T-cell Prolymphocytic Leukemia Cells Research Articles

Preclinical Evaluation of T-Cell Prolymphocytic Leukemia Demonstrates Heterogeneous BCL2-Family Dependency That May be Effectively Targeted with Small Molecule Inhibitors

T-cell prolymphocytic leukemia (T-PLL) is a rare, aggressive, mature T-cell leukemia with very few treatment options and adverse prognosis. There are currently no approved therapies for patients with relapsed T-PLL, where the median OS is less than 6 months. There are limited reports suggesting clinical activity of the BCL2 inhibitor venetoclax in patients with T-PLL. We aimed to characterize the dependencies of T-PLL on members of the BCL2-family of antiapoptotic proteins using BH3 profiling, a functional assay to assess the propensity of a cell to undergo apoptosis (priming) and the relative dependence of a cell on different antiapoptotic proteins. We then sought to experimentally exploit these dependencies with targeted therapies alone, and in combination, designed to inhibit the antiapoptotic proteins towards a therapeutic goal. Cell lines of mature T cell lymphomas/leukemias (TCL/L) including SUPT11 (T-cell leukemia with rearranged TCL1) (n=10) and bone marrow and peripheral blood samples from untreated patients with T-PLL (n=7) were analyzed by BH3 profiling. We also used a relapsed/refractory T-PLL patient sample that were able to engraft and expand in a PDX model. Finally, scRNA/ATAC seq has also been performed in a set of naïve-treated T-PLL patients. In contrast to TCL/L that demonstrated heterogeneity on BCL2 family member dependency, T-PLL samples were primed and consistently exhibited dependency on BCL2/BCL-xL and MCL1. Integration of scRNA/ATAC seq confirmed high expression of BCL2 and MCL1 in T-PLL cells from four patients supporting the premise that BCL2 and MCL1 could potentially be targeted for therapeutic benefit. Therefore, SUPT11 cells (used as a model of T-PLL) were exposed to venetoclax (25 and 50nM; BCL-2inh), fadraciclib (25 and 50nM; CDK2/9inh - indirect inhibitor of MCL-1 as it decreases mRNAs with high decay rates including MCL1 and MYC) or a combination of venetoclax and fadraciclib. We demonstrate that exposure to fadraciclib caused a rapid decline in the levels of phosphorylation on Serine 2 of RNA polymerase II C-terminal domain, as well as decline in the levels of MCL1 within 4 hours and completely depleted these proteins by 8-12 h of exposure. We also show that there was a synergistic increase both in mitochondrial dysfunction as measured by cytochrome C release by 18h and in cell death in cells exposed to the combination at 24h. These results suggest that combining venetoclax and fadraciclib might represent a potential therapeutic approach for T-PLL. In conclusion, preclinical evaluation of a T-PLL cell line and primary patient samples demonstrate BCL2-family dependency that can be effectively targeted with small molecule inhibitors of BCL2 and CDK2/9. In vivo studies in PDX models of T-PLL are underway to form the basis for clinical trials to study these combinations.

Advanced Pathogenetic Concepts in T-Cell Prolymphocytic Leukemia and Their Translational Impact

T-cell prolymphocytic leukemia (T-PLL) is the most common mature T-cell leukemia. It is a typically aggressively growing and chemotherapy-resistant malignancy with a poor prognosis. T-PLL cells resemble activated, post-thymic T-lymphocytes with memory-type effector functions. Constitutive transcriptional activation of genes of the T-cell leukemia 1 (TCL1) family based on genomic inversions/translocations is recognized as a key event in T-PLL’s pathogenesis. TCL1’s multiple effector pathways include the enhancement of T-cell receptor (TCR) signals. New molecular dependencies around responses to DNA damage, including repair and apoptosis regulation, as well as alterations of cytokine and non-TCR activation signaling were identified as perturbed hallmark pathways within the past years. We currently witness these vulnerabilities to be interrogated in first pre-clinical concepts and initial clinical testing in relapsed/refractory T-PLL patients. We summarize here the current knowledge on the molecular understanding of T-PLL’s pathobiology and critically assess the true translational progress around this to help appraisal by caregivers and patients. Overall, the contemporary concepts on T-PLL’s pathobiology are condensed in a comprehensive mechanistic disease model and promising interventional strategies derived from it are highlighted.

Preliminary Study of Ruxolitinib and Venetoclax for Treatment of Patients with T-Cell Prolymphocytic Leukemia Refractory to, or Ineligible for Alemtuzumab

Preliminary Study of Ruxolitinib and Venetoclax for Treatment of Patients with T-Cell Prolymphocytic Leukemia Refractory to, or Ineligible for Alemtuzumab

CDK9 Inhibition with LDC526 Is an Effective Treatment for T-Cell Prolymphocytic Leukemia (T-PLL) in Vitro

▪IntroductionT-cell prolymphocytic leukemia (T-PLL) is a rare leukemia with an aggressive disease course, characterized by chemotherapy resistance and a median overall survival of less than two years. Apart from defects in DNA damage response genes like ATM and genes of JAK/STAT signaling, overexpression of MYC, often caused by gain of chromosomal material, is frequently observed in T-PLL. Intravenous application of the anti-CD52 antibody Alemtuzumab has been demonstrated to improve progression-free survival and is currently considered the standard of care. In younger patients remissions can be consolidated by allogeneic stem cell transplantation, which results in long-term remission in a small subgroup of patients. The prognosis in the majority of patients relapsing from their disease is dismal and thus novel treatment options are urgently needed. Cyclin-dependent kinase 9 (CDK9), a serine/ threonine kinase which is ubiquitously expressed, is the catalytic unit of the positive transcription elongation factor (P-TEFb). In the presence of cyclin T, p-TEFb stimulates transcription elongation by phosphorylation of the carboxy-terminal domain (CTD) of RNA polymerase II (RNA Pol II). CDK9 inhibition by LDC526, a specific CDK9 inhibitor, leads to reduced phosphorylation of CTD-Ser2, Ser5 and Ser7 of RNA Pol II and subsequently to the downregulation of MYC and MCL-1.Methods</spanCD3+CD4+ tumor cells from cryopreserved peripheral blood samples of 11 T-PLL patients were enriched to a purity of >95% by fluorescence-activated cell sorting (FACS) or magnetic activated cell separation (MACS). RNA and DNA were isolated using the RNeasy Mini Kit® or the QIAamp DNA blood midi kit® (Qiagen, Hilden, Germany), respectively. To determine IC50 values, we performed cell viability assays for T-PLL cells of 11 patients treated with LDC526 and a panel of reference compounds compared to DMSO controls (CellTiter-Glo Luminescent Cell Viability Assay; Promega, Fitchburg, WI, USA). Gene expression profiling was performed in primary T-PLL cells treated with the CDK9 inhibitor LDC526 in a concentration of 10µM compared to DMSO (n=3) on Clariom S Pico Assays (Affymetrix, Santa Clara, CA, USA) and analyzed by gene set enrichment analysis (GSEA) and GeneTrail analysis. Gene expression measurement was performed with quantitative real-time PCR (qPCR) for MYC (Taqman, Thermo Fisher Scientific, Waltham, MA, USA). Protein expression was analyzed for pCTD Ser2, Ser5 and Ser7, MYC, p-STAT3 and MCL-1 by Western Blot (n=4) after LDC526-treatment in three different concentrations (10, 30 and 100 µM).ResultsLDC526 inhibited T-PLL cell survival in vitro in a dose-dependent manner. The median IC50 was 1.7 µM (range: 0.9 -3.5) which compared favourably to a panel of reference compounds including fludarabine, clofarabine, methotrexate, cytarabine and ruxolitinib (median IC50 values 6.4, 6.2, 29.9, 8.3, and 29.2, respectively). Similar experiments with BAY1143572, a structurally closely related CDK9 inhibitor, which is currently investigated in phase I studies of acute myeloid leukemia, revealed an even lower median IC50 of 0.9 µM (range: 0.5 - 1.7). Of note, the purine analogues fludarabine and clofarabine were only active in some of the patients tested, whereas both CDK9 inhibitors showed activity in all samples analyzed.Gene expression profiles of LDC526 treated T-PLL cells compared to DMSO controls revealed an enrichment of genes in gene sets associated with interferon and TNFA signatures, MYC targets as well as JAK/STAT genes, which were subsequently confirmed by qPCR experiments. At the protein level LDC526 dose-dependently reduced phosphorylation of CTD-Ser2, Ser7 and to a lesser extent of Ser5 compared to DMSO controls resulting in efficient down-regulation of MYC, MCL-1 and p-STAT3.ConclusionsOur experiments identify CDK9 as a novel therapeutic target in T-PLL which can be efficiently inhibited by LDC526 and BAY1143572 at clinically meaningful concentrations. CDK9 inhibition resulted in both reduced T-PLL cell survival and down-regulation of MYC and JAK/STAT signaling, which have a key role in the molecular pathogenesis of T-PLL. DisclosuresDürig:Lead Discovery Center: Research Funding.

BH3 profiling identifies ruxolitinib as a promising partner for venetoclax to treat T-cell prolymphocytic leukemia

Conventional therapies for patients with T-cell prolymphocytic leukemia (T-PLL), such as cytotoxic chemotherapy and alemtuzumab, have limited efficacy and considerable toxicity. Several novel agent classes have demonstrated preclinical activity in T-PLL, including inhibitors of the JAK/STAT and T-cell receptor pathways, as well as histone deacetylase (HDAC) inhibitors. Recently, the BCL-2 inhibitor venetoclax also showed some clinical activity in T-PLL. We sought to characterize functional apoptotic dependencies in T-PLL to identify a novel combination therapy in this disease. Twenty-four samples from patients with primary T-PLL were studied by using BH3 profiling, a functional assay to assess the propensity of a cell to undergo apoptosis (priming) and the relative dependence of a cell on different antiapoptotic proteins. Primary T-PLL cells had a relatively low level of priming for apoptosis and predominantly depended on BCL-2 and MCL-1 proteins for survival. Selective pharmacologic inhibition of BCL-2 or MCL-1 induced cell death in primary T-PLL cells. Targeting the JAK/STAT pathway with the JAK1/2 inhibitor ruxolitinib or HDAC with belinostat both independently increased dependence on BCL-2 but not MCL-1, thereby sensitizing T-PLL cells to venetoclax. Based on these results, we treated 2 patients with refractory T-PLL with a combination of venetoclax and ruxolitinib. We observed a deep response in JAK3-mutated T-PLL and a stabilization of the nonmutated disease. Our functional, precision-medicine–based approach identified inhibitors of HDAC and the JAK/STAT pathway as promising combination partners for venetoclax, warranting a clinical exploration of such combinations in T-PLL.

Mutations in PIGA cause a CD52-/GPI-anchor-deficient phenotype complicating alemtuzumab treatment in T-cell prolymphocytic leukemia.

Infusional alemtuzumab followed by consolidating allogeneic hematopoietic stem cell transplantation in eligible patients is considered a standard of care in T-cell prolymphocytic leukemia (T-PLL). Antibody selection against CD52 has been associated with the development of CD52-negative leukemic T cells at time of relapse. Clinical implications and molecular mechanisms underlying this phenotypic switch are unknown. We performed flow cytometry and real-time-PCR for CD52-expression and next generation sequencing for PIGA mutational analyses. We identified loss of CD52 expression after alemtuzumab treatment in two of 21 T-PLL patients resulting from loss of GPI-anchor expression caused by inactivating mutations of the PIGA gene. One patient with relapsed T-PLL exhibited a single PIGA mutation, causing a CD52-negative escape variant of the initial leukemic cell clone, preventing alemtuzumab-retreatment. The second patient with continued complete remission after alemtuzumab treatment harbored three different PIGA mutations that affected either the non-neoplastic T cell or the mononuclear cell compartment and resulted in symptomatic paroxysmal nocturnal hemoglobinuria. Next generation sequencing of T-PLL cells collected before the initiation of treatment revealed PIGA wild-type sequence reads in all 16 patients with samples available for testing. These data indicate that PIGA mutations were acquired during or after completion of alemtuzumab treatment.

Anti-leukemic effect of CDK9 inhibition in T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is an aggressive malignancy characterized by chemotherapy resistance and a median survival of less than 2 years. Here, we investigated the pharmacological effects of the novel highly specific cyclin-dependent kinase 9 (CDK9) inhibitor LDC526 and its clinically used derivate atuveciclib employing primary T-PLL cells in an ex vivo drug sensitivity testing platform. Importantly, all T-PLL samples were sensitive to CDK9 inhibition at submicromolar concentrations, while conventional cytotoxic drugs were found to be largely ineffective. At the cellular level LDC526 inhibited the phosphorylation at serine 2 of the RNA polymerase II C-terminal domain resulting in decreased de novo RNA transcription. LDC526 induced apoptotic leukemic cell death through down-regulating MYC and MCL1 both at the mRNA and protein level. Microarray-based transcriptomic profiling revealed that genes down-modulated in response to CDK9 inhibition were enriched for MYC and JAK-STAT targets. By contrast, CDK9 inhibition increased the expression of the tumor suppressor FBXW7, which may contribute to decreased MYC and MCL1 protein levels. Finally, the combination of atuvecliclib and the BCL2 inhibitor venetoclax exhibited synergistic anti-leukemic activity, providing the rationale for a novel targeted-agent-based treatment of T-PLL.

Characterizing the Anti-Apoptotic Dependencies of T-Cell Prolymphocytic Leukemia Identifies HDAC and JAK/STAT Pathway Inhibitors As Promising Combination Partners to Augment Bcl-2 Targeted Killing By Venetoclax

Characterizing the Anti-Apoptotic Dependencies of T-Cell Prolymphocytic Leukemia Identifies HDAC and JAK/STAT Pathway Inhibitors As Promising Combination Partners to Augment Bcl-2 Targeted Killing By Venetoclax

Genomic Profiling of T-Cell Neoplasms Reveals Frequent JAK1 and JAK3 Mutations With Clonal Evasion From Targeted Therapies.

PurposeThe promise of precision oncology is that identification of genomic alterations will direct the rational use of molecularly targeted therapy. This approach is particularly applicable to neoplasms that are resistant to standard cytotoxic chemotherapy, like T-cell leukemias and lymphomas. In this study, we tested the feasibility of targeted next-generation sequencing in profiles of diverse T-cell neoplasms and focused on the therapeutic utility of targeting activated JAK1 and JAK3 in an index case.Patients and MethodsUsing Foundation One and Foundation One Heme assays, we performed genomic profiling on 91 consecutive T-cell neoplasms for alterations in 405 genes. The samples were sequenced to high uniform coverage with an Illumina HiSeq and averaged a coverage depth of greater than 500× for DNA and more than 8M total pairs for RNA. An index case of T-cell prolymphocytic leukemia (T-PLL), which was analyzed by targeted next-generation sequencing, is presented. T-PLL cells were analyzed by RNA-seq, in vitro drug testing, mass cytometry, and phospho-flow.ResultsOne third of the samples had genomic aberrations in the JAK-STAT pathway, most often composed of JAK1 and JAK3 gain-of-function mutations. We present an index case of a patient with T-PLL with a clonal JAK1 V658F mutation that responded to ruxolitinib therapy. After relapse developed, an expanded clone that harbored mutant JAK3 M511I and downregulation of the phosphatase, CD45, was identified. We demonstrate that the JAK missense mutations were activating, caused pathway hyperactivation, and conferred cytokine hypersensitivity.ConclusionThese results underscore the utility of profiling occurrences of resistance to standard regimens and support JAK enzymes as rational therapeutic targets for T-cell leukemias and lymphomas.

Abstract 370: T-PLL cells resemble memory-type T-cells with aberrant effector functions implicating a leukemogenic cooperation of TCL1A and TCR signaling

Abstract The pathogenesis of the rare and aggressive T-cell prolymphocytic leukemia (T-PLL) is poorly understood, which particularly applies to a mechanistic concept around its hallmark oncogene TCL1A. Existing data implicate TCL1A as a catalytic enhancer of the oncogenic kinase AKT, a central node in a T-cell’s antigen receptor (TCR) signaling cascade, which mediates proliferation and differentiation. The levels and role of TCR activation in T-PLL’s pathogenesis are not known. To first clarify which physiological T-cell subset T-PLL cells most resemble, we performed comprehensive global gene expression profiling and immunophenotyping of primary T-PLL (n=79) in comparison to healthy-donor derived T-cell populations. Principle component analyses and gene signature alignments revealed a high similarity of T-PLL cells to (central) memory T-lymphocytes over naïve T-cells. Surface markers revealed a spectrum of memory-type differentiation (n=69/79; 87%) with predominant central-memory stages (n=35/79; 44%). The usually TCR and/or CD28-coreceptor positive T-PLL cells revealed no restrictions to genetic or surface TCR-clonotypes. The abnormally high basal activation levels (surface CD25, CD38, CD69) correlated in their degree with inferior clinical outcomes (med. survival 20.8 vs 58.3 mo.; p=0.0012). In parallel, T-PLL cells lost expression of negative-regulatory TCR-co-receptors (e.g. CTLA-4, LAG3). Fittingly, TCR engagement of primary T-PLL cells revealed a trend to hyperactive intracellular responses and interleukin(IL)-2 release alongside a prominent Th1-cytokine program. T-PLL cells also showed a robust resistance to stimulation-induced cell death and agonistic CD95 ligation. TCR-derived signals (phospho-kinase induction, IL-2 release) were enhanced in vitro by the modulated presence of TCL1A with kinetics indicative of a sensitizer relationship, mainly in the CD3 axis as opposed to the CD28 branch. A mouse model with TCL1A-initiated protracted development of T-PLL (Lckpr-TCL1Atg) revealed congruent findings with the aberrant T-cell phenotype of human T-PLL. TCL1A expressing T-cells of this model, that were further equipped with monoclonal epitope-defined TCRs against ovalbumine or a chimeric-antigen-receptor (CAR) against carcinoembryonic antigen, gained a pre-leukemic growth advantage in scenarios of pulsed or continuous low-level receptor stimulation. Overall, we establish that T-PLL cells resemble antigen-experienced memory T-cells. Retention of functional effector responses to TCR stimulation and loss of restricting activation regulators underlie a highly activated phenotype and a marked resistance to death-inducing signals. TCL1A proactively enhances TCR responses and we postulate that this leukemogenic cooperation drives accumulation of memory-type cells that utilize amplified, hence permissive, low-level cognate antigen input. Note: This abstract was not presented at the meeting. Citation Format: Alexandra Schrader, Kathrin Warner, Sebastian Oberbeck, Giuliano Crispatzu, Petra Mayer, Sabine Pützer, Hans Diebner, Stephan Stilgenbauer, Georg Hopfinger, Jan Dürig, Torsten Haferlach, Mark Lanasa, Ingo Roeder, Michael Hallek, Dimitrios Mougiakakos, Michael von Bergwelt-Baildon, Monika Brüggemann, Sebastian Newrzela, Hinrich Abken, Marco Herling. T-PLL cells resemble memory-type T-cells with aberrant effector functions implicating a leukemogenic cooperation of TCL1A and TCR signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 370. doi:10.1158/1538-7445.AM2017-370

Comparison of clinicopathological characteristics between T-cell prolymphocytic leukemia and peripheral T-cell lymphoma, not otherwise specified.

T-cell prolymphocytic leukemia (T-PLL) is a very rare, aggressive T-cell neoplasm. Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is also a highly aggressive lymphoma. These two diseases can often be confused with each other; therefore, we aimed to determine the clinical and pathological differences between T-PLL and PTCL-NOS. We analyzed 15 T-PLL and 91 PTCL-NOS patients and also compared clinical features between T-PLL and PTCL-NOS with leukemic presentation. Peripheral blood images and biopsy specimens were analyzed, and treatment responses were determined via imaging modalities. The clinicopathological characteristics were statistically compared. T-PLL cells were smaller in size than those of PTCL-NOS with leukemic presentation (P=.0068); moreover, PTCL-NOS cells with leukemic presentation were smaller than those of PTCL-NOS without leukemic presentation (P=.0017). Immunophenotypic patterns in T-PLL and PTCL-NOS were similar. Five-year overall survival rates of T-PLL and all PTCL-NOS patients were 57.5% and 36.8%, respectively. No significant differences were found in clinical manifestations or prognoses; T-PLL and PTCL-NOS with leukemic presentation had essentially equivalent characteristics. T-PLL and PTCL-NOS may share common biological and clinical characteristics in Japanese patients.

Targeting Interleukin-2-inducible T-cell Kinase (ITK) and Resting Lymphocyte Kinase (RLK) Using a Novel Covalent Inhibitor PRN694

Interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK or TXK) are essential mediators of intracellular signaling in both normal and neoplastic T-cells and natural killer (NK) cells. Thus, ITK and RLK inhibitors have therapeutic potential in a number of human autoimmune, inflammatory, and malignant diseases. Here we describe a novel ITK/RLK inhibitor, PRN694, which covalently binds to cysteine residues 442 of ITK and 350 of RLK and blocks kinase activity. Molecular modeling was utilized to design molecules that interact with cysteine while binding to the ATP binding site in the kinase domain. PRN694 exhibits extended target residence time on ITK and RLK and is highly selective for a subset of the TEC kinase family. In vitro cellular assays confirm that PRN694 prevents T-cell receptor- and Fc receptor-induced cellular and molecular activation, inhibits T-cell receptor-induced T-cell proliferation, and blocks proinflammatory cytokine release as well as activation of Th17 cells. Ex vivo assays demonstrate inhibitory activity against T-cell prolymphocytic leukemia cells, and in vivo assays demonstrate durable pharmacodynamic effects on ITK, which reduces an oxazolone-induced delayed type hypersensitivity reaction. These data indicate that PRN694 is a highly selective and potent covalent inhibitor of ITK and RLK, and its extended target residence time enables durable attenuation of effector cells in vitro and in vivo. The results from this study highlight potential applications of this dual inhibitor for the treatment of T-cell- or NK cell-mediated inflammatory, autoimmune, and malignant diseases.

Integrated genomic sequencing reveals mutational landscape of T-cell prolymphocytic leukemia

AbstractThe comprehensive genetic alterations underlying the pathogenesis of T-cell prolymphocytic leukemia (T-PLL) are unknown. To address this, we performed whole-genome sequencing (WGS), whole-exome sequencing (WES), high-resolution copy-number analysis, and Sanger resequencing of a large cohort of T-PLL. WGS and WES identified novel mutations in recurrently altered genes not previously implicated in T-PLL including EZH2, FBXW10, and CHEK2. Strikingly, WGS and/or WES showed largely mutually exclusive mutations affecting IL2RG, JAK1, JAK3, or STAT5B in 38 of 50 T-PLL genomes (76.0%). Notably, gain-of-function IL2RG mutations are novel and have not been reported in any form of cancer. Further, high-frequency mutations in STAT5B have not been previously reported in T-PLL. Functionally, IL2RG-JAK1-JAK3-STAT5B mutations led to signal transducer and activator of transcription 5 (STAT5) hyperactivation, transformed Ba/F3 cells resulting in cytokine-independent growth, and/or enhanced colony formation in Jurkat T cells. Importantly, primary T-PLL cells exhibited constitutive activation of STAT5, and targeted pharmacologic inhibition of STAT5 with pimozide induced apoptosis in primary T-PLL cells. These results for the first time provide a portrait of the mutational landscape of T-PLL and implicate deregulation of DNA repair and epigenetic modulators as well as high-frequency mutational activation of the IL2RG-JAK1-JAK3-STAT5B axis in the pathogenesis of T-PLL. These findings offer opportunities for novel targeted therapies in this aggressive leukemia.

C-Terminal Defect of CD20 on T-Cell Leukemia/Lymphoma Cells: Implications for Resistance to Rituximab

Rituximab is commonly used for treatment of B-cell leukemia/lymphoma and its efficiency on CD20+ B-cell malignancies has been established. On the other hand, the effect of rituximab on CD20+ T cell leukemia/lymphoma is unclear. Two patients CD20+ T-cell malignancy (1 with T-cell prolymphocytic leukemia (T-PLL) and 1 with peripheral T-cell lymphoma, unspecified (PTCL-u)) were recently treated with rituximab without appreciable effects. The treatment failure prompted a study of the mechanisms underlying the resistance to rituximab therapy using leukemia/lymphoma cells of these patients and another CD20+ T-PLL patient. Patient 1: A 68-year old Japanese male was diagnosed as having T-PLL (small cell variant) characterized by CD2+CD3+CD4− CD5+CD8+CD20+TCR-Vβ8+. Southern blotting of these T-PLL cells showed a TCR-β gene rearrangement. The systemic lymphadenopathy and high-fever of the patient were ameliorated by the administration of prednisolone (20 mg/d). Rituximab was administered weekly, but no clinical improvement was observed. In vitro treatment of the T-PLL cells with rituximab did not show any complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Patient 2: A 75-year old Japanese male was diagnosed as having T-PLL (small cell variant) with a phenotype of CD2+CD3+CD4+CD5+CD8+CD20+. Southern blotting of these T-PLL cells showed a TCR-β gene rearrangement. The patient is being followed without treatment because his pancytopenia is non-progressive. Patient 3: A 67-year old Japanese male developed a left thigh tumor which proved to be PTCL-u. The phenotype of lymphoma cells was CD2+CD3−CD4−CD5−CD8+CD20+ and Southern blotting of these cells showed a TCR-β gene rearrangement. The patient's PTCL-u was refractory to CHOP and rituximab plus EPOCH therapy. The reactivity of various monoclonal Abs specific to CD20 molecules against these patients' leukemia/lymphoma cells is summarized in Table 1. CD20 molecules of these 3 patients' leukemia/lymphoma cells were detectable by flow cytometry using anti-CD20 mAbs (L27 and rituximab) and also by Western blotting using anti-CD20 mAbs which recognize N-terminal region of CD20. However, the CD20 molecules could not be revealed either by immunohistochemistry using anti-CD20 mAbs (L26) or by Western blotting using anti-CD20 mAbs which recognize C-terminal region of CD20. The sequence of the full-length CD20 cDNA derived from Patient 1's T-PLL cells proved to be intact. These results indicate that the CD20 molecules of the 3 patients' leukemia/lymphoma cells have defects in the cytoplasmic C-terminal region which may impair the cytotoxic effect of rituximab such as CDC and ADCC and the defect may be due to post translational changes such as phosphorylation of serine and threonine residues.Table 1. Reactivity to different Abs specific to CD20PtAgeGenderL27 (FACS)Rituximab (FACS)L26 (IHC)C-terminal Ab (WB)N-terminal Ab (WB)168M++−−+275M++−−+367M+ND−−+FACS, flow cytometry; IHC, immunohistochemistry; WB, Western blotting; +, positive; −, negative; ND, not determined

Detection of Epstein-Barr virus in T-cell prolymphocytic leukemia cells in vitro

Background Epstein-Barr virus (EBV) is closely associated with the development of a number of tumors. During latent infection, EBV continuously expresses a number of viral genes which are essential for cell transformation and maintenance of the malignant phenotype of EBV-related tumors. There has been no previous link between EBV and T-cell prolymphocytic leukemia (T-PLL), a distinctive form of leukemia derived from T-cells at an intermediate stage of differentiation between a cortical thymocyte and a mature peripheral blood T-cell. Objective To determine if EBV was present in the T-PLL cells collected. Study design T-PLL cells were isolated from the peripheral blood of a patient diagnosed with T-PLL and continuously cultured for about 1 year. The existence of EBV in these cells was detected using multiple strategies including PCR, Western blotting, immunofluorescent assay and flow cytometry analysis. Results The EBV genome was present in these T-PLL cells by PCR analysis across multiple sites in the viral genome. In addition, these T-PLL cells expressed a number of EBV latent antigens. The EBV oncoproteins LMP1, EBNA1 and EBNA3C were expressed in the majority of the infected cells. Conclusion This report suggests a potential link between EBV infection and T-PLL and provides new information about the potential contribution of EBV in the initiation or maintenance of T-PLL.

Extremely short telomeres and high telomerase activity in T-cell prolymphocytic leukemia (T-PLL): An optimal target for telomerase inhibition with GRN163L?

7061 Background: T-cell prolymphocytic leukemia (T-PLL) is an uncommon aggressive lymphoproliferative disease characterized by the expansion of a T-cell clone. We recently reported that leukemic T-PLL cells have extremely short telomeres (n=11, mean ± sd: 1.53 kb ± 0.65 kb) and high levels of telomerase activity (Roeth et al., 2007). We hypothesized that such cells have telomeres that are very close to critically short lengths and thus could be sensitive to telomerase inhibition with high sensitivity and short lag times. Methods: Our aim was to investigate the effect of the selective telomerase inhibitor GRN163L on survival of leukemic cells from T-PLL patients in short-term culture with detection of cell apoptosis by FACS-analysis using Annexin V-PI double staining. Telomere lengths of subsets of leukocytes from the peripheral blood of patients with T-PLL were determined by the automated flow-FISH and telomerase activity was measured by the TRAP-assay. Results: The leukemic cells of these T-PLL patients had short telomere lengths and high telomerase activity. We observed a dose-dependent cytotoxicity in T-PLL cell cultures with GRN163L. Viable cells as a percentage of untreated controls (viability index) after 7 days of cell culture with 0, 1, 3, 10 μM GRN163L were as follows (% mean ± sd, (t-test p-value versus control)): 100 ± 0, 57.3 ± 17.5 (p=0.024), 37.4 ± 25.0 (p=0.022), 25.3 ± 20.0 (p=0.008), respectively, (n=4). No effect on viability was observed in normal, unstimulated T-cells after 7 days with 0, 1, 3, 10 μM GRN163L. Viability indices for normal T- cells were as follows: (% mean ± sd) 100 ± 0.0, 97.9 ± 1.7, 102.4 ± 9.4, 95.1 ± 4.7, respectively, (n=3). In addition, a dose-dependent increase of apoptotic cells with the telomerase inhibitor GRN163L by Annexin V-PI double staining was observed. A mismatch control oligonucleotide at 10 μM had no significant effect on viability of leukemic cells. Conclusions: GRN163L is active in T-PLL cells in vitro and leads to rapid cytotoxicity. Targeting telomerase and telomeres therefore seems an attractive strategy for the future treatment of this disease. These results provide strong rationale for a planned clinical trial of GRN163L in patients with T-PLL. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Geron Corporation Geron Corporation Geron Corporation

T-Cells with Extremely Short Telomeres and High Telomerase Activity in T-Cell Prolymphocytic Leukemia (T-PLL): The Ideal Target for Telomerase Inhibition.

T-cell prolymphocytic leukemia (T-PLL) is a rare aggressive lymphoproliferative disease characterized by the expansion of a T-cell clone derived from immuno-competent post-thymic T-lymphocytes. Important mechanisms involved in expansion of human malignant cells are reactivation of telomerase, an enzyme complex, which is able to compensate the loss of telomere repeats by cell division, and maintenance or elongation of telomere length.To investigate the role of telomeres and telomerase we measured telomere length by automated multicolor flow-FISH and telomerase activity by telomeric repeat amplification protocol in subsets of peripheral blood leukocytes from 11 newly diagnosed or relapsed patients with sporadic T-PLL. In addition, we analyzed the effect of the selective telomerase inhibitor BIBR1532 on T-PLL cells in short-term culture assays.The average telomere length in the clonal T-cells of all samples analyzed was extremely short (mean ± std: 1.53 kb ± 0.65 kb) compared to the non-clonal T-cells (5.03 kb ± 0.71 kb; p=0.012). The average telomere length for B-cells in these patients was 6.37 kb ± 0.71 kb (n=6). Telomere length values of the clonal T-cells were all below the 1st percentile of telomere length values observed in T-cells from healthy aged-matched controls whereas non-clonal T-cells and B-cells fell between the 1st and 99th percentile of the normal distribution. Interestingly, telomere length in the clonal T-cells remained stably short at 1.0 kb ± 0.6 kb without further telomere loss in one patient over a period of 18 months. No cell doublets indicative of fused or bridged chromosomes and telomere dysfunction were observed. Clonal T-cells exhibited high levels of telomerase activity almost comparable to levels of the positive control K562 whereas there was no measurable telomerase activity in normal, unstimulated T-cells. Telomerase levels even correlated inversely with telomere length in clonal T-cells (r=−0.91, n=6). In addition, we could induce a dose-dependent cytotoxicity of T-PLL cells with the telomerase inhibitor BIBR1532 (viable cells as percentage of untreated controls (viability index) in % after 10 days of cell culture (mean ± std) with 0, 10, 40, 100 μM BIBR1532: 100 ± 0, 54 ± 14.3, 27.5 ± 11.7, 2.6 ± 1.6, n=6) whereas no effect was observed in normal, unstimulated T-cells (viability index in % (mean ± std) after 10 days with 0, 10, 40, 100 μM BIBR1532: 100 ± 0, 100.8 ± 8.6, 96.9 ± 7.9, 103.1 ± 22.4, n=3).In summary, clonal T-cells in T-PLL exhibit extremely short telomeres which could explain the genomic instability with cytogenetic aberrations. The high levels of telomerase found in T-PLL cells are sufficient to stably maintain the critically short telomeres and allow clonal expansion. In addition, we can demonstrate that inhibition of telomerase in vitro in the situation of T-PLL cells with already very short telomeres and high telomerase levels leads to rapid cytotoxicity of the T-cell clone without a time delay before telomeres get critically short as observed in the situation of longer telomeres. Targeting telomerase and telomeres seems therefore an attractive strategy for the future treatment of this devastating disease.

Histopathology of the Spleen in T-Cell Large Granular Lymphocyte Leukemia and T-Cell Prolymphocytic Leukemia

We review retrospectively the spleen histology in 8 patients with T-cell large granular lymphocyte (LGL) leukemia and 4 with T-cell prolymphocytic leukemia (T-PLL) to identify characteristic patterns of involvement and to distinguish such patterns from those described in other low grade B- and T-cell malignancies. Moderate splenic enlargement with red pulp expansion due to lymphocytic infiltration was characteristic of LGL leukemia. Abnormal lymphocytes expressed cytotoxic granule proteins and were consistently CD45RO and CD5 negative in contrast to normal red pulp T cells. This infiltration respected anatomic boundaries with encroachment but no invasion of white pulp areas. Unlike in hairy cell leukemia, the main differential diagnosis for red pulp lymphocytosis, the white pulp was not only preserved in T-cell LGL leukemia but showed germinal center hyperplasia with expansion of the mantle zones. By comparison, T-PLL spleens showed marked red pulp lymphoid infiltration by medium-sized cells with irregular nuclei and prominent eosinophilic nucleoli. T-PLL lymphocytes, unlike LGLs, were more invasive, infiltrating the spleen capsule as well as white pulp areas. T-cell prolymphocytes did not express cytotoxic granule proteins or NK-cell markers, were CD5+, CD45RO+ like normal spleen T cells, were CD2+, CD3+, CD45+, CD43+, TCRbeta+, but CD25-, CD30-, ALK-1-, TRAP-, DBA44-, and TdT-. Expression of CD4 and CD8 in these cells mirrored that of circulating T-PLL cells. These observations on the morphologic and immunohistochemical appearances of the spleen in T-cell LGL leukemia and T-PLL may aid diagnosis of these uncommon T-cell disorders, particularly T-cell LGL leukemia, where presentation may be cryptic and where unique pathognomonic features, are absent.

T-Cell Prolymphocytic Leukemia Involving Extramedullary Sites

T-cell prolymphocytic leukemia (T-PLL) can involve extramedullary sites, but the diagnosis is usually established by examination of blood and bone marrow. As a result, the histologic findings at extramedullary sites are poorly documented in the literature. We describe 19 extramedullary biopsy specimens from 14 patients with T-PLL. Skin (n = 10) was the most common site biopsied. T-PLL surrounded dermal blood vessels and appendages (n = 6), diffusely replaced dermis (n = 3), or formed a subcutaneous mass (n = 1). Other extramedullary sites included liver and lymph nodes (3 each) and spleen, lung, and cecum (1 each). In liver and lymph nodes, the neoplasm predominantly involved portal tracts and paracortex, respectively. Cytologically, the T-PLL cells were round (n = 16) or Sezary cell-like (n = 3). Nucleoli were observed in a subset of cells in 8 specimens and were prominent in 3 specimens. Immunostaining for T-cell leukemia-1 (TCL-1) was positive in specimens from 9 (64%) of 14 patients. We conclude that the prolymphocytoid features of T-PLL cells can be difficult to detect in routinely stained sections of extramedullary biopsy specimens. TCL-1 expression can aid in diagnosis at extramedullary sites.

IL-2-induced growth of CD8 + T cell prolymphocytic leukemia cells mediated by NF-κB induction and IL-2 receptor α expression

The binding of interleukin-2 (IL-2) to its receptor on normal T cells induces nuclear expression of nuclear factor κB (NF-κB), activation of the IL-2 receptor (IL-2R) α chain gene, and cell proliferation. In the present study, the role of IL-2R signaling in the growth of CD8 + T cell prolymphocytic leukemia (T-PLL) cells has been investigated. Flow cytometry revealed that primary leukemia cells from a patient with CD8 + T-PLL expressed IL-2Rα and β chains, and the cells showed a proliferative response and an increase in IL-2Rα expression on culture with exogeneous IL-2. Northern blot analysis failed to detect IL-2 mRNA, suggesting that IL-2 may act in a paracrine manner in vivo. Electrophoretic mobility-shift assays revealed that recombinant IL-2 increased NF-κB binding activity in nuclear extracts of the leukemia cells, and Northern blot analysis showed that IL-2 increased the abundance of mRNAs encoding the NF-κB components c-Rel and KBF1 in these cells. IL-2 binding analysis demonstrated that IL-2 markedly increased the number of low affinity IL-2Rs on the leukemia cells, without an effect on the number of high-affinity IL-2Rs. These results show that IL-2 is capable of inducing the nuclear expression of NF-κB in primary CD8 + T-PLL cells, and that this effect is mediated, at least in part, at a pretranslational level.