T-cell Prolymphocytic Leukemia Patients Research Articles

Subgroups of T-Cell Prolymphocytic Leukemia (T-PLL) Discovered By High-Throughput Ex Vivo Drug Testing and Genetic Profiling

T-cell prolymphocytic leukemia (T-PLL) is a rare disease with an aggressive clinical course and a median overall survival of less than three years. Although almost 75% of T-PLL patients are reported to harbor translocations causing the activation of the proto-oncogene TCL1A, T-PLL is genetically heterogenous: most T-PLL patients also have mutations or deletions in the ATM gene and the genes involved in the JAK-STAT pathway are mutated in 76% of cases. There is an urgent need for more rational based therapies, but clinical trials are difficult to perform due to the rareness of the disease. Here, we systematically explored the diversity of drug responses in T-PLL patient samples ex vivo using a drug sensitivity and resistance testing (DSRT) system including 306 oncology drugs (approved or investigational). We also aimed to determine any associations between the genetic aberrations and drug sensitivities in T-PLL patients.Primary mononuclear cells were gathered from 30 T-PLL patients for drug testing. Cells were plated in 384-well plates and subjected to the 306 substances using a 10,000-fold concentration range. After 72 hours, cell viabilities were measured, the results were depicted as dose-response curves for each compound, and differential drug sensitivity scores (sDSS), representing leukemia-specific responses, were computed by comparing patient samples to healthy donors. Drug response profiles across patients were clustered and visualized by hierarchical clustering. The subgroups resulting from the clustering were statistically compared using a two-sample t-test to understand which drug classes were driving the grouping.To delineate heterogeneous pathway dependencies, drug sensitivities were correlated with somatic genetic variants and recurrent chromosomal aberrations. Genetic characterization was performed by targeted amplicon sequencing of tumor cells to profile known recurrent genetic variants (STAT5b, IL2RG, JAK1, JAK3, ATM). Information on chromosomal aberrations (TCL1A translocations, ATM deletions) was derived from parallel clinicopathologic databases.Amplicon sequencing revealed that 70% of T-PLL patients (21/30) harbored a mutation in genes involved in the JAK-STAT pathway (JAK1, JAK3, STAT5b or IL2RG). The most prevalent mutation led to an M511I amino acid exchange in the JAK3 protein (26% of patients). Interestingly, the STAT5b mutations (5/30) did not coexist with any of the JAK mutations in our cohort.Based on DSRT analysis, all T-PLL samples were sensitive to the CDK-inhibitor SNS-032 and the anti-cancer antibiotic actinomycin D. Next, we clustered patients using sDSS values for all 306 different substances, and this showed that patient samples could be divided into 3 main groups, based on their drug responses (Figure). According to two-sample t-test, the grouping was driven by the selective sensitivities of Groups II and III to HDAC inhibitors (belinostat, panobinostat, quisinostat, CUDC-101 and vorinostat) and the selective sensitivity of Group III to PI3K/AKT/mTOR inhibitors (AZD-8055, MK-2206, apitolisib, dactolisib, PF-04691502, ZSTK474, and omipalisib), HSP90 inhibitors (BIIB021, luminespib, alvespimycin, and tanespimycin) as well as JAK inhibitors (ruxolitinib, momelotinib, tofacitinib, gandotinib). Group I samples were on the other hand relatively resistant to these classes of drugs. Surprisingly, despite the prevalence of the signature event of activation of TCL1 (the established AKT coactivator) in nearly all cases, only a subset of cases (group III) responded to PI3K/AKT/mTOR inhibitors. Strikingly, the grouping of selective responses to HDAC, JAK, PI3K/mTOR/Akt and HSP90 inhibitors did not link to the presence of JAK/STAT mutations, TCL1A translocations, or ATM deletion status.Ex vivo drug screening of primary T-PLL samples revealed heterogenous selective drug responses in specific drug classes (such as HDAC-, JAK-, HSP90- and PI3K/Akt/mTOR-inhibitors). Surprisingly, the drug response patterns did not correlate with known recurrent genetic aberrations suggesting that sequencing for recurrent genetic biomarkers cannot easily be turned into effective therapeutic strategies in T-PLL, and that further elucidation of the biological pathways driving T-PLL is needed. [Display omitted] DisclosuresKoschmieder:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences, Research Funding; Novartis Foundation: Research Funding; Baxalta/CTI: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Other: Travel reimbursement for scientific conferences ; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences. Wennerberg:Pfizer: Honoraria, Research Funding. Ding:Merek: Research Funding. Mustjoki:Pfizer: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, 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. .

Comprehensive Hybrid Capture-Based Genomic Profiling of T-Cell Leukemias and Lymphomas Reveals Targetable JAK1 and JAK3 Co-Existing Mutations

Background: T-cell leukemias and lymphomas (TL&Ls) are rare when compared with other hematologic malignancies, and pose a challenge for clinical management. TL&Ls are characteristically resistant to chemotherapeutic regimens used to treat other hematologic malignancies (PMID 17369126, 22649104, 25042790). Recently, activating JAK1 and JAK3 mutations have been described in a subset of mature TL&Ls including adult T-cell leukemias/lymphoma (ATLL), NK/T-cell lymphoma, and T-cell prolymphocytic leukemia (T-PLL). We used a comprehensive hybrid capture-based sequencing assay to identify genomic alterations that might suggest benefit from targeted therapy.Methods and Results.Using FoundationOne® and FoundationOne® Heme assays, we performed genomic profiling on 28 consecutive TL&Ls (leukemias, n=10; lymphomas, n=18) for all alterations in 370 or 405 cancer-related genes and select introns of 19 or 31 genes commonly rearranged in cancer, or both DNA and RNA were extracted and captured using custom baits for 405 genes and 265 frequently rearranged genes, respectively. The samples were sequenced to high uniform coverage, averaging >500X for DNA and >8M total pairs for RNA (PMID 24142049). JAK3 alterations were the most common at 25% (7/28) with an enrichment in T-cell derived leukemias (2 T-PLL, 3 T-ALL, and 1 cutaneous T-cell lymphoma (CTCL)). Other altered genes in the 28 cases included CDKN2A/B (21%), TP53 (18%), and TET2 , DNMT3A , and STAT3 (11% each). A complex rearrangement involving FCHO1 and JAK3 resulting from a ~70kb genomic segment deletion was also identified in a CTCL. The most frequently co-altered genes identified in the JAK3 mutated cases were relevant to the chromatin remodeling pathway. Focal STAT3, STAT5A, and STAT5B amplifications were identified in 2 cases (CTCL and ALCL) as well as one STAT3 D661Y in a T-cell large granular lymphocytic leukemia (T-LGL). All 3 cases with STAT gene alterations were mutually exclusive with JAK3 alterations. Additionally, 2 patients with T-PLL and T-ALL respectively were both found to harbor multiple co-existing JAK1 and JAK3mutations (2/28; 7%).One heavily pre-treated T-PLL patient harbored a major JAK1 V658F clone (40%) and a minor JAK3 M5111 clone (5%), and loss of heterozygosity for mutant ATM and TP53. Based on assay results, the patient was treated with ruxolitinib, a potent and specific inhibitor of JAK1 and JAK2. The patient had an impressive clinical response of duration of 4 months after which her T-PLL cell count rose markedly to pre-treatment levels and she developed worsening thrombocytopenia, indicating acquired resistance to therapy. The resistant T-PLL was assayed and was found to harbor an increased frequency of JAK3 M5111 (28%), decrease of JAK1 V658F (18%) and a new EZH2mutation. The patient’s disease progressed through another line of cytotoxic chemotherapy and she succumbed to her disease. Signal transduction analysis was conducted which showed in T-PLL, the JAK1 and JAK3 mutations were functionally significant as they induced constitutive phosphorylation of downstream STAT proteins. Importantly, these phosphorylations could be inhibited by JAK inhibitors. Collection of clinical outcome on additional cases is ongoing.Conclusion. In this study, we discovered a high frequency of JAK-STAT alterations in 28 T-cell neoplasms with 25% of cases harboring JAK3 missense mutations and two cases with concordant JAK1 and JAK3 mutations. In one case, treatment with the JAK1/2 inhibitor, ruxolitinib, resulted in an impressive clinical response. Our data argue for oncogene dependence upon the JAK-STAT pathway in diverse T-cell neoplasms and underscore the importance of genomic profiling by FoundationOne Heme® of rare and difficult to treat cases by revealing alterations that may be targeted by specific inhibitors, such as ruxolitinib, and may also be predictive of treatment resistance. DisclosuresOff Label Use: Ruxolitinib is FDA approved for myeloproliferative disorders.. Palma:Foundation Medicine, Inc.: Employment, Equity Ownership. Wang:Foundation Medicine Inc: Employment. Ali:Foundation Medicine, Inc.: Employment, Equity Ownership. Stephens:Foundation Medicine: Employment, Equity Ownership. Miller:Foundation Medicine, Inc.: Employment, Equity Ownership.

Recurrent mutation of JAK3 in T‐cell prolymphocytic leukemia

T-cell prolymphocytic leukemia (T-PLL) is an aggressive post-thymic T-cell malignancy characterized by the recurrent inv(14)(q11q32)/t(14;14)(q11;q32) or t(X;14)(q28;q11) leading to activation of either the TCL1 or MTCP1 gene, respectively. However, these primary genetic events are insufficient to drive leukemogenesis. Recently, activating mutations in JAK3 have been identified in other T-cell malignancies. Since JAK3 is essential for T-cell maturation, we analyzed a cohort of 32 T-PLL patients for mutational hot spots in the JAK3 gene using a step-wise screening approach. We identified 14 mutations in 11 of 32 patients (34%). The most frequently detected mutation in our cohort was M511I (seen in 57% of cases) previously described as an activating change in other T-cell malignancies. Three patients carried two mutations in JAK3. In two patients M511I and R657Q were simultaneously detected and in another patient V674F and V678L. In the latter case we could demonstrate that the mutations were on the same allele in cis. Protein modeling and homology analyses of mutations present in other members of the JAK family suggested that these mutations likely activate JAK3, possibly by disrupting the activation loop and the interface between N and C lobes, increasing the accessibility of the catalytic loop. In addition, four of the 21 patients lacking a JAK3 point mutation presented an aberrant karyotype involving the chromosomal band 19p13 harboring the JAK3 locus. The finding of recurrent activating JAK3 mutations in patients with T-PLL could enable the use of JAK3 inhibitors to treat patients with this unfavorable malignancy who otherwise have a very poor prognosis.

Clinical and laboratory features of T-cell prolymphocytic leukemia in China

To investigate the clinical and laboratory characteristics and survival of Chinese patients with T- cell prolymphocytic leukemia (T-PLL). Eleven patients with T-PLL admitted in our hospital from Jan 2006 to Oct 2012 were retrospectively analyzed. Of the 11 patients, nine were males and two females, with the median age of 56.0(19-69) years old. All the patients, except for three, presented with leukocytosis. The incidence of hyperleukocytosis (1/11) was less frequent than that in the British series (75%) (P=0.000). Lymphocyte counts in peripheral blood were increased in 9 of the 11 patients with the median absolute lymphocyte count (ALC) of 17.22(0.58-148.83)×10⁹/L. Superficial lymphadenopathy and splenomegaly were the most common physical signs. It was common that serum lactate dehydrogenase (LDH) and beta 2 microglobulin(β2-MG)were higher than normal level. All cases were positive for CD2/CD3/CD5/TCRαβ, negative for CD1a /HLA-DR and TdT, and most of them were strong positive for CD7 expression. By chromosome analyses, most cases. (9/10) have normal chromosome. This rate is significantly higher than that of the British and American series (3% and 25%, respectively) (P=0.000, P=0.001). The 14q11 abnormality and trisomy 8q, which are common among Western cases, were not observed in any of our cases. With a median follow-up of 23.0 months, three patients died. Two year progress free survival (PFS) and overall survival (OS) were 53.3% and 50%, respectively. There were 3 patients with PFS over a number of years, whether it should be considered as the T-chronic lymphocytic leukemia (T-CLL) is worthy of further studies. The common clinical manifestations of T-PLL patients were increased lymphocyte counts and lymphadenopathy as well as splenomegaly. And most cases have high level of blood LDH and β2- MG and normal chromosome karyotype.

Epigenetic Modulation of CD30 Gene Expression in Alemtuzumab Resistant T Cell Malignancy:Rationale and Efficacy of Brentuximab Vedotin.

Abstract 2454▪▪This icon denotes a clinically relevant abstract Background:T-Cell prolymphocytic leukemia (T-PLL) is an aggressive hematological disease mainly affecting adults. It is characterized by skin involvement and high lymphocyte counts. It affects mature T-cells mainly of the CD4+/CD8- phenotype. Although treatment with alemtuzumab, an anti-CD52 monoclonal antibody (mAb), induces complete remission (CR) in >50% of patients, it is short lived (median: 6 mos) and resistance is common. Epigenetic therapy with the histone deacetylase inhibitors (HDACi) vorinostat (SAHA) or romidepsin and the hypomethylating agent cladribine (2-CdA), can circumvent lack of response to alemtuzumab. Brentuximab Vedotin (SGN-35) is an antibody drug conjugate ADC) targeted against CD30 (TNFRSF8) and we have show here this ADC has activity in combination with epigenetic agents in T-PLL. Methods:Five T-PLL patients were studied. Diagnosis was made by hematopathology, flow cytometry and TCR rearrangement studies. Alemtuzumab (30 mg SQ d1,3,5) and 2-CdA (5mg/m2 IVd1–5), followed by alemtuzumab, 2-CdA and SAHA (400mg po daily X 5 days) or romidepsin (14mg/m2IV d 1,8,15) was given. After, epigenetic therapy with 2-CdA, SAHA, and romidepsin expression of CD30 in skin and blood was assayed where applicable.SGN-35 was administered at 1.8 mg/kg IV q 3 weeks. Peripheral blood was obtained and assayed under an IRB approved protocol. Genes of interest were analyzed by qRT-PCR. Chromatin Immunoprecipitation (ChIP) assays were used to study epigenetic changes at the CD30 promoter. Results:Of the 5 patients studied, three were alemtuzumab refractory with disease in blood and/or skin. With the addition of 2-CdA and SAHA or romidepsin, all three patients’ peripheral blood counts normalized and flow cytometry and PCR assays were negative for residual disease. One patient at the time of relapse developed skin lesions that were unresponsive to treatment with alemtuzumab, romedpsin, 2-CdA, or palatrexate. Lack of response to alemtuzumab in this patient was circumvented through induction of the cell surface receptor CD30, the target for the potent ADC, brentuximab vedotin. qRT-PCR assays on leukemic blood demonstrated that 4/5 of the T-PLL patients demonstrated increased CD30 mRNA following epigenetic therapy (mean: 14; range: 1.3–69 fold). These increases in CD30 mRNA expression correlated with increases in CD30 protein levels by Western blot analysis. CD30 and CD52 expression in the skin was confirmed by immunohistochemical analysis. In two patients, ChIP analysis of the CD30 promoter showed decreases in histone methylation markers and increases in Pol II binding at the coding sequence after treatment with epigenetic agents, consistent with increased CD30 expression. The patient with skin lesions had a dramatic response of his skin and normalization of his WBC count, with flow and PCR studies of skin and blood negative for residual disease after two cycles of SGN35.In contrast, a second patient with T-PLL who was refractory to up front alemtuzumab (IV) responded and achieved remission with the addition of 2-CdA, Her leukemic cells 2 days post treatment expressed CD30 mRNA in. At relapse 16 months later, her leukemic cells did not express CD30 and treatment with 2-CdA and romedepsin was unable to derepress CD30 gene expression. Correlative studies with ChiP assays differed from the previous patient. Further elaboration and follow up will be presented with both patients. Conclusions:Therapy with HDACi and 2-CdA epigenetically augments clinical activity of alemtuzumab and SGN-35, as evidenced by de-repression of TNFSFR8 (CD30) and increased response to treatment in one patient. While CD30 is a gene that can be epigenetically regulated in T cell malignancies, further complexities in epigenetic regulation of CD30 require additional investigation. Correlative studies are underway to elucidate the regulation of CD30 by epigenetic mechanisms. We present a rationale for the use of epigenetic drugs in combination with mAbs such as alemtuzumab and ADCs in T-PLL. A prospective multicenter clinical trial is warranted. Disclosures:Off Label Use: Brentuximab vedotin for PLL. Epner:Seattle Genetics: Speakers Bureau.

Epigenetically induced expression of CD30 in T-PLL: A rationale for the use of brentuximab vedotin.

6616 Background: T-cell prolymphocytic leukemia (T-PLL) is characterized by high lymphocyte counts, skin involvement and enlarged organs. Treatment with alemtuzumab (alem) induces complete remission (CR) in >50% of patients, duration is short (median: 6 mos). Therapy with the histone deacetylase inhibitors (HDACi) vorinostat (SAHA) or romidepsin (romi) and cladribine (2-CdA), may augment mAb activity. Methods: Five T-PLL patients were studied (Table). Diagnosis was made by pathology, flow cytometry and TCR studies. Alem (30 mg sq d1,3,5) and 2-CdA (5mg/m2 d1-5), followed by alem, 2-CdA and SAHA (400mg q d X 5 days) or romi (14mg/m2d 1,8,15) was given. One patient with skin lesions expressed CD30 and received brentuximab vedotin (SGN-35). Results: All patients achieved CR (mean: 10; median: 8 mos). Relapsed patients responded with repeat dosing. Genes assayed indicate involvement of the MAPK pathway (p53, DUSP2, TRIB1, C/EBP). mRNA expression of the membrane receptor CD30 (TNFRSF8) increased following therapy in 4/5 patients (mean: 14; Range: 1.3-69 fold). CD30 protein was confirmed by IHC and Western blotting. One patient with relapsed T-PLL involving skin had a dramatic clearing of lesions and blood counts after one dose of SGN35. Conclusions: Therapy with HDACi and 2-CdA overcomes resistance and augments clinical activity of alem and SGN-35, as evidenced by derepression of CD30 and response to treatment. We present a rationale for epigenetic resensitization to mAbs and their drug conjugates (ADC) in T-PLL. A prospective multicenter clinical trial is warranted. [Table: see text]

GeneXpert Essay for BCR-ABL Compared to RT-PCR for Clinical Management of CML

Abstract 4416Reverse transcriptase-polymerase chain reaction (RT-PCR) is considered the most sensitive method available for detecting low copy numbers of the BCR-ABL gene fusion. The difference between the BCR-ABL Ct (threshold cycle) and ABL Ct is expected to represent the ratio of the two populations of mRNAs and ultimately the percentage of neoplastic cells present.An international scale (IS) has been proposed for BCR-ABL RQ-PCR measurements. To enable testing centers to gain access to the IS, the Adelaide laboratory initiated a process to develop and validate laboratory-specific conversion factors (CFs) that can be used to convert local values to IS values. Recently, Cepheid introduced its GeneXpert-based assay for the identification of the BCR-ABL gene fusion in cells from blood samples. This system comprises a walkaway self-contained instrument that combines cartridge- based microfluidic sample preparation with reverse transcriptase-polymerase chain reaction-based fluorescent signal detection and BCR-ABL and ABL Ct determination. The CF provided by Cepheid for this procedure is 0.47. We tested this BCR-ABL fusion detection system, compared with a classical RT-PCR analysis as a clinical diagnostic tool for CML patients.We tested 19 patient peripheral blood samples by both methods. The negative control group included 3 blood samples, obtained from 3 patients with hematological disorders unrelated to BCR-ABL gene fusion: one with essential thrombocythemia, one with chronic myelomonocytic leukemia, one with T-cell-prolymphocytic leukemia. The remaining 16 clinical samples belonged to 12 patients with an established diagnosis of CML in chronic phase and to one patient with Philadelphia chromosome-positive acute lymphoblastic leukemia (p210).GeneXpert software reported as negative 2 samples with a numerical threshold limit calculated on Ct (i.e. BCR-ABL was not detected at a detection limit of 0.00046%); the 3rd negative control was indicated as BCR-ABL invalid: this result was probably due to the high proportion of ABL detected as consequence of hyperleukocytosis (WBC 559,6 × 109/L) in T-Cell prolymphocytic leukemia patients. Therefore, the performance of GeneXpert test in this series was: Sensitivity 100%, Specificity 0,66%, Positive Predictive Value 100%, Negative Predictive Value 100%.Among the series of 16 true positive samples GeneXpert revealed mean 8,67; mean standard deviation 27,55; median 1,48. Whereas in the same series RT-PCR results were: mean 37,09; mean standard deviation 59,33; median 3,37.In conclusion GeneXpert essay for BCR-ABL could be an important tool for diagnose and monitor CML but require complete clinical data for the correct interpretation of results. Disclosures:No relevant conflicts of interest to declare.

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

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

Haploinsufficiency of CDKN1B contributes to leukemogenesis in T-cell prolymphocytic leukemia

T-cell prolymphocytic leukemia (T-PLL) is consistently associated with inactivation of the ATM gene and chromosomal re-arrangements leading to an overexpression of MTCP1/TCL1 oncoproteins. These alterations are present at the earliest stage of malignant transformation, suggesting that additional events are required for overt malignancy. In this study, we pursued the investigation of the 12p13 deletion, previously shown to occur in approximately half of T-PLLs. We refined the minimal region of deletion by single nucleotide and microsatellite polymorphism allelotyping. We defined a 216-kb region containing the CDKN1B gene that encodes the cyclin-dependent kinase inhibitory protein p27(KIP1). Sequencing this gene in 47 T-PLL patient samples revealed a nonsense mutation in one case without 12p13 deletion. The absence of biallelic inactivation of CDKN1B for most patients suggested a haploinsufficiency mechanism for tumor suppression, which was investigated in an animal model of the disease. In a Cdkn1b(+/-) background, MTCP1 transgenics had consistent and multiple emergences of preleukemic clones not observed in control cohorts. The second Cdkn1b allele was maintained and expressed in these preleukemic clones. Altogether, these data strongly implicate CDKN1B haploinsufficiency in the pathogenesis of T-PLL.

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

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.

Small Cell Variant of T-Cell Prolymphocytic Leukemia with a 33 Immunophenotype

T-cell prolymphocytic leukemia (T-PLL) is a rare postthymic T-cell disorder. The disease is characterized by lymphadenopathy, splenomegaly, skin lesions, a high white blood cell count, and an aggressive clinical course. The small cell variant of T-PLL occurs in approximately 20% of patients. Most T-PLL patients express membrane T-cell receptors (TCR) of the alphabeta phenotype. The diagnosis of small cell variant T-PLL in a 56-year-old woman was based on the findings of abnormal lymphocytosis, immunophenotype, lymphadenopathy, and aggressive clinical behavior. Immunophenotype analysis showed that lymphocytes were positive for CD2, CD3, CD5, CD7, CD8, and TCR gammadelta antigens and negative for CD1a, CD4, and TCR alphabeta antigens. Southern blot analysis revealed rearrangement of the TCR Jgamma and Jdelta-1 genes. A cytogenetic study of peripheral blood showed a normal karyotype. T-PLL with a TCR gammadelta phenotype is very rare. This case was typical T-PLL except for the morphologically small cell type and the lack of the typical chromosome aberration. If cases accumulate in the future, the specific features of the gamma8 type of T-PLL will become clearer.

T-cell Prolymphocytic Leukemia: Update and Focus on Alemtuzumab (Campath-1H)

T-cell prolymphocytic leukemia (T-PLL) is a rare mature T-cell lymphoproliferative disorder. While the etiology of T-PLL is unknown, recent progress in unraveling the molecular basis of leukemogenesis has been substantial and may yield novel therapeutic targets. T-PLL is a distinct disease entity and the diagnosis can be readily made based on characteristic clinical features and laboratory findings. Prior to the appearance of pentostatin and alemtuzumab in clinical protocols, outcome for T-PLL patients was exceedingly poor with median survival measured in months. While the use of alemtuzumab in particular has improved remissions, the disease remains incurable. Future collaborative efforts investigating novel treatment approaches will be crucial to improving survival for patients with this disease.

Human T-Lymphotropic Virus Type I Provirus and T-Cell Prolymphocytic Leukemia

T-cell prolymphocytic leukemia (T-PLL) is a rare type of post-thymic T-cell neoplasm, the etiology of which is unknown. Patients with T-PLL have been found to be seronegative for human T-lymphotropic virus type-I (HTLV-I), and their leukemia cells do not retain mono-clonally integrated HTLV-I provirus. Recently, we have demonstrated the presence of defective HTLV-I provirus by polymerase chain reaction in the DNA extracted from peripheral blood cells or affected lymph nodes of T-PLL patients. Although there is a possibility, from our observation, that an alternative mechanism is operating in HTLV-associated leukemogenesis, it is still unknown whether and how HTLV-I can contribute to the leukemogenesis of T-PLL. In this review, we describe controversial issues and discuss a role of HTLV-I in the leukemogenesis of T-PLL.

Serum levels of parathyroid hormone-related protein are not elevated in patients with T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is a rare post-thymic T-cell neoplasm which shares most clinical features with adult T-cell leukemia (ATL). We measured serum level of C-terminal parathyroid hormone-related protein (C-PTHrP) in patients with T-PLL and ATL. Serum C-PTHrP levels of eight patients with T-PLL (median 36.8 pmol/l; range 27.0-50.2 pmol/l) did not differ from those of 30 human T-lymphotropic virus type I (HTLV-I)-seronegative blood donors (median 37.0 pmol/l; range 22.6-54.0 pmol/l). The C-PTHrP levels in ten ATL patients (median 69.6 pmol/l; range 42.5-899.4 pmol/l) were significantly higher than those in healthy controls (p<0.0001) or T-PLL patients (p=0.001). We suggest that the serum level of PTHrP can provide useful information for differentiating between T-PLL and ATL.

Abnormalities of the short arm of chromosome 12 in T cell prolymphocytic leukemia.

Abnormalities of the short arm of chromosome 12 are nonrandom events in T cell prolymphocytic leukemia (T-PLL). Fluorescence in situ hybridization (FISH) studies were performed in three patients with T-PLL and one patient with T cell peripheral lymphoma and rearrangement of 12p. Whereas the rearrangements of 12p were different in the four patients, a breakpoint centromeric to the ETV6 gene was present in the three T-PLL patients. In addition, loss of heterozygosity for a chromosomal segment telomeric to ETV6 with loss of the RAD52 locus was also shown by FISH studies. In contrast, the breakpoint was telomeric to ETV6 in the patient with peripheral lymphoma.

T-cell Prolymphocytic Leukemia.

BACKGROUND: T-cell prolymphocytic leukemia (T-PLL) is a post-thymic T-cell malignancy with aggressive clinical course. Although T-PLL has been referred to under different designations, it is a distinct clinico-biological entity and should be distinguished from other T-cell disorders. METHODS: The literature on T-PLL is reviewed. Experience on the clinical and laboratory features, differential diagnosis, and therapy on a large series of T-PLL patients is presented. RESULTS: T-PLL affects adults and occurs more frequently in men. The principal disease characteristics are organomegaly, skin lesions, and a raised lymphocyte count. Immunological markers show a post-thymic T-cell phenotype (TdT- CD2+ CD5+ CD3ñ) with strong expression of CD7. A CD4+ CD8- phenotype is seen in two thirds of cases. CD4 and CD8 are coexpressed in 25%, and a CD4- CD8+ phenotype is rare. Cytogenetics show a recurrent abnormality inv(14)(q11;q32) that is always associated to other aberrations (particularly iso8q or trisomy 8). Differential diagnosis between T-PLL and other T-cell malignancies is based on a constellation of clinical and laboratory features. Generally, T-PLL patients are refractory to the therapy used in lymphoid disorders. Median survival is short but is improving with the use of 2'-deoxycoformycin and the humanized monoclonal antibody, anti-CDw52 (Campath-1H). CONCLUSIONS: T-PLL is a distinct T-cell disorder with characteristic clinical and laboratory features and a poor prognosis. A precise diagnosis of this disease is important in determining patient management and treatment.