Case Of T-cell Prolymphocytic Leukemia Research Articles

Morphology, immunophenotype, and suggested diagnostic criteria of TCL1 family–negative T-prolymphocytic leukemia

Abstract Objectives We sought to investigate the morphologic and immunophenotypic characteristics of TCL1 family–negative T-cell prolymphocytic leukemia (T-PLL) Methods Twenty cases of TCL1 family–negative T-PLL were studied. Results The doubling time of leukemic cells ranged from less than 2 days to more than 5 years, with a median of 5.5 months. Leukemic cells were small to medium-sized, with round to irregular nuclei, variably condensed chromatin, and small amounts of agranular cytoplasm. A visible nucleolus was identified in 11 (55%) cases. Cytoplasmic blebs/protrusions were identified in all cases, but their occurrence was highly variable from case to case. Bone marrow biopsy showed an interstitial pattern in 90% of cases and a diffuse pattern in the remaining 10% of cases. Flow cytometric immunophenotypic analysis showed that the leukemic cells in all cases were CD4 positive; 3 (15%) also showed concurrent CD8 expression. All cases were positive for CD2 and CD5. Surface CD3 and CD7 were positive in 19 of 20 (95%) cases, and all CD3-positive cases expressed the T-cell receptor αβ. Compared with prototypic T-PLL cases, these 2 groups shared many immunophenotypic findings, except CD8 and CD26, both of which were more commonly expressed in prototypic T-PLL cases. Conclusions TCL1 family–negative T-PLL cases have morphologic and immunophenotypic features that are similar to prototypic T-PLL. They are characterized by neoplastic proliferation of small to medium-sized mature T cells with CD4-positive T-cell receptor αβ phenotype. Tumor cells frequently maintain pan-T antigen expression. Recognizing these morphologic and immunophenotypic features will aid in accurately diagnosing this rare subset of T-PLL.

Une présentation particulière de leucémie à grands lymphocytes granuleux

T cell prolymphocytic leukemia (T-PLL) is a rare, aggressive neoplasm derived from post-thymic T cells. Patients are typically middle-aged with a slight male predominance who present with a high white blood cell count, hepatosplenomegaly, lymphadenopathy, and other symptoms typically associated with leukemia. Although cutaneous involvement has been reported in up to 30% of cases of T-PLL, to our knowledge, none have presented with a presentation resembling livedoid vasculopathy. In the correct clinical context, an underlying hematolymphoid neoplasm should be included in the differential diagnosis of a patient presenting with livedoid vasculopathy.

P1194: A PHASE IB TRIAL OF ITACITINIB COMBINED WITH ALEMTUZUMAB IN PATIENTS WITH T-CELL PROLYMPHOCYTIC LEUKEMIA (T-PLL)

Background: T-cell prolymphocytic leukemia (T-PLL) is a mature T-cell neoplasm with an aggressive clinical course including rapid lymphocyte proliferation, progressive organomegaly, lymphadenopathy, pleural and pericardial effusions, skin manifestations, and severe B-symptoms. Overall prognosis has been poor, with inadequate response to conventional chemotherapy and limited efficacy of allo stem cell transplant (SCT). The CD52 monoclonal antibody alemtuzumab (CAMPATH) has been the most effective therapy for T-PLL, but responses are not durable. Three quarters of cases of T-PLL harbor activating mutations in the JAK-STAT pathway (JAK1, JAK3, STAT5b, IL2RG). Aims: We designed astudy to evaluate the combination of the JAK1 inhibitor itacitinib with CAMPATH to target activated JAK-STAT signaling. Methods: 15 pts were enrolled (Table 1). 8 pts (53%) were newly diagnosed and 7 pts (47%) were previously treated, with a median of 1 (1-3) prior therapies, including 2 (33%) with prior SCT. 10 pts (67%) had chromosome 14 abnormality by conventional cytogenetics, and 6/6 (100%) pts tested by FISH had TCL1 positivity. 5/7 (71%) tested pts having activating mutations in the JAK-STAT pathway. Pts received a median of 4 cycles (1-12) of therapy, including a median of 2 (1-4) combination cycles and a median of 3 (0-10) maintenance cycles. Of 8 evaluable frontline pts, there were 4 (50%) complete remissions (CR), all MRD (-) by flow cytometry, and 3 (38%) partial remissions (PR) for an overall response rate of 88%. Of 7 evaluable previously-treated pts, there were 3 (43%) CR, all MRD (-) by flow cytometry, 1 (14%) PR, 2 non responders (29%), and 1 (17%) early death in a pt that declined transfusions, for an ORR 57%. The median number of cycles to best response were 2 (1-4) and 1 (1-2) in the frontline and salvage cohorts, respectively. While no objective responses were observed in WBC or lymph nodes during the 2 week window of single-agent itacitinib, pts experienced improvement in B-symptoms, splenomegaly, and rash during this period prior to CAMPATH. The regimen was well tolerated. The most common grade 3/4 AEs on study were lung infection (4), allergic reaction (3), other infection (2), neutropenic fever (1), and kidney injury (1). No AEs were attributed to single-agent itacitinib. 3 of the 6 (50%) responding frontline pts went on to allogeneic SCT, 1 patient (81 years old) died after 11 cycles on protocol of unknown cause, and 2 pts are ongoing on protocol. The median OS was 18.6 m and 8.5 m in the frontline and salvage cohorts, respectively. Results: Single-agent itacitinib was associated with patient reported improvements in B-symptoms, rash, appetite, and reduced splenomegaly. Response rates with the combination were encouraging in frontline and salvage, with favorable OS. Further study of the combination without a 2-week window (of itacitinib alone) and with objective measurement of patient reported symptoms is planned. Image:Summary/Conclusion: The combination of itacitinib 200 mg daily and CAMPATH in pts with T-PLL was safe and well tolerated with no dose-limiting toxicities. Single-agent itacitinib was associated with patient reported improvements in B-symptoms, rash, appetite, and exam findings of reduced splenomegaly. Response rates with the combination were encouraging in frontline and salvage, with favorable OS. Further study of the combination without a 2-week window (of itacitinib alone) and with objective measurement of patient reported symptoms is planned.

TCL-445: Case of T-Cell Prolymphocytic Leukemia Successfully Treated with Fludarabine, Mitoxantrone, and Cyclophosphamide

Context T-cell prolymphocytic leukemia (T-PLL) is an extremely rare mature T-cell neoplasm associated with an aggressive clinical course, poor response to conventional chemotherapy, and a dismal prognosis. Current therapeutic strategies conventionally employ the anti-CD52 antibody alemtuzumab as the most active agent. Alemtuzumab, originally marketed for use in cancer (Mabcampath®), is currently licensed and formulated for the treatment of relapsing multiple sclerosis (Lemtrada®), causing a lack of drug availability, with potentially grave consequences for T-PLL patients’ prognosis. Design We present the case of a 68-year-old male patient diagnosed with T-PLL in December 2020, who underwent four cycles of fludarabine 25 mg/m2, mitoxantrone 8 mg/m2, and cyclophosphamide 200 mg/m2, and achieved complete remission (CR), according to the criteria established by the T-PLL International Study Group. Results A 68-year-old Caucasian male presented to our center with fatigue and weakness for several months and a history of persistent, progressive lymphocytosis for the past 18 months. A computed tomography scan revealed bilateral cervical, axillary, and intra-abdominal lymph nodes, measuring approximately 3 cm each; an enlarged spleen of 180 mm in length; and mild hepatomegaly, measuring 177 mm on the midclavicular line. Laboratory studies revealed leukocytosis (282 × 109/L) and an absolute lymphocyte count of 255 × 109/L, with no anemia or thrombocytopenia. The metabolic profile was normal, except for an elevated lactate dehydrogenase of 974 U/L. The bone marrow aspirate showed 86%–88% polymorphic lymphoid cells, while the flow cytometry analysis indicated T cells positive for CD2 and surface and cytoplasmic CD3, CD5, and CD7, 70% of which were CD4+CD8+, and 30% CD4+. The core biopsy revealed a markedly hypercellular marrow (80%), with a lymphoid infiltrate made up of small/medium-sized cells, some with cerebriform nuclei, some with prominent nucleoli, with weakly positive immunohistochemistry staining for CD52. Conclusions The patient presented in this case study was treated with conventional chemotherapy alone and achieved CR of T-PLL, maintaining the treatment response to date (3 months after completion of the therapy). Despite this, alemtuzumab remains a crucial element in the arsenal of treatment for T-PLL, and its lack of availability poses a serious challenge for clinicians.

Poster: TCL-445: Case of T-Cell Prolymphocytic Leukemia Successfully Treated with Fludarabine, Mitoxantrone, and Cyclophosphamide

Poster: TCL-445: Case of T-Cell Prolymphocytic Leukemia Successfully Treated with Fludarabine, Mitoxantrone, and Cyclophosphamide

Cutaneous involvement by T‐cell prolymphocytic leukemia presenting as livedoid vasculopathy

T-cell prolymphocytic leukemia (T-PLL) is a rare, aggressive neoplasm derived from post-thymic T-cells. Patients are typically middle aged with a slight male predominance who present with a high white blood cell count, hepatosplenomegaly, lymphadenopathy, and other symptoms typically associated with leukemia. Although cutaneous involvement has been reported in up to 30% of cases of T-PLL, to our knowledge, none have presented with a presentation resembling livedoid vasculopathy. In the correct clinical context, an underlying hematolymphoid neoplasm should be included in the differential diagnosis of a patient presenting with livedoid vasculopathy.

Inmunoglobulina intravenosa en el síndrome de activación macrofágica asociado a lupus eritematoso sistémico

BACKGROUND T-cell prolymphocytic leukemia (T-PLL) is a T-cell lymphoproliferative disorder that frequently involves the skin. The objective was to describe two cases of T-PLL with cutaneous involvement and to present a review of the literature concerning the clinical characteristics, differential diagnosis and treatment of these patients. CASE REPORTS 1) 79 year-old man, with a previous diagnosis of T-PLL based on a laboratory incidental finding. He had been treated with alemtuzumab, but it had to be interrupted due to recurrent infections. After interrupting the treatment, the patient developed a symmetrical rash on his extremities. The skin biopsy demonstrated TPLL infiltration. 2) 28 year-old man that presented with asthenia and lymphocytosis. He also showed a purpuric rash on his trunk and facial erythema. Histopathology of the skin and bone marrow confirmed the diagnosis of T-PLL with cutaneous involvement. CONCLUSIONS T-cell prolymphocytic leukemia accounts for 2% of mature leukemias in adults. Skin involvement is reported in 20-50% of the patients. The characteristic features are facial involvement, purpuric lesions and symmetry of the rash, although there are atypical manifestations as well. Differential diagnosis includes other T-cell lymphoproliferative disorders with hematologic and skin involvement, such as Sezary syndrome. Patients with T-PLL may show cutaneous infiltration at the moment of debut or relapse of the disease. The skin is an accessible organ for taking samples to study and diagnose these patients.

The miR-200c/141-ZEB2-TGFβ axis is aberrant in human T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is mostly characterized by aberrant expansion of small- to medium-sized prolymphocytes with a mature post-thymic phenotype, high aggressiveness of the disease and poor prognosis. However, T-PLL is more heterogeneous with a wide range of clinical, morphological, and molecular features, which occasionally impedes the diagnosis. We hypothesized that T-PLL consists of phenotypic and/or genotypic subgroups that may explain the heterogeneity of the disease. Multi-dimensional immuno-phenotyping and gene expression profiling did not reveal clear T-PLL subgroups, and no clear T-cell receptor a or b CDR3 skewing was observed between different T-PLL cases. We revealed that the expression of microRNA (miRNA) is aberrant and often heterogeneous in T-PLL. We identified 35 miRNA that were aberrantly expressed in T-PLL with miR-200c/141 as the most differentially expressed cluster. High miR- 200c/141 and miR-181a/181b expression was significantly correlated with increased white blood cell counts and poor survival. Furthermore, we found that overexpression of miR-200c/141 correlated with downregulation of their targets ZEB2 and TGFbR3 and aberrant TGFb1- induced phosphorylated SMAD2 (p-SMAD2) and p-SMAD3, indicating that the TGFb pathway is affected in T-PLL. Our results thus highlight the potential role for aberrantly expressed oncogenic miRNA in T-PLL and pave the way for new therapeutic targets in this disease.

Analysis of T-Cell Receptor Beta Chain (Tß) Gene Rearrangements Demonstrates the Monoclonal Nature of T-Cell Chronic Lymphoproliferative Disorders

We investigated the rearrangement patterns of the gene coding for the beta chain of the T cell receptor (T beta) in 11 patients with T-cell derived chronic lymphoproliferative disorders, including T-cell prolymphocytic leukemia (T-PLL) and T-cell chronic lymphocytic leukemia (T-CLL). We found that all five cases of T-PLL, and five of six cases of T-CLL, displayed T beta-gene rearrangements, clearly establishing their monoclonal nature. Clonality could not be determined in one case of T-CLL where the T beta gene was found unrearranged. Our results demonstrate that the majority of cases of both clinically aggressive T-PLL and clinically indolent T-CLL are monoclonal. These results suggest that the analysis of T beta gene rearrangements represents a valid tool for the differential diagnosis and clinical monitoring of T-cell derived chronic lymphoproliferative diseases.

Reconstruction of rearranged T-cell receptor loci by whole genome and transcriptome sequencing gives insights into the initial steps of T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is an aggressive tumor with leukemic presentation of mature T-lymphocytes. Here, we aimed at characterizing the initial events in the molecular pathogenesis of T-PLL and particularly, at determining the point in T-cell differentiation when the hallmark oncogenic events, that is, inv(14)(q11q32)/t(14;14)(q11;q32) and t(X;14)(q28;q11) occur. To this end, we mined whole genome and transcriptome sequencing data of 17 and 11 T-PLL cases, respectively. Mapping of the 14q32.1 locus breakpoints identified only TCL1A, which was moreover significantly overexpressed in T-PLL as compared to benign CD4+ and CD8+ T-cells, as the only common oncogenic target of aberrations. In cases with t(14;14), the breakpoints mapped telomeric and in cases with inv(14) centromeric or in the 3'-untranslated region of TCL1A. Regarding the T-cell receptor alpha (TRA) locus-TCL1A breakpoint junctions, all 17 breakpoints involved recombination signal sequences and 15 junctions contained nontemplated (N-) nucleotides. All T-PLL cases studied carried in-frame TRA rearrangements on the intact allele, which skewed significantly toward usage of distal/central TRAV/TRAJ gene segments as compared to the illegitimate TRA rearrangements. Our findings suggest that the oncogenic TRA-TCL1A/MTCP1 rearrangements in T-PLL occur during opening of the TRA locus, that is, during the progression from CD4+ immature single positive to early double positive thymocyte stage, just before physiologic TCL1A expression is silenced. The cell carrying such an oncogenic event continues maturation and rearranges the second TRA allele to achieve a functional T-cell receptor. Thereafter, it switches off RAG and DNTT expression in line with the mature T-cell phenotype at presentation of T-PLL.

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

CD161 (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. Retrospective review of T-PLL cases with complete flow cytometry data including CD161. We 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. These 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.

Next-generation amplicon TRB locus sequencing can overcome limitations of flow-cytometric Vβ expression analysis and confirms clonality in all T-cell prolymphocytic leukemia cases.

T-cell receptor (TCR) β repertoire analysis can distinguish monoclonal from polyclonal T-cell proliferations and crucially aid in the diagnosis of T-cell malignancies. TCR repertoire can be assessed either by flow cytometry (FCM), or by molecular genetic techniques. We compared the results of parallel analyses of Vβ expression by FCM and TRB rearrangements by DNA-based next-generation sequencing (NGS) in 80 diagnostic peripheral blood samples of patients with T-cell prolymphocytic leukemia (T-PLL) for (1) the diagnosis of clonality and (2) the assessment of dominant Vβ usage. FCM-based analysis of the surface expression was performed using the IOTest Beta Mark kit. The NGS-based analysis employed the multiplex Biomed-2 VB-JB primers. In all the samples, one or two clonal TRB rearrangements were detected by NGS. Although a dominant Vβ domain usage was detected by FCM in only 41/80 (51%) samples, clonality was suspected in all of them. In a total of 12 cases, the FCM missed the clone detected by NGS, despite theoretical coverage by the antibodies, the functionality of the rearrangement, and the expression of TCRαβ on the cell surface. Partly overlapping with those cases, FCM discovered predominant Vβ usage in the T-PLL population that differed from the one detected by NGS in 10 cases. Overall, the concordant NGS and FCM results were obtained on 61/80 (76%) of samples. We conclude that NGS-based TRB analysis can overcome certain limitations of FCM-based analysis by the identification of both productive and nonproductive rearrangements and by covering the whole Vβ spectrum. Currently available FCM analysis of Vβ expression lacks this breadth but has advantages, such as parallel immunophenotyping and a more accurate quantification of the Vβ usage. © 2018 International Society for Advancement of Cytometry.

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.

Small Cell Variant of T-Cell Prolymphocytic Leukemia with Trisomy of the TCL1A Locus Via a Novel Translocation

Small-cell variant of T-cell prolymphocyticleukaemia (T-PLL) is a rare entity, with only 32 cases described1. A 64 year old asymptomatic female presented with lymphocytosis (15.6 x 10^9/L), already present 9 years earlier. Clinical examination was normal. Flow cytometry demonstrated 94% of lymphocytes were CD3+/CD4+/CD8- T cells with normal expression of CD2/5/7. V-beta T cell receptor repertoire and T cell gene rearrangement confirmedmonoclonality. Peripheral blood morphology showed small mature lymphocytes without cytoplasmic granulation and only a rare form showing indistinct nucleoli [arrowhead, figure 1]. Lymphocyte count initially increased slowly (to 23.7 x 10^9/L 10 months after diagnosis), then rapidly (47.2 x 10^9/L, 90.8 x 10^9/L, 151.6 x 10^9/L, 201.6 x 10^9/L at 16 months, 22 months, 28 months and 29 months after diagnosis respectively). By 29 months thrombocytopenia (platelet count 24 x 10^9/L), elevated LDH and spontaneous bruising developed. CT scan did not showadenopathyor splenomegaly. Bone marrow biopsy showed extensive infiltration by small, mature lymphocytes with an identicalimmunophenotypeto that seen at diagnosis. Treatment with CHEOP for 2 cycles did not achieve any response and causedmyelosupression, GI upset and fatigue. Progressive disease was seen during 2 cycles ofpanobinostatwith development ofadenopathy, splenomegaly and rising lymphocyte count.Alemtuzumabproduced rapid disease response with normalisation of lymphocyte count and resolution of theadenopathyand splenomegaly.Bone marrow biopsy after 28 doses ofalemtuzumabshowed no morphological orimmunophenotypicevidence of residual lymphoma.The patient declined to undertake consolidative autologous peripheral blood stem cell transplant at best response. Unfortunately the patient relapsed 4 months later with progressive lymphocytosis, splenomegaly and high LDH. Gemcitabine was given without response and the patient passed away 45 months after diagnosis. Comprehensive genetic analysis was undertaken using G-banded karyotyping, M-FISH, allele-specific FISH for TCL1A and SNP array. Multiple complex rearrangements were detected with abnormalities involving chromosomes 2, 3, 7, 8, 9, 10, 11, 12, 13, 14, 17, 18, 20 and 22, but lacking the inv(14)(q11.2q32) commonly seen in typical T-PLL. In total, 58 copy number changes were identified, but the sole region of copy number gain was a 21Mb duplication of chromosome 14 distal to band 14q31.3. It was present in approximately 90% cells, consistent with the presence of a der(21)t(14;21)(q31;p11.2) observed by metaphase cytogeneticsand resulting in trisomy of the TCL1A locus seen on allele-specific FISH for TCL1A [arrowheads, figure 2]. Some changes detected by SNP array were present in virtually all cells and were thus likely to be have occurred early during tumour evolution. Other subclonalchanges were seen in approximately 90%, 75% or 60% cells respectively, indicating the presence of four distinct cell lines in total. Deletion of the distal portion of 12p was present in virtually all cells, in keeping with the presence of either the dicentric12;13or the dicentric12;14 chromosome in all malignant cells. Of note, a cytogenetically cryptic 1.5Mb deletion at 2p23.2 resulting in deletion of DNMT3A was also an early event, as was a heterozygous deletion of DNMT3B at 20q11.2. The deletion of DNMT3B was seen in association withchromothripsis-like changes on chromosome 20. Despite demonstrated rearrangement of both copies of chromosome 20 by metaphasecytogenetics, no regions of homozygous deletion on chromosome 20 were identified. Typically cases of T-PLL, small cell variant, possess rearrangements of 14q11, often theparacentricinversion typically seen in standard T-PLL, but with other translocations involving the 14q11 locus also described. Thet(14;21) seen in this case demonstrates a novel mechanism of TCL1A dysregulation, not previously described in T-PLL. 1Rashidi, A. and Fisher, S. I. (2015), T-cell chronic lymphocyticleukemia or small-cell variant of T-cellprolymphocyticleukemia: a historical perspective and search for consensus.Eur JHaematol, 95: 199-210.doi:10.1111/ejh.12560 Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Novel Mutations in NOTCH and Altered Wnt/β-Catenin Pathway Indicate a Role of Embryonic Signals in the Pathogenesis of T-Cell Prolymphocytic Leukemia

Background:Recurrent mutations in ATM, JAK1/3 and STAT5B genes have been identified in T-cell prolymphocytic leukemia (T-PLL), a mature T cell leukemia with poor survival. However, much remains to be understood regarding the dysregulated pathways involved in the pathogenesis of this aggressive disease. Here, we integrated the analyses of whole exome sequencing (WES) and transcriptomes of a cohort of 12 T-PLL samples to gain further insights into molecular mechanisms of this malignancy.Methods: Leukemic cells were isolated from peripheral blood or bone marrow samples of T-PLL patients. Tumor DNA and RNA, and germline DNA were extracted from purified leukemic T cells and bone marrow derived stromal cells, respectively. WES (n=12) and transcriptome analysis (n=10) were performed and data analyzed with published standard methods (Robinson et al. 2010 and DePristo et al. 2011)Results: 7 paired tumor and germline DNA and 5 tumor-only DNA samples were used for WES analysis. Mutations in ATM and TET2 were detected in 4 (33%) and 1 (8%) patients, respectively. JAK3 (p.M511I, p.Q501H and p.Q503H) or STAT5B (2 p.T628S and 1 p.N642H) mutations each occurred in 3 (25%) cases and were mutually exclusive, consistent with prior reports. Importantly, we found several novel mutations in genes with regulatory roles in the NOTCH signaling pathway. Specifically, 2 somatic mutations p.A3V and p.A3S in NOTCH2 and 2 other mutations (p.S244L and p.A1414V) in NOTCH4 were identified in 4 (33%) patients. One mutation was in the EGF- like and another one in the NODP domain critical for NOTCH-mediated cellular differentiation. Two (16.7%) novel missense mutations (p.G174D and p.V836I) were found in MAML2. Three missense mutations (2 p.S583N and 1 p.R190H) occurred in MAML1. Both MAML2 and MAML1 are essential cofactors of the NOTCH signaling pathway. In addition, a somatic mutation in the NOTCH signal regulator DTX2 (p.G268D) was detected. Altogether 7 (58.3%) patients harbored mutations in genes with a regulatory role in the NOTCH pathway. Among the epigenetic modifiers, we found deletions and point mutations in KDM6B and KDM6A in 4 (30%) patients including 2 codon deletions in KDM6B (p.X751Val) and 1 missense mutation each in KDM6B (p.R1016Q) and KDM6A (p.V510G). KDM6A and KDM6B are histone H3 lysine 27 demethylases known to be involved in regulation of DNA repair and play an important role in embryonic differentiation.Transcriptome analyses of 10 T-PLL cases versus normal CD3+ T cells from 5 healthy donors revealed 706 up-regulated and 655 down-regulated genes. Consistent with prior data, TCL1A and TCL1B were significantly overexpressed (23.7 and 13.1 fold, respectively). Using Ingenuity Pathway Analysis, we found PTEN Signaling, glutamate receptor, and axonal guidance signaling to be among the top 3 upregulated pathways. The top upstream regulators implicated for the up-regulated signals were FGF, Wnt/β-catenin and TNF. The top 3 down-regulated pathways were granulocyte adhesion and diapedesis, IL-17A in psoriasis and IL-10 signaling. The top regulators for the down-regulated pathways were LPS, TNF and IFNg. Wnt/β-catenin and Human embryonic stem cell pluripotency signaling were among the top 20 up-regulated pathways. Many genes involved in the regulation of Wnt signal pathway were included in the above pathways, including Wnt ligands (Wnt9A, Wnt4), transmembrane receptors (CDH1, LRP5, FZD6, FZD7, SMO), cytoplasmic factor (AXIN2), and nuclear factors (MYC, SOX12, SOX8, SOX18, CCND1).Summary: Our study identified recurrent mutations in the NOTCH signaling pathway as well as dysregulated embryonic developmental pathway, e.g. Wnt/NOTCH as newly implicated molecular mechanisms in T-PLL pathogenesis for further functional studies. Additionally, novel (KDM6A and KDM6B) and known mutations (ATM, TET2) in chromatin regulatory genes underscore the importance of epigenetic dysregulation in the tumorigenesis of T-PLL. DisclosuresParikh:Pharmacyclics: Honoraria, Research Funding. Mustjoki:Ariad: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Ding:Merck: Research Funding.

Novel Phenotypic and Genetic Analysis of T-Cell Prolymphocytic Leukemia (T-PLL)

IntroductionT-cell prolymphocytic leukemia (T-PLL) is a rare mature T-Cell malignancy typically associated with aggressive clinical course. Leukemic T-cells demonstrate post-thymic T-cell phenotypes (Tdt-, CD1a-, CD5+, CD2+ and CD7+) and commonly are CD4+/CD8- T-cells, but co-expression of CD4+/CD8+ or CD8+/CD4- have also been observed. Rearrangement of chromosome 14 involving TCL1 locus is the cornerstone for T-PLL diagnosis which leads to overexpression of oncogene TCL1. As a binding partner for TCL1, AKT expression correlates with worse prognosis. Despite advances in identification of novel mutations, therapeutic option is limited with most patients having very short survival. Herein, we performed a detailed analysis of specific T-cell subsets affected in T-PLL and a comprehensive genomic analysis by whole exome sequencing (WES) and RNA sequencing. We also explored the preclinical efficacy of targeting AKT activation by AKT inhibitor MK2206 on leukemic T-cells isolated from T-PLL patients.MethodsT-PLL leukemic cells were isolated from blood or marrow samples of T-PLL patients (n=9) and were tested for involved T-cell subsets by multicolor flow cytometric analysis. Leukemic T-cells were treated with escalating doses of MK2206 (0 to 10 µM) for about 80 hours and were assessed for their apoptosis levels using Annexin V flow cytometric analysis. Whole exome sequencing (WES) and RNA sequencing were conducted using genomic DNA and RNA isolated from purified leukemic T cells from T-PLL patients.ResultsPhenotypic analysis of T-cell subsets in T-PLL patients showed that leukemic T-cells in 4 untreated T-PLL patients are consistent with naïve T-cell phenotype (CD45RA+, CD45RO-, CCR7+). Leukemic T-cells in 3 of the untreated patients and in 2 of the treated patients have central memory T (CD45RA-, CD45RO+, CCR7+) and effector memory T phenotypes (CD45RA-, CD45RO+,CCR7-) respectively. MK2206 treatment was able to induce dose-dependent apoptosis on the isolated PBMC (containing > 90% leukemic T-cells) of T-PLL patients (n=4) (IC50: 5 µM). FISH analysis found a rearrangement of TCL1 locus on chromosome 14 in all T-PLL cases (n=9). Three cases have also been detected to have del (11q) involving ATM locus and one patient has both del(11q) and del(17p). Subsequent WES (n=7) and RNA sequencing (n=6) analysis revealed recently reported mutations in ATM (frame shift mutation and early stop mutation W579*) in 2 cases, JAK3 (M511I) in 2 cases and STAT5B (T628S and N642H) in 2 cases. Importantly, novel somatic mutations in gene IKZF1 (N159S), NTRK1 (R33W), AP2A2 (P514L), HDAC8 (I115R), RARB (G90W) and TNIP2 (K104Q) were detected by WES. Mutations in EML4 (L548W and F304S) and VAV3 (C282Y and splice site mutation) were also identified in 2 different T-PLL cases. RNA sequencing revealed several fusion transcripts resulting in early stop of several different genes including PTPRT, L3MBTL1, UCKL1 in one T-PLL case.ConclusionT-PLL leukemic T-cells are consistent with either naïve or central memory T-cell subsets in untreated patients. The AKT inhibitor MK-2206 was capable of inducing apoptosis and could be a potential therapeutic agent for T-PLL patients. In addition, we detected known mutations in ATM and JAK-STAT pathways. Novel mutations in genes involving DNA binding and chromatin remodeling (IKZF1, HDAC8) or kinase signal pathway (NTRK1, TNIP2, VAV3, EML4) were uncovered. These results suggest that further therapeutic approaches could be developed to target DNA binding factors or JAK-STAT or AKT-TCL1 signal pathway with an ultimate goal to improve survival of T-PLL patients. DisclosuresOff Label Use: Off label use of MK2206: MK-2206 is an AKT inhibitor obtained from Selleck Chemicals and is used to treat leukemic cells in vitro to test the leukemic sensitivity to AKT inhibition. .

A Comprehensive Cytogenetic and Molecular Genetic Characterization of Patients with T-PLL Revealed Two Distinct Genetic Subgroups and JAK3 Mutations As an Important Prognostic Marker

Background: T-cell prolymphocytic leukemia (T-PLL) is a rare, mature T-cell neoplasm with poor prognosis. Only few T-PLL cases have been analyzed with regard to cytogenetic and molecular genetic aberrations so far. Therefore, we performed a comprehensive characterization of patients with T-PLL, including the identification of potential correlations between the respective markers and their impact on prognosis.Methods: The cohort comprised 47 T-PLL cases (32 male, 15 female). Median age was 69.8 years (range: 32.7-86.6 years). Diagnosis of T-PLL was assigned by immunophenotyping and cytomorphology. All 47 patients were further investigated using (i) chromosome banding analysis (CBA), (ii) interphase FISH to determine the copy number state for TP53 and ATM and chromosomal rearrangements of TCRA/D and TCL1 and (iii) array CGH. Next-generation amplicon deep-sequencing was performed to analyze mutations in ATM,BCOR, TP53 (n=47, respectively); JAK1 (n=44) and JAK3 (n=45) were analyzed by Sanger sequencing. Clinical follow-up data was available for 43 patients.Results: In all 47 cases, chromosomal abnormalities and/or molecular mutations were detected. Combining CBA and FISH data, an inv(14)(q11q32)/t(14;14)(q11;q32) was observed in 37/47 (78.7%) cases, a t(X;14)(q27;q11) in 3 cases (6.4%) and an i(8)(q10) in 17/47 (36.2%) cases. ATM deletions were detected in 27/47 (57.5%), TP53 deletions in 11/47 (23.4%) patients. Array CGH analyses revealed additional gains and losses of specific chromosomal regions, mainly affecting 7q (deletions in region 7q34-7q36; n=16), 12p (deletions in 12p12-12p13; n=11) and 22q (deletions in 22q11-q12 with a concomitant gain of 22q12-q13; n=8). Regarding molecular analyses, the most frequently mutated gene was ATM (34/47; 72.3%). Mutations in TP53 were found in 7/47 (14.9%) and in BCOR in 4/47 (8.5%) patients. Mutations of JAK1 were found in 3/44 (6.8%), and of JAK3 in 8/45 (17.8%) cases.ATM and TP53 frequently carried a mutation of one allele and a deletion of the other: 23/34 (67.6%) cases with ATM mutation also showed an ATM deletion and in 5/7 (71.4%) cases with TP53 mutation also a TP53 deletion was detected. Regarding chromosomal aberrations, all cases with i(8)(q10) harbored a TCRA/D rearrangement and an ATM mutation, whereas TP53 mutations were not present in any case with i(8)(q10). ATM mutations were found to be correlated to TCRA/D rearrangements (33/40 TCRA/D+ cases, 82.5%; 1/7 TCRA/D- cases, 14.3%; p<0.001). In contrast, TP53 mutations were predominantly observed in patients without TCRA/D rearrangement (4/7 TCRA/D- cases, 57.1%; 3/40 TCRA/D+ cases, 7.5%; p=0.008). Additionally, all three JAK1 mutations were detected in cases with a TCRA/D rearrangement. When splitting the cohort into patients ≤60 years (n=13) and >60 years (n=34), JAK1 mutations (0/12 vs. 3/32) and mutations/deletions in the TP53 gene were detected exclusively in patients >60 years (TP53mut: 0/13 vs. 7/34; TP53del: 0/13 vs. 11/34). JAK3 mutations were also found predominantly in older patients (1/12; 8.3% vs. 7/33; 21.2%).Median overall survival (OS) was 27.4 months. No influence on OS was found for mutations and/or deletions of ATM, TP53, BCOR orJAK1 or aberrations of chromosomes 8 or 14. The age of patients was found to impact OS (median OS, ≤60 years: 29.0 months vs. >60 years: 15.9 months), although this was not significant (p=0.077). However, OS was found to be significantly shorter in patients with JAK3 mutation compared to patients without JAK3 mutation (median OS, 5.1 months vs. 29.1 months; p=0.009).Conclusions: Genetic abnormalities were revealed in all 47 cases with T-PLL. Two distinct genetic subgroups of T-PLL were identified: A large subset, comprising 81% of patients, showed abnormalities involving the TCRA/D locus activating the proto-oncogenes TCL1 (14q32) or MTCP1 (Xq28). This subgroup had higher frequencies of i(8)(q10) and of ATM mutations, while the second group was characterized by a higher frequency of TP53 mutations (figure). Further, JAK3 mutations were identified as an important prognostic marker, showing a significant negative impact on OS. [Display omitted] mut=mutation, del=deletion, TCRA/D=rearrangements involving TCRA/D, TCL1=rearrangements involving TCL1, MTCP1= rearrangements involving MTCP1 DisclosuresStengel:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Characterization of Spontaneous Malignant Lymphomas in Japanese Macaques (Macaca fuscata)

Lymphomas are common spontaneous tumors in nonhuman primates but remain poorly characterized in Japanese macaques (Macaca fuscata). This study examined 5 cases of spontaneous malignant lymphoma in Japanese macaques, focusing on the immunophenotypes and presence of simian lymphocryptoviruses, which are Epstein-Barr virus-related herpesviruses in nonhuman primates. The macaques with lymphoma were 5 to 28 years old, indicating that lymphomas develop over a wide age range. The common macroscopic findings were splenomegaly and enlargement of lymph nodes. Histologic and immunohistochemical analyses revealed that all cases were non-Hodgkin type and exhibited a T-cell phenotype, positive for CD3 but negative for CD20 and CD79α. The lymphomas exhibited diverse cellular morphologies and were subdivided into 3 types according to the World Health Organization classification. These included 3 cases of peripheral T-cell lymphoma, not otherwise specified; 1 case of T-cell prolymphocytic leukemia; and 1 case of an unclassifiable T-cell lymphoma. Positive signals were detected by in situ hybridization in 2 of the 4 examined cases using probes for the Epstein-Barr virus-encoded small RNA (EBER). Furthermore, the presence of M. fuscata lymphocryptovirus 2, a macaque homolog of Epstein-Barr virus, was demonstrated in EBER-positive cases by polymerase chain reaction amplification followed by direct sequencing. Immunohistochemistry using antibody to the Epstein-Barr virus-encoded nuclear antigen 2 was negative, even in the EBER-positive cases. The present study suggests that T-cell lymphoma is more common than B-cell lymphoma in Japanese macaques and that M. fuscata lymphocryptovirus 2 is present in some cases.

Identification of a novel SEPT9-ABL1 fusion gene in a patient with T-cell prolymphocytic leukemia

T-cell prolymphocytic leukemia (T-PLL), a rare type of peripheral T-cell leukemia, is characterized by marked splenomegaly with rapidly progressive lymphocytosis and a poor prognosis. Nine kinds of ABL1 chimeric genes have been identified in various kinds of hematological malignancies, such as chronic myeloid leukemia and B- or T-lymphoblastic leukemia. However, there have been no reports describing T-PLL cases with ABL1 rearrangements. We herein report a case of T-PLL with a novel SEPT9-ABL1 fusion gene which induced strong resistance to tyrosine kinase inhibitors such as imatinib and dasatinib.

T-cell prolymphocytic leukemia revealed by spinal cord compression

T-cell prolymphocytic leukemia is a lymphoid hematologic malignancy of T origin, mature, individualized well by the clinical, morphological, and immunophenotypic as well as cytogenetic aspects. We report the case of a 39-year-old man admitted in our department because of a spinal cord compression. The cerebrospinal fluid analysis had revealed an increased albuminemia level; MRI evoked a vertebral infiltration by a malignancy. Evolution was characterized by the appearance of a tumor syndrome and a peripheral lymphocytosis. Immunophenotyping and the lymph node biopsy concluded of a diagnosis of a T-cell prolymphocytic leukemia. To our knowledge, we report here the first case of T-cell prolymphocytic leukemia revealed by a spinal cord compression.