Intra-Lineage Phenotypic Switch in T-Cell Prolymphocytic Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2005-2005
Author(s):  
Nnenna Osuji ◽  
Ilaria Del Giudice ◽  
Estella Matutes ◽  
Vasantha Brito-Babapulle ◽  
Alison Morilla ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Clinical Features ◽  
Residual Disease ◽  
Leukemic Cells ◽  
Phenotypic Change ◽  
Phenotypic Switch ◽  
Acute Leukemias ◽  
Prolymphocytic Leukemia ◽  
Cytogenetic Abnormalities

Abstract T-cell prolymphocytic leukemia (T-PLL) is a rare T-cell disorder characterized by an aggressive clinical course and short survival. Immunophenotypically, the leukemic cells show heterogeneous CD4 and CD8 expression; most cases are CD4+CD8− but, CD4+CD8+ and CD4−CD8+ cases are recognized. CD4+CD8+ phenotype is conventionally associated with cortical thymocytes, however, double positive T-prolymphocytes, are TdT −, CD1a − and CD3+ suggesting a post-thymic origin. Phenotypic changes in malignant cells while recognized in acute leukemias, are rare in mature T-cell disorders. In T-PLL, there is only 1 report describing 2 cases of phenotypic change (Tuset et al, Leukemia and Lymphoma 2001.42:1379-83). We retrospectively investigate the frequency of this phenomenon amongst 20 patients with T-PLL for whom sequential immunophenotyping of leukemic cells was available. This included the 2 cases previously reported. Diagnosis of T-PLL was based on clinical features, morphology, immunophenotype and karyotype. Membrane markers were sequentially evaluated by flow cytometry on peripheral blood and bone marrow. Sequential chromosome analysis of metaphases derived from T-mitogen stimulated metaphases cultures was available in a proportion of cases. Intra-lineage phenotypic switch occurred at relapse in 5 cases (25%) (table 1). No characteristic pattern of switch was seen. Serial karyoptying was available in 3 of these 5 patients (60%) and showed acquisition of additional cytogenetic abnormalities at the time of relapse. Change in morphology was documented in 1 case where cells displayed a more ‘blastic’ appearance with loss of CD52. Change in phenotype was mirrored in clinical features with increasingly progressive disease and refractoriness to treatment. In all cases, a mature TdT negative phenotype was maintained. Splenectomy was not performed in the interval between initial and documented change in phenotype and LGL’s were not apparent morphologically or by flow cytometry in these 5 patients Change in phenotype in T-PLL Subsequent phenotype Initial phenotype CD4+CD8− CD4+CD8+ CD4−CD8+ Total * change in phenotype CD4+CD8− 8 1* 0 9 (45%) CD4+CD8+ 0 2 2* 4 (20%) CD4−CD8+ 1* 1* 5 7 (35%) Total 9 (45%) 4 (20%) 7 (35%) 20 . All patients received alemtuzumab treatment and while it is possible that this agent selectively depleted sub-clones of the disease, the lack of a consistent emergent phenotype, together with acquisition of additional cytogenetic abnormalities as opposed to replacement by a different abnormal karyotype, favors clonal evolution as a likely cause. Such change in phenotype was not observed in the remaining 15 patients despite alemtuzumab treatment in the majority. Our findings indicate that intra-lineage phenotypic switch in T-PLL occurs in up to 25% of cases. This indicates the need to use comprehensive T-cell panels in monitoring for minimal residual disease and/or relapse. Sequential evaluation of phenotype and cytogenetics may provide valuable insights into mechanisms of relapse and resistance and may impact on treatment selection in T-PLL.

scholarly journals CD161 Is Expressed in a Subset of T-Cell Prolymphocytic Leukemia Cases and Is Useful for Disease Follow-up

2019 ◽  
Vol 152 (4) ◽  
pp. 471-478
Author(s):  
Scott R Gilles ◽  
Sophia L Yohe ◽  
Michael A Linden ◽  
Michelle Dolan ◽  
Betsy Hirsch ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Nk Cells ◽  
Retrospective Review ◽  
T Cell Subsets ◽  
Prolymphocytic Leukemia ◽  
Flow Cytometry Data ◽  
Flow Cytometric ◽  
Flow Cytometric Evaluation

AbstractObjectivesCD161 (NKRP1) is a lectin-like receptor present on NK cells and rare T-cell subsets. We have observed CD161 expression in some cases of T-cell prolymphocytic leukemia (T-PLL) and found it to be useful in follow-up and detection of disease after treatment.MethodsRetrospective review of T-PLL cases with complete flow cytometry data including CD161.ResultsWe identified 10 cases of T-PLL with flow cytometric evaluation of CD161 available. Six of these cases were positive for CD161 expression. All CD161-positive cases were positive for CD8 with variable CD4 expression, whereas all CD161-negative cases were negative for CD8. In a case with two neoplastic subsets positive and negative for CD8, only the former expressed CD161.ConclusionsThese novel results suggest that CD161 is often aberrantly expressed in a defined subset of T-PLL positive for CD8. We are showing the utility of this immunophenotype in diagnosis and follow-up.

scholarly journals Detection of minimal residual disease in T-cell acute lymphoblastic leukemia using polymerase chain reaction predicts impending relapse

Blood ◽  
1991 ◽  
Vol 78 (3) ◽  
pp. 739-747 ◽  
Author(s):  
GA Neale ◽  
J Menarguez ◽  
GR Kitchingman ◽  
TJ Fitzgerald ◽  
M Koehler ◽  
...  
Keyword(s):  
T Cell ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Pcr Amplification ◽  
Leukemic Cells ◽  
Chain Reaction ◽  
Leukemic Clone ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Polymerase Chain

Abstract After achieving remission, approximately one-third of patients with T- cell acute lymphoblastic leukemia (T-ALL) relapse due to the resurgence of residual leukemic cells that cannot be detected in remission by morphologic methods. Thus, the early detection of residual disease is highly desirable to monitor the efficacy of therapy, or to institute an alternative mode of therapy. Toward this aim, we have examined the applicability of polymerase chain reaction (PCR) amplification in the detection of minimal residual disease (MRD) in bone marrow samples from patients with T-ALL in morphologic remission. Two different approaches were taken to identify leukemic clone-specific sequences that could be used as targets for PCR amplification. The first technique used T-cell receptor-delta (TCR-delta) gene rearrangements that were sequenced directly after PCR amplification of leukemic DNA. This method was successful in generating clone-specific probes for 76% of T-ALL patients screened. An alternative method was used to clone and sequence a TCR-beta chain gene from leukemic cells to generate a specific probe. The PCR assays that we used were specific for each patient's leukemic clone, and were capable of routinely detecting one leukemic cell in 10(4) normal cells. Using these sensitive PCR-based assays, we found no evidence for persistence of the leukemic clone in any of the bone marrow samples from four T-ALL patients who are in long-term (3.9 + to 8.1 + years) remission. In contrast, we detected residual disease in clinical remission samples from two patients who subsequently relapsed. In one patient, where we had appropriate samples, we observed a dramatic expansion of the leukemic clone 3 months before clinical relapse. These results suggest that PCR-based assays for detection of MRD in T-ALL patients have great potential in predicting impending relapse, and in determining the efficacy of the anti-leukemic therapy. These methods may also allow the identification of long-term survivors.

scholarly journals Leukopenic chronic T cell leukemia mimicking hairy cell leukemia: association with human retroviruses

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 949-956 ◽  
Author(s):  
CC Sohn ◽  
DW Blayney ◽  
JL Misset ◽  
G Mathe ◽  
G Flandrin ◽  
...  
Keyword(s):  
T Cell ◽  
Hairy Cell Leukemia ◽  
Sheep Erythrocyte ◽  
The Other ◽  
Leukemic Cells ◽  
Type I ◽  
T Cell Leukemia ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
Prolymphocytic Leukemia

Abstract We report two cases of a T cell lymphoproliferative disease not previously described, with cytologic and clinical features similar to those associated with Galton's “prolymphocytic” leukemia (PL). Our patients, like those with Galton's PL, had massive splenomegaly and minimal or absent hepatomegaly and lymphadenopathy. In contrast, however, our patients had leukopenia, as well as low percentages of leukemic cells in the peripheral blood and in the bone marrow. In splenic imprints, the nuclear chromatin pattern of most of the leukemic cells was intermediate between those of mature lymphocytes and those of lymphoblasts, and the nuclei contained single, centrally located, conspicuous nucleoli. In sections of the spleen, the leukemic cells diffusely infiltrated the red pulp in a pattern strikingly similar to that of hairy cell leukemia; however, when the leukemic cells were studied cytochemically, the cytoplasmic acid phosphatase positivity was punctate and tartrate-sensitive. The leukemic cells were sheep erythrocyte rosette-positive and expressed T cell-associated antigens. Initially, both patients responded well to therapeutic splenectomy. One patient received combination chemotherapy after splenectomy and is alive and well 24 months after diagnosis. The other patient was in complete clinical remission for one year after splenectomy and received chemotherapy at relapse. He died, however, 23 months after splenectomy, with disseminated disease. IgG antibody titers against human T lymphotropic virus type I (HTLV-I) were detected in one patient and against HTLV-II in the other. The leukemia in these patients represents a distinct clinicopathologic entity within the spectrum of peripheral T cell lymphoproliferative diseases that includes Galton's PL of T cell derivation, T cell chronic lymphocytic leukemia, T cell hairy cell leukemia, and adult T cell leukemia/lymphoma.

Adoptive T-Cell Therapy for B-Cell Acute Lymphoblastic Leukemia: Preclinical Studies

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3531-3540 ◽  
Author(s):  
Angelo A. Cardoso ◽  
J. Pedro Veiga ◽  
Paolo Ghia ◽  
Hernani M. Afonso ◽  
W. Nicholas Haining ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Adoptive T Cell Therapy ◽  
Leukemic Cells ◽  
Acute Leukemias ◽  
T Cell Lines

We have previously shown that leukemia-specific cytotoxic T cells (CTL) can be generated from the bone marrow of most patients with B-cell precursor acute leukemias. If these antileukemia CTL are to be used for adoptive immunotherapy, they must have the capability to circulate, migrate through endothelium, home to the bone marrow, and, most importantly, lyse the leukemic cells in a leukemia-permissive bone marrow microenvironment. We demonstrate here that such antileukemia T-cell lines are overwhelmingly CD8+ and exhibit an activated phenotype. Using a transendothelial chemotaxis assay with human endothelial cells, we observed that these T cells can be recruited and transmigrate through vascular and bone marrow endothelium and that these transmigrated cells preserve their capacity to lyse leukemic cells. Additionally, these antileukemia T-cell lines are capable of adhering to autologous stromal cell layers. Finally, autologous antileukemia CTL specifically lyse leukemic cells even in the presence of autologous marrow stroma. Importantly, these antileukemia T-cell lines do not lyse autologous stromal cells. Thus, the capacity to generate anti–leukemia-specific T-cell lines coupled with the present findings that such cells can migrate, adhere, and function in the presence of the marrow microenvironment enable the development of clinical studies of adoptive transfer of antileukemia CTL for the treatment of ALL.

Complete Cytogenetic Response in a Case of T-PLL with a Complex Karyotype after Treatment with Alemtuzumab.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4535-4535
Author(s):  
André F. Marinato ◽  
Fábio Morato de Oliveira ◽  
Rafael H. Jácomo ◽  
Edgar G. Rizzatti ◽  
Roberto P. Falcão ◽  
...  
Keyword(s):  
T Cell ◽  
Lymph Nodes ◽  
Peripheral Blood ◽  
Leukemic Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Cell Phenotype ◽  
Complex Karyotype ◽  
Prolymphocytic Leukemia ◽  
Human T Cell ◽  
Cytogenetic Remission

Abstract T-prolymphocytic leukemia (T-PLL) is an aggressive T-cell leukemia characterized by the proliferation of prolymphocytes with a mature postthymic T-cell phenotype and commonly involves the blood, bone marrow, lymph nodes, liver, spleen, and skin. Leukemic cells appear as small to medium-sized prolymphocytes or small variant cells, occasionally with cerebriform nuclei. T prolymphocytes are CD2+, CD3+, and CD7+ and may express CD4+CD8−, CD4+CD8+, or CD4−CD8+ markers. The most frequent chromosomal abnormalities are t(14;14)(q11;q32), inv(14)(q11q32), t(X;14)(q28;q11), i(8)(q10), and t(8;8)(p12;q11). Although patients treated with conventional chemotherapy generally have a poor prognosis, alemtuzumab (Campath®) has been associated with good clinical responses in patients with T-PLL. Routine cytogenetic analysis to determine the response to alemtuzumab has not been performed in patients with T-PLL. This study reports the complete cytogenetic remission achieved following alemtuzumab treatment in a single patient with T-PLL. The 41-year old male patient presented with weight loss, night sweats, diffuse lymphadenopathy and hepatosplenomegaly, in the absence of any skin rash. Peripheral blood counts were: platelets 128 x 109/L and leukocytes 93.6 x 109/L, and hemoglobin values were 12.5 g/dL. The lymphocytes were mostly medium-sized with round to irregular nuclei; some cerebriform cells were also observed, with intermediate chromatin, single evident nucleoli, and a basophilic cytoplasm. Human T-cell lymphotrophic virus-1 serology was negative. Immunophenotypic studies showed CD2+, CD3+, CD5+, CD7+, CD4−, CD8+, CD1−, terminal deoxynucleotidyl transferase-negative (TdT-), and T-cell receptor a/b+. Classic cytogenetics and spectral karyotyping (SKY) were performed and an abnormal karyotype was observed in 18 metaphases analyzed: 46, t(X;14)(q28;q11), t(Y;14)(q12;q11), r[i(8)(q10)]. These abnormalities have not been previously reported in cases of T-PLL. The patient then underwent chemotherapy with CHOP, and subsequent therapy with fludarabine. The patient was then treated with alemtuzumab, 30 mg twice a week, for a total of 7 doses. The patient achieved complete clinical, hematological, and cytogenetic remission, characterized by lack of symptoms, reduction of lymph nodes, liver, and spleen size to normal volumes, and the disappearance of prolymphocytes from peripheral blood. Cytogenetic studies performed 7 months after treatment by classic cytogenetics and SKY revealed a karyotype of 46,XY[20]. This study is the first to demonstrate a complete cytogenetic remission following treatment with alemtuzumab in a patient with T-PLL that was refractory to standard chemotherapy. This is especially impressive in a patient with a complex karyotype and with different cytogenetic alterations not previously described.

Analysis of In Vitro Effects of Bortezomib (Velcade) on T Cell Prolymphocytic Leukemia (T-PLL).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4426-4426
Author(s):  
Fulya Ozpuyan ◽  
Paul N. Meyer ◽  
Hytham Al-Masri ◽  
Hongyu Ni ◽  
Serhan Alkan
Keyword(s):  
T Cell ◽  
Lymphoproliferative Disorder ◽  
Flow Cytometric Analysis ◽  
Leukemic Cells ◽  
Cell Phenotype ◽  
Base Line ◽  
Prolymphocytic Leukemia ◽  
Bortezomib Treatment ◽  
Flow Cytometric

Abstract T-cell prolymphocytic leukemia (T-PLL) is an aggressive lymphoproliferative disorder with postthymic T cell phenotype and prolymphocytic morphology. In the majority of patients, the leukemic process progresses rapidly and patients die shortly after diagnosis (median survival of 7 months). Bortezomib, the first proteasome inhibitor to be approved for use in haematological malignancies such as multiple myeloma, is beginning to be utilized as an effective anti-neoplastic agent in other hematopoietic and non-hematopoietic neoplastic disorders. We report here the in vitro apoptotic effects of bortezomib on leukemic cells isolated from three T-PLL patients. Interestingly, one of the patient’s leukemia developed in the setting of immunosupression due to transplant therapy (post-transplant lymphoproliferative disorder). Flow cytometric analysis of leukemic cells of the three patients showed CD8, double CD4+CD8+ and double CD4−CD8− immunophenotypic features. All cases showed monoclonal band pattern by T-cell receptor (TCR) gene rearrangement as analyzed by the PCR amplification of the TCR gamma heavy chain gene. Freshly isolated leukemic cells with the CD8 phenotype T-PLL analyzed for apoptosis after ficoll hypaque separation and cultured in the presence of various concentration of Bortezomib (0.001, 0.01, 0.1, 1, and 10 uM) for dose curve analysis. Apoptosis of the leukemic cells was determined by Annexin-V and 7-AAD staining and flow cytometric analysis after incubation at 24 and 72 hours, respectively. Samples treated for 72 hours showed higher rate of apoptosis compared to 24 hours: 10 uM (62% increase above the base line of control cells), 1 uM (58%), 0.1 uM (55%), 0.01 uM (40%) and 0.001 uM (0%) concentrations while samples treated for 24 hours with 10 uM showed (42% increase above the base line of control cells) and 1 uM (33% increase above the base line). Light microscopic analysis of leukemic cells treated with Bortezomib confirmed that the majority of cells undergo apoptosis with Bortezomib treatment as it revealed nuclear fragmentation and apoptotic bodies. Leukemic cells recovered from cryopreservation from the second and third T-PLL patient samples analyzed also showed significant increase in early and late apoptosis at 24 hours with Bortezomib treatment (10nm). These results suggest that Bortezomib may provide an alternate therapy in the treatment of T-PLL. Future collaborative efforts investigating efficacy with Bortezomib as a single agent or in combination with other therapeutic agents will be crucial to improving survival for patients with this disease.

Sequencing-Based Minimal Residual Disease Assessment In T-Cell Malignancies: T-Cell Prolymphocytic Leukemia Case Study

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1392-1392
Author(s):  
Katherine Sutherland ◽  
Katherine Kong ◽  
Aaron C. Logan ◽  
Malek Faham ◽  
David B. Miklos
Keyword(s):  
T Cell ◽  
Peripheral Blood ◽  
Residual Disease ◽  
Cell Receptor ◽  
Employment Equity ◽  
Prolymphocytic Leukemia ◽  
Blood Samples ◽  
Leukemia Cutis ◽  
Equity Ownership

Abstract Background The prognostic significance of minimal residual disease (MRD) quantification in the post-transplant setting has been demonstrated in multiple lymphoid malignancies, including acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). Previous studies support the clinical utility of molecular MRD quantification of tumor burden after allogeneic hematopoietic cell transplantation (allo-HCT) (Logan et al, Leukemia 2013). We have developed the ClonoSIGHT™ test, which is based on the deep sequencing of immunoglobulin and T-cell receptor rearrangements and has a detection limit of one cancer cell per million leukocytes in peripheral blood or bone marrow (Faham et al, Blood 2012; Armand et al, Brit J Haematol 2013). In this report, we will discuss the technical performance of the ClonoSIGHT test for routine MRD quantification after allo-HCT and present a case study on a patient with T-cell prolymphocytic leukemia (T-PLL). Methods A 55 year old female presented with T-PLL including symptomatic CNS disease, received 12 weeks of Alemtuzumab therapy and then 12 weeks following her last Alemtuzumab treatment received an unrelated donor myeloablative allo-HCT using Fludarabine, BCNU and Melphalan conditioning with antithymoglobulin, Mycophenolate mofetil and cyclosporine primary immune prophylaxis. Peripheral blood samples were collected for MRD assessment before and serially after allo-HCT. Using universal primer sets, we amplified T-cell receptor beta (TRB), delta (TRD) and gamma (TRG) variable, diversity, and joining gene segments from genomic DNA isolated from peripheral blood mononuclear cells (PBMC). Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination, and leukemia-specific clonotypes were identified based on their frequency within the T-cell repertoire (>5%). The leukemia-specific clonotype was then quantified in serial peripheral blood samples and reported as the absolute number of leukemic-specific clones among total leukocytes. Results A single clonal TRG gene rearrangement accounting for 26.1% WBC in the pre-transplant sample was identified and quantified in serial peripheral blood samples. A 4-log decline in MRD levels occurred post allo-HCT (Figure 1) thru 56 days following graft infusion; however, serial MRD monitoring demonstrated increasing levels of leukemia-specific clonotypes in the peripheral blood over time (Figure 1). Immunosuppression tapering strategies were employed in response to clinical events and MRD levels. Specifically, the patient developed an EBV+ post-transplant lymphoproliferative disease (PTLD) 60 days post allo-HCT, and cyclosporine was tapered in addition to instituting anti-CD20 rituximab treatment. As per institutional practice, a bone marrow biopsy 84 days post-HCT showed full donor engraftment with normal cellularity and no evidence of PLL was detected by flow cytometry when ClonoSIGHT detected 0.013% PLL in the patient's blood. Unfortunately, in the setting of immune suppression taper at 100 days post allo-HCT, the patient developed Grade II skin GVHD and was treated with 0.5mg/kg prednisone daily and tapered as indicated. At 160 days post allo-HCT, the patient presented with new skin papules suspected to be leukemia cutis. The PLL clonotype was detected in the skin biopsy; however, it was present at lower frequency in the TRG repertoire than in the blood, thus not supporting a diagnosis of leukemia cutis. In agreement, skin pathology revealed Verruca Vulgaris (warts). However, the patient's MRD continued to increase in the blood while immunosuppression was tapered and stopped completely 6 months post-HCT. Conclusions MRD assessment can be used to monitor a patient's disease progression after immune cellular therapy and aids immune suppression management following allo-HCT. Further, as presented in this case study, ClonoSIGHT detection of the leukemia clone in the blood compared with other tissues can sensitively and specifically assess extramedullary relapse. Disclosures: Kong: Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

N-Myc Is Overexpressed In Both Murine and Human Early T-Cell Precursor Leukemia and Is Sufficient To Initiate this Leukemia In Multipotent Primitive Arf-/- thymocytes

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 348-348
Author(s):  
Louise M. Treanor ◽  
Sheng Zhou ◽  
Yu Fukuda ◽  
Nandakumar Satish ◽  
David Finkelstein ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Murine Model ◽  
Leukemic Cells ◽  
Control Element ◽  
Mrna Levels ◽  
Cell Precursor ◽  
Double Positive ◽  
Fluorescent Intensity ◽  
Cell Counts

Abstract Early T-cell precursor–ALL (ETP-ALL) is a subtype of T-cell acute lymphoblastic leukemia that displays both myeloid and lymphoid phenotypic features and has a relatively poor prognosis. Our previous work has shown that activating mutations in the Il7 receptor (Il7r) and Lmo2 overexpression in CD4-CD8- thymocytes cause ETP-ALL in mice via DN2 thymocyte differentiation block. To further understand the molecular mechanisms responsible for initiation and maintenance of ETP-ALL, we compared expression profiles of seven murine ETP leukemias induced by Lmo2 and Il7r mutant vectors and four preleukemic blocked DN2 Arf-/- thymocyte populations to normal murine DN2 Arf-/- thymocytes. This analysis and qRT-PCR identified N-Myc as one of the top upregulated genes in either Lmo2 or Il7r mutant samples. Importantly in pediatric ETP-ALL N-Myc RNA is expressed at high levels in 11/13 human cases compared to 26/72 T-ALL cases corroborating our murine model with the human disease. To investigate if N-Myc itself was sufficient to induce ETP leukemia, immature murine Arf-/- thymocytes were transduced with a retroviral vector expressing the N-Myc cDNA and YFP. After 20 days in culture a block at the DN2 stage of thymocyte development was observed that recapitulated the Lmo2 and the Il7r receptor mutant phenotype. When sublethally irradiated Rag2-/-, γc-/- recipient mice were transplanted with N-Myc transduced Arf-/- thymocytes 5/5 of the primary recipients developed ETP leukemia originating from the thymocyte graft with a median latency of 60 days (Figure 1). These mice had elevated white blood cell counts between 17-85x103/µl and enlarged spleens ranging in weight from 0.42g-0.67g. Flow cytometry analysis showed that their spleen, thymus and peripheral blood contained greater than 70% YFP+ Gr1+ cells. These N-Myc induced leukemias expressed both T-cell (cytoplasmic CD3) and myeloid (Gr1 and Mac1) markers and displayed other features typical of ETP disease. Having established that N-Myc is sufficient to initiate ETP leukemia in the murine model, we next tested if N-Myc is critical in the maintenance of ETP leukemia induced by Il7r mutants. ETP leukemic cells arising from the Il7r 241-242TC mutant were transduced with a mir30 lentiviral vector that contains a short hairpin N-Myc inhibiting sequence and mCherry as a reporter gene (shN-Myc). This vector has been shown to reduce N-Myc mRNA levels by five fold in N-Myc overexpressing cells. The transduced cells were analyzed and sorted for leukemic+ (GFP) scrambled+ (mCherry) or leukemic+(GFP) shN-Myc+(mCherry) double positive cells. Analysis of the cells showed the mCherry mean fluorescence intensity (MFI) of the leukemic cells transduced with shN-Myc was approximately double that of the scrambled transduced cells, demonstrating at least equal transduction efficiency of the two vectors. Sorted double positive leukemic cells were transplanted into sublethally irradiated WT recipients. All animals succumbed to leukemia and were subsequently analyzed by flow cytometry for expression of scrambled and shN-Myc vector expression. Analysis of tumor cells showed a significant decrease in the mCherry expression of the cells containing the shN-Myc compared to the scrambled control indicating that sustained N-Myc expression is selected for during the maintenance of ETP leukemia and in the future a tetracycline control element will be incorporated into the shN-Myc to confirm this (Figure 2). Furthermore we plan to test therapeutics against N-Myc such as bromodomain and Aurora kinase A inhibitors in vitro and plan to expand this in vivo. This demonstrates the utility of this novel mouse model combined with gene expression profiling to elucidate the signaling networks in ETP leukemia to develop targeted therapy for this aggressive disease.Figure 1Survival curve of recipient mice transplanted with CD4-CD8- thymocytes transduced with N-Myc and Il7r mutants.Figure 1. Survival curve of recipient mice transplanted with CD4-CD8- thymocytes transduced with N-Myc and Il7r mutants.Figure 2Mean fluorescent intensity (MFI) of mCherry in mice transplanted with leukemic+ (GFP) Scrambled+(mCherry) or leukemic+(GFP) shN-Myc+(mCherry) double positive cells.Figure 2. Mean fluorescent intensity (MFI) of mCherry in mice transplanted with leukemic+ (GFP) Scrambled+(mCherry) or leukemic+(GFP) shN-Myc+(mCherry) double positive cells. Disclosures: No relevant conflicts of interest to declare.

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