachey. Targeting the Jak/Stat signaling pathway is highly effective in xenograft models of early T cell precursor (ETP) acute lymphoblastic leukemia (ALL). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 997. doi:10.1158/1538-7445.AM2014-997

Defined, Serum-Free Conditions for in Vitro culture of Primary Human T-ALL Blasts

AbstractAbstract 3537Acute lymphoblastic leukemia (ALL) is the most common malignancy diagnosed in children, accounting for 30% of cancer diagnoses in patients under the age of 15 years. Of these cases, roughly 15% derive from the T-cell lineage (T-ALL). Steady improvements in treatment protocols have recently led to survival rates near 80%; however, the remaining 20% have uniformly poor outcomes. Thus far, the therapeutic strategies implemented have focused on intensifying multiagent chemotherapy. As undesirable toxicity arises with additional intensification of standard chemotherapy agents, more targeted molecular agents for T-ALL are urgently needed to further improve outcomes of this disease.Preclinical studies have largely relied on a relatively small number of established T-ALL cell lines that have been maintained in culture for long periods of time, as well as primary T-ALLs xenografted in immunodeficient mice. Recently, research efforts have focused on the development of robust in vitro culture models for efficient testing and validation of novel therapeutics on primary human T-ALL blasts. A relatively simple and efficient in vitro culturing system would in part eliminate the concerns of using cell lines that might not reflect the biology of a typical disease in patients, and mitigate the costly and time-consuming endeavor of xenografting T-ALL blasts. Moreover, personalized medicine initiatives would be dramatically improved by the ability to assess drug sensitivity profiles on patients' own tumor cells in direct in vitro culture assays.Several groups have devised methods for in vitro culture of patient T-ALL cells, the most successful of which have utilized stromal feeders. These methods have relied also upon serum-containing medium and thus have yielded variable results due in part to lot-to-lot variation in serum quality. Using a defined, serum-free media based on the WIT formulation initially described for in vitro propagation of normal and transformed human mammary epithelial cells, we have now optimized culture conditions required for primary human T-ALL samples. We demonstrate this serum-free culture system to consistently outperform serum-containing medium, supporting expansion of primary T-ALL samples 2- to 200-fold within 3 weeks of initial explantation from either xenografted mice or patient biopsy material and with only limited cell death. When carried for longer periods of time (up to 6 weeks), cultures maintained at higher cell densities attained maximal expansions of 40- to 100-fold while those maintained at lower cell densities expanded up to 10,000-fold. Importantly, T-cell receptor γ heteroduplex analysis confirmed expansion of the original clone throughout the culture period and flow cytometric analysis confirmed cultures to be composed of immature human T-lineage cells. Of note, cultures tended to regress after 30–40 days in vitro, suggesting that long-term renewing cells are not supported under these conditions.The use of defined, serum-free medium in this culture system has facilitated systematic characterization of growth factor requirements. We show that supplemental IL-7 and insulin/IGF1 are critical for short-term culture expansion, whereas SCF and Flt3L are dispensable. This culture system will also enable us to assess other “stem” supporting factors and genes (by lentiviral transduction) for their contribution to long-term culture maintenance. Finally, we demonstrate the facility of this culture system for testing of novel therapeutic compounds. By allowing direct study of primary samples in vitro, this culture system will advance basic mechanistic studies in T-ALL biology as well as preclinical studies aimed at developing individually tailored therapy protocols. Disclosures:Ince:Stemgent (2008–10): Consultancy, TAI receives royalties for WIT-P, -I, and -T media Other.

Siglec‐7 tetramers characterize B‐cell subpopulations and leukemic blasts

Cell surface glycosylation has important regulatory functions in the maturation, activation, and homeostasis of lymphocytes. The family of human sialic acid-binding immunoglobulin-like lectins (siglecs) comprises inhibitory as well as activating receptors intimately involved in the regulation of immune responses. Analyses of the interaction between siglecs and glycans are hampered by the low affinity of this interaction. Therefore, we expressed siglec-7 in eukaryotic cells, allowing for glycosylation, and oligomerized the protein in analogy to MHC tetramers. Using this tool, flow cytometric analysis of lymphocytes became possible. Sialic acid-dependent binding of siglec-7 tetramers was confirmed by glycan array analysis and loss of siglec tetramer binding after neuraminidase treatment of lymphocytes. In contrast to most lymphocyte subpopulations, which showed high siglec-7 ligand expression, B-cell subpopulations could be further subdivided according to different siglec-7 ligand expression levels. We also analyzed blasts from acute lymphoblastic leukemias of the B-cell lineage as well as the T-cell lineage, since malignant transformation is often associated with aberrant cell surface glycosylation. While pediatric T-ALL blasts highly expressed siglec-7 ligands, siglec-7 ligands were barely detectable on cALL blasts. Taken together, oligomerization of recombinant soluble siglec-7 enabled flow cytometric identification of physiologic lymphocyte subpopulations and malignant blasts.

High Mir-221 Expression Is Associated with Poorer Treatment Outcome of Patients with T-Cell Acute Lymphoblastic Leukemia

Abstract 1439T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cells that accounts about 15% of pediatric and 25% of adult ALL cases. In the last years, several clinical and laboratory features have been described as prognostic markers; nevertheless, with intensification of therapy most of them have lost their predictive value. MicroRNA (miRNA) expression analysis has proved to be an useful tool for identifying specific subsets of cancer patients with relevant cytogenetic, laboratorial and clinical features. The aim of the present study was to determine if miRNAs may be useful markers in T-ALL. First, we performed a supervised analysis comparing the miRNA expression profile of T-ALL blasts from 36 T-ALL/CD56− and 12 T-ALL/CD56+. We selected CD56 as prognostic marker based on our previous report showing that the disease-free survival (DFS) of T-ALL/CD56+ patients was of 28.5 months compared to 69.8 in the CD56− group. Also patients tended to be older and to present normal platelet counts in the T-LLA/CD56+ group. We used the Taqman MicroRNA Assay Human Panel (Applied Biosystems) to perform a screening of 164 knowledge mature miRNA sequences using specific primers and probes according to manufacturer instructions. Total RNA input was normalized based on the geometric means of Ct values obtained from four endogenous RNAs. All reactions were run in duplicate and a coefficient of variation greater than 5% was used as an exclusion factor (seven miRNAs were excluded). The fold change was calculated using comparative 2−δCt method. We have identified a set of 14 miRNAs differentially expressed, of which miR-374 and miR-221 best distinguished T-ALL/CD56+ from T-ALL/CD56− blasts. Based on this profile, we selected miR-221 and miR-374 as potential markers and quantified their expression in the same samples using RQ-PCR. Patients were stratified as high and low expression using the median value as cut off. We detected a significant association between the miR-221 high expression and poorer treatment outcome. On the contrary, miR-374 expression levels were not associated with treatment outcome. We evaluate the impact of age, white blood cell counts, CD56 and miR221 expression on overall survival (OS). Age and miR-221 were the only ones found to be significant. The estimate 5-year OS (mean and confidence interval 95%) was of 67.0 ± 10.3% in the group of patients expressing miR-221 below the cut-off value, whereas this value was of 28.5 ± 14.5% in the alternative group. Even among T-ALL/CD56− patients, the higher expression of miR-221 was significantly associated with poorer outcome. Our data suggest that miR-221 play an important role in T-ALL and its regulation may represent a potential therapeutic intervention. Disclosures:No relevant conflicts of interest to declare.

Enhancement of TRAIL cytotoxicity by AG-490 in human ALL cells is characterized by downregulation of cIAP-1 and cIAP-2 through inhibition of Jak2/Stat3

The ability of death-inducing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to selectively kill a variety of cancer cells has been largely described, but one of the major concerns with the treatment is the occurrence of drug resistance and possible toxic side effects. Here, we report that TRAIL induces apoptosis in Jurkat and SUPT1 T cell lines and in human T-ALL blasts but not in healthy subject-derived peripheral blood mononuclear cells. In parallel, the treatment with TRAIL and Tyrphostin (AG-490), a selective Janus kinase 2 inhibitor, produces an evident enhancement of cytotoxicity, characterized by a significant inhibition of Stat3 phosphorylation compared to controls or to TRAIL alone-treated samples, and associated with a dramatic decrease of both cIAP-1 and cIAP-2 mRNA levels. Downregulation of cIAP-1 and cIAP-2 by specific small interference RNAs significantly amplifies TRAIL-reduced cytotoxicity. All together, these findings strongly indicate that cIAP-1 and cIAP-2 downregulation is a fundamental step in the signaling pathways mediating the combinatorial effect of TRAIL and AG-490 on T cell leukemia. These findings may help to open new routes for the development of less toxic pharmacological strategies in the treatment of patients affected by TRAIL-sensitive leukemias.

Epigenetic control of differential expression of specific ERG isoforms in acute T-lymphoblastic leukemia

Expression of ERG is of prognostic significance in acute myeloid leukemia (AML) and T-lymphoblastic leukemia (T-ALL) pointing to its role in leukemogenesis. To unravel its transcriptional regulation we analyzed the expression of ERG specific isoforms. Expression of the two main isoforms ERG2 and ERG3 was found in AML and normal CD34+ cells, whereas T-ALL blasts only expressed ERG isoforms harboring exon 5 ( ERG3) lacking expression of ERG2. Bisulfite sequencing revealed hypermethylation of a CpG island within the ERG2 promoter region in T-ALL. Treatment of the T-lymphoblastic cell line BE13 with decitabine led to re-expression of ERG2 and pyrosequencing showed concordant DNA hypomethylation, thus confirming a methylation regulated expression of ERG2. Moreover, the identification of a new ERG isoform ( ERG3Δ ex12) suggests the association with different interaction partners and adds to the complexity of downstream pathways mediated by the expression of specific ERG transcripts in acute leukemia.

NKX3.1 Is a Direct TAL1 Target Gene That Mediates the TAL1 Dependent Proliferation of Human T-Cell Acute Lymphoblastic Leukemia (T-ALL)

The TAL1/SCL gene encodes a bHLH (basic Helix-Loop-helix) protein that acts as a master gene in hematopoiesis. The TAL1/SCL gene is also the most frequently activated gene in human T-ALL but the oncogenic transcriptional programs, downstream of TAL1 in human T-ALL, are not well characterized. Using RNA interference to knockdown TAL1 expression, we show that TAL1 regulates both cell proliferation and death of human T-ALL cells. To determine the TAL1 target genes in human T-ALL, we combine TAL1 knockdown and gene expression profiling and show that TAL1 activates and repress a common subset of genes in cell lines. This subset includes known TAL1 target genes but also the NKX3.1 gene that is a homeobox gene, specifically expressed in the prostate epithelium during prostate development and in adulthood. NKX3.1 gene inactivation is one of the earliest events that occur in prostate cancer initiation, defining NKX3.1 as a major tumor suppressor gene of this cancer. TAL1 expression is associated with NKX3.1 activation in human T-ALL cell lines and NKX3.1 is expressed in TAL1 expressing human T-ALL blasts. TAL1 and GATA-3 are specifically bound in vivo to the [−870/−570] region of the human NKX3.1 gene promoter, and ex vivo, TAL1 can either directly binds an E-box [position −738] or be recruited by GATA-3 on a GATA binding site [position −697]. Finally, functional analyses of the NKX3.1 promoter indicate that these binding sites mediate the transcriptional activity of this promoter in T-cell lines. Sequences analysis of the human and mouse NKX3.1 promoters show that the regulatory sequences involved in the TAL1 activation of the human NKX3.1 gene are not conserved in the mouse gene, indicating why the NKX3.1 gene is not expressed in mouse models of TAL1 mediated leukemogenesis. NKX3.1 knockdown shows that NKX3.1 is necessary for the proliferation of TAL1 expressing T-ALL cell lines and NKX3.1 overexpression can complement the proliferation defects associated with TAL1 knockdown in T-ALL cell lines. Microarray analyses show that TAL1 and NKX3.1 regulate a common subset of genes in T-ALL that includes numerous genes encoding proteins known to be involved in T-cell proliferation and/or signaling. Finally, using a new culture system that enables proliferation of primary human leukemic cells, we show that the NKX3.1 gene is specifically activated in human TAL1 expressing T-ALL together with the defined potential TAL1 and/or NKX3.1 target genes. These results characterize NKX3.1 as the first gene directly activated by TAL1 and involved in the TAL1 dependent proliferation of human T-cell Acute Lymphoblastic Leukemia.

Pediatric T-ALL but Not cALL Blasts Express Ligands for Siglec-7 Inhibiting NK Cell Mediated Lysis.

Changes in the glycosylation pattern of cell surface proteins occur during early stages of malignant transformation. These alterations in the glycan repertoire may contribute to numerous processes of tumor cell survival such as adhesion, migration and immune evasion. Sialic acid is the most common terminal carbohydrate moiety of glycan structures in humans binding to a family of eleven receptors termed sialic acid binding Ig-like lectins (siglecs). Siglecs are expressed on virtually all effector cells of the immune system and several members function as inhibitory receptors. Siglec-7 is known as an inhibitory receptor expressed by NK cells and T cells. Therefore, siglec-7 ligands may contribute to immune evasion of malignant cells.In order to analyze the expression of siglec-7 ligands by malignant cells, we cloned the extracellular domain of human siglec-7. The recombinant protein was expressed in 293 cells to allow glycosylation, which is supposed to be important for specificity. As the interaction of a single glycan and lectin is of low affinity, we tetramerized siglec-7 in analogy to MHC-tetramers to increase avidity. This strategy allowed for reliable use of siglec-7 fusion protein in flow cytometry. Analysis of PBMC from healthy donors revealed that siglec-7 ligands are expressed on all subpopulations of PBMC: expression was detected on CD4+ and CD8+ T-cells as well as on monocytes and NK cells. B cells could be subdivided into a positive and negative population after staining with siglec-7 tetramers.Subsequently, we analyzed primary lymphatic blasts from childhood leukemias. Interestingly, T-ALL blasts expressed ligands for siglec-7, whereas these ligands were absent from cALL blasts. As cALL blasts are known to be better targets for NK cell-mediated cytotoxicity than T-ALL blasts, we hypothesized that siglec-7 ligands on the surface of leukemic blasts inhibit NK cells. To test the functional relevance of siglec-7 ligand expression for NK cell-mediated cytotoxicity, we used soluble monomeric siglec-7 as competitive inhibitor in cytotoxicity assays. In these competition assays, NK cell-mediated specific lysis of leukemic cells increased roughly twofold in the presence of soluble siglec-7.These results show that engagement of inhibitory siglecs on the surface of effector cells might contribute to immune escape mechanisms of malignant cells. It underlines the function of siglec-7 as a non-MHC-specific inhibitory receptor on NK cells and the important role of altered glycans expression on malignant cells in tumor immunology.

Clinical flow cytometry, a hypothesis-driven discipline of modern cytomics.

Recently, two major books have been published that summarize the historical aspects and recent achievements of practical flow cytometry (1,2). Both emphasize the role played by this newly developed technical discipline in the development of scientific (1) and diagnostic platforms during late 20th-century medicine (2). Indeed, the gray box called a flow cytometer is the result of a multidisciplinary collaboration between engineers, biophysicists, biochemists, histopathologists, molecular cytologists, hematologists, immunologists, and quality controllers, with a more recent contribution from physicians specializing in the human immunodeficiency virus (HIV), oncologists, and epidemiologists (Table 1). The foresight by the “fathers” has been astonishing (1). In his book, Howard Shapiro reminds us that flow cytometry, used to investigate cells in a flow system instead of on a static microscope, was put into practice by Coulter when he used impedance to count red cells. Rapid cell spectroscopy was introduced by Kamentsky to leukocyte differential counting with computer-assisted displays. Then optical cell counters, fluorescence dies, lasers, and photomultiplier tubes were added by Fulwyler and Jones. Immunologic concepts and reagents were introduced by the Herzenbergs and were fully primed to harvest the gems of the incipient monoclonal antibody revolution. The first commercial cytometer with precision engineering of the flow cells was launched by Goehde. These initial steps were duly followed by the release of a series of cytometers that showed increasing sensitivity, computerization, and practicality in research and routine laboratory work. This is illustrated by science historians Keating and Cambrosio in their book that blends the sociological aspects of medical technological developments with witness accounts (2). Flow cytometry currently is a colorful, practical discipline that has become available to medicine at the appropriate time for various pressing clinical applications, thus rewarding the scientists who had the vision to foresee these needs. These contributions established cytometry, including flow technology, image analysis, and advanced microscopy, among the leading trends of modern biomedicine and health care. After the launch of the Human Genome Project (3), two areas, genomics and proteomics, have been majestically promoted on both sides of the Atlantic and in the East (4). Genomics includes the identification of genes and gene regulatory processes, and proteomics investigates the abundance of proteins simultaneously with the changes associated with alterations of the functional state of the cell. Such a “pseudo-functional” approach aims to extend the study of quantitative changes during differentiation, proliferation, and signaling of different cell types (5). Clearly, there is an enormous, newly generated influx of information here, but it is not certain that a mere analysis of genes and protein structure, even in its extended format that includes the interaction of various biomolecules, will provide all of the necessary information to understand function and regulation at the level of living cells and organisms. Hence, the concept of cytomics has been introduced recently (6,7), for two reasons. First, cytomics is the cell-oriented analysis of molecules and their functions in cellular systems and/or organs, referred to as cytomes. Second, cytomics is the new, absolutely essential, interface between biosciences and clinical medicine. Importantly, advanced cytometry, including the traits defined in Table 1, represent the driving engine of cytomics. A new paradigm is that cytomics is the combined, interrelated force for cytometry, proteomics, and genomics (6,7). There are, however, significant differences between these three modern trends. First, it is in the realm of cytometry, with the major contribution of flow systems, where the appropriate functional analysis of cells, the display and regulation of functional molecules, and the differentiation pathways are successfully carried out. Animal models and human diseases provide targets for these studies. Second, it is the cytometric field where the diagnostic tests, for malignancies (cf. 8,9) and the cellular analysis of infectious diseases (10), currently harvest the richest crop of practical results, whereas in the fields of genomics and proteomics only the seeds are sown for the practically useful multiarray systems. Third, these two fields at their current developmental state, are essentially observational sciences that primarily document the observed heterogeneity. As a marked contrast, the research by flow cytometry has been, from the beginning, primarily

Phenotypic heterogeneity of TDT+ cells in the blood and bone marrow: implications for surveillance of residual leukemia [see comments

Terminal deoxynucleotidyl transferase (TdT) is a useful marker for normal lymphocyte precursors and acute lymphoblastic leukemia (ALL). Our previous studies, however, have shown that for monitoring minimal residual disease in the circulation, assay for TdT alone is not sufficiently specific to distinguish leukemia cells from the background of rare normal blood TdT+ cells. In an attempt to increase specificity for leukemic cells, we have used double and triple immunophenotypic analysis to characterize normal circulating and bone marrow TdT+ cells. Overall, normal TdT+ cells were about 1000-fold more frequent in the marrow than in the blood. More than 75% of TdT+ cells in both the blood and marrow expressed the CD34, CD22, and HLA-DR antigens. However, circulating TdT+ cells infrequently expressed CD19 (4.5%) and CD9 (2.3%), compared with their marrow counterparts (74% and 47%, respectively). The brightly staining CD10+ phenotype, frequently associated with ALL blasts, was significantly less common among normal blood (5.7%) than marrow (31%) TdT+ cells. Although T-lineage markers were rarely expressed on TdT+ cells in either site, CD7+ cells were far more prevalent within the circulating TdT+ subset (4%) than among the marrow population (less than 0.2%). The results suggest a selective release of lineage-uncommitted and/or thymus-destined TdT+ cells from the marrow into the circulation. Moreover, since CD19, CD9, and high- density CD10 are frequently found on ALL blasts, staining for these markers on TdT+ cells in the circulation should improve the specificity of assay for residual common ALL cells. Likewise, assay for CD5+ and possibly CD7+ TdT+ cells in either marrow or blood should provide a very sensitive method of detection of T-ALL blasts.