T-prolymphocytic Leukemia Patients Research Articles

The miR-141/200c-STAT4 Axis Contributes to Leukemogenesis by Enhancing Cell Proliferation in T-PLL.

T-prolymphocytic leukemia (T-PLL) is a rare and mature T-cell malignancy with characteristic chemotherapy-refractory behavior and a poor prognosis. Molecular concepts of disease development have been restricted to protein-coding genes. Recent global microRNA (miR) expression profiles revealed miR-141-3p and miR-200c-3p (miR-141/200c) as two of the highest differentially expressed miRs in T-PLL cells versus healthy donor-derived T cells. Furthermore, miR-141/200c expression separates T-PLL cases into two subgroups with high and low expression, respectively. Evaluating the potential pro-oncogenic function of miR-141/200c deregulation, we discovered accelerated proliferation and reduced stress-induced cell death induction upon stable miR-141/200c overexpression in mature T-cell leukemia/lymphoma lines. We further characterized a miR-141/200c-specific transcriptome involving the altered expression of genes associated with enhanced cell cycle transition, impaired DNA damage responses, and augmented survival signaling pathways. Among those genes, we identified STAT4 as a potential miR-141/200c target. Low STAT4 expression (in the absence of miR-141/200c upregulation) was associated with an immature phenotype of primary T-PLL cells as well as with a shortened overall survival of T-PLL patients. Overall, we demonstrate an aberrant miR-141/200c-STAT4 axis, showing for the first time the potential pathogenetic implications of a miR cluster, as well as of STAT4, in the leukemogenesis of this orphan disease.

Advances in the understanding and management of T-cell prolymphocytic leukemia.

T-prolymphocytic leukemia (T-PLL) is a rare T-cell neoplasm with an aggressive clinical course. Leukemic T-cells exhibit a post-thymic T-cell phenotype (Tdt−, CD1a−, CD5+, CD2+ and CD7+) and are generally CD4+/CD8−, but CD4+/CD8+ or CD8+/CD4− T-PLL have also been reported. The hallmark of T-PLL is the rearrangement of chromosome 14 involving genes for the subunits of the T-cell receptor (TCR) complex, leading to overexpression of the proto-oncogene TCL1. In addition, molecular analysis shows that T-PLL exhibits substantial mutational activation of the IL2RG-JAK1-JAK3-, STAT5B axis. T-PLL patients have a poor prognosis, due to a poor response to conventional chemotherapy. Monoclonal antibody therapy with antiCD52-alemtuzumab has considerably improved outcomes, but the responses to treatment are transient; hence, patients who achieve a response to therapy are considered for stem cell transplantation (SCT). This combined approach has extended the median survival to four years or more. Nevertheless, new approaches using well-tolerated therapies that target growth and survival signals are needed for most patients unable to receive intensive chemotherapy.

Impact of Alemtuzumab Therapy and Route of Administration in T-Prolymphocytic Leukemia: A Single-Center Experience.

We conducted a single-center retrospective analysis to determine the impact of the anti-CD52 monoclonal antibody alemtuzumab including route of administration compared to non-alemtuzumab-containing regimens in T-prolymphocytic leukemia (T-PLL). The study was a retrospective analysis of a consecutive cohort of adult patients diagnosed with T-PLL at Mayo Clinic Rochester from January 1, 1997, through September 30,2014. A total of 41 patients were diagnosed with T-PLL per the World Health Organization 2008 classification. The median age was 66 years, and 23 (56%) were male. After a median follow-up of 18 months (range, 0.4-66.1 months), 32 patients (78%) had died, with a median overall survival of 16.9 months. Approximately half the cohort was treated with alemtuzumab, almost exclusively after 2004. Median survival for patients receiving intravenous alemtuzumab-based therapy was 40.5 versus 10.3 months for all other therapies (P= .0004). A significant survival difference between intravenous versus subcutaneous alemtuzumab administration of 40.5 versus 13.7 months was noted (P= .0014). Only 4 (14%) of 28 patients aged< 70 years underwent hematopoietic stem cell transplantation, with a median survival after transplantation of 4 months. In this large series of T-PLL patients treated at a single tertiary-care center, we confirmed the prior observation of the superiority of intravenous alemtuzumab over other therapies. Hematopoietic stem cell transplantation was feasible in a minority of potentially eligible patients. Early transplant referral should be considered for all eligible patients.

Discovery of Novel Drug Sensitivities in T-Prolymphocytic Leukemia (T-PLL) By High-Throughput Ex Vivo Drug Testing and Genetic Profiling

IntroductionT-PLL is a mature post-thymic T-cell neoplasm with an aggressive clinical course (5-year overall survival 21%). Almost 75% of T-PLL cases harbor chromosome 14 translocations resulting in aberrant activation of the proto-oncogene TCL1A. Furthermore, in the majority of T-PLL cases the ATM gene is mutated or deleted, and recently it was reported that mutations in genes involved in the JAK-STAT pathway were found in 76% of T-PLL cases. Due to the rareness and aggressive nature of the disease, clinical trials are difficult to execute. By using a high-throughput ex vivo drug sensitivity and resistance testing (DSRT) platform covering 306 approved and investigational oncology drugs we systematically investigated the heterogeneity of drug responses in PLL-patients. As the impact of mutations on drug sensitivity is not well understood we aimed to identify relevant associations between the drug responses and genetic lesions in T-PLL patients.MethodsPrimary cells (MNCs) from seven T-PLL patients were obtained for drug screening. Samples were seeded in 384-well plates and 306 active substances were tested using a 10,000-fold concentration range resulting in a dose-response curve for each compound. Cell viability was measured after 72 h incubation and differential drug sensitivity scores (sDSS), representing leukemia-specific responses, were calculated by comparing patient samples to healthy donors. Hierarchical clustering of the drug responses was performed with Cluster 3.0 and Java Tree View. To assess the performance of the drug screening platform we also exchanged six samples with the German Cancer Research Center in Heidelberg for a comparison of results between two independent drug screening systems.To understand heterogeneous pathway dependencies, drug sensitivities were correlated with somatic genetic variants and recurrent chromosomal aberrations. Genetic characterization was performed by exome sequencing of tumor and matched healthy cells to profile known recurrent genetic variants (ATM, STAT5b, IL2RG, JAK1, JAK3) as well as CNVs (TCL1A translocations, ATM deletions, recurrent chromosomal aberrations).ResultsFour out of seven patient samples showed high sensitivity to small molecule BCL2 inhibitors navitoclax (IC50: 10-68nM) and ABT-199 (IC50: 14-45nM) and to HDAC inhibitors panobinostat and belinostat (IC50: 2-65nM). Intriguingly, the CDK inhibitor SNS-032 was effective in 6/7 patient samples (IC50: 7-95nM). SNS-032 inhibits Cdk2, Cdk7 and Cdk9, which control transcription of anti-apoptotic proteins including MCL1 and XIAP. As the AKT1/MTOR pathway is activated in many T-PLL patients due to expression of the TCL1A oncoprotein, it was interesting to observe that patient samples did not show any response to AKT inhibitors (MK-2206 and GDC-0068 IC50 values >1000 nM) nor to MTOR inhibitors (rapalogs temsirolimus and everolimus). Similarly, T-PLL cells were insensitive to JAK-inhibitors.Clustering of drug responses from T-PLL patients with primary AML and ALL patient samples revealed the drug response profiles to be specific for T-PLL patients (Figure). 6/7 patients clustered together while the only patient (PLL4) in our cohort with confirmed mutations in the JAK-STAT pathway genes STAT5b (P702S) and IL2RG (K315E) exhibited a non-sensitive response pattern when compared to other samples (Figure). Interestingly, exome sequencing did not reveal any JAK mutations in our PLL-cohort (n=5) nor additional STAT5b or IL2RG mutations in other patients except in this unresponsive patient.In the comparison between the platforms the correlation of the censored IC50 values from the 60 overlapping drugs was r=0.75. Similar fits of dose-response curves were seen for most drugs, although there were notable exceptions, which may be due to divergent culture conditions and day of read-out.ConclusionsEx vivo drug testing of primary patient cells has the potential to provide novel personalized drug candidates (such as BCL2, HDAC and CDK inhibitors) for T-PLL. The drug response pattern was T-PLL specific warranting further clinical testing. Drug screening, mutation analysis and RNA sequencing of additional patients is currently ongoing (n=20) to validate whether drug responses can be predicted based on the mutation profile or aberrant gene expression. [Display omitted] DisclosuresKallioniemi:Medisapiens: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Primary T-Prolymphocytic Leukemia (T-PLL) Cells Are Sensitive To BCL-2 and HDAC Inhibitors: Results From High-Throughput Ex Vivo Drug Testing

IntroductionT-PLL is a rare mature post-thymic T-cell neoplasm with an aggressive clinical course and median overall survival of less than one year. Almost 75% of T-PLL cases harbor chromosome 14 translocations involving the T-cell receptor A/D locus resulting in aberrant activation of the proto-oncogenes TCL1A or MTCP1. T-PLL patients are difficult to treat as the leukemic cells are often resistant to most available chemotherapeutic drugs. Due to the rareness and aggressive nature of the disease, large clinical trials are difficult to execute. We therefore aimed to discover novel potential therapeutic targets using a high-throughput ex vivo drug sensitivity and resistance testing (DSRT) platform covering 306 approved and investigational oncology drugs. MethodsPrimary T-PLL cells were available from two patients. The first patient had a double positive CD4+CD8+CD3+ Vβ.14+ T-cell phenotype (patient 1) and cells underwent DSRT twice during a 5-month time-period (no treatment during that time). The second patient had a CD4+CD3+ phenotype (patient 2) and the cells were assayed once by DSRT. Fresh blood mononuclear cells (MNCs) were separated by Ficoll centrifugation from the patient samples (over 85 % leukemic cells in the MNC fraction) and healthy controls. Cells were seeded in 384-well plates and 306 active substances were tested using a 10,000-fold concentration range resulting in a dose-response curve for each compound. Cell viability was measured after 72 h incubation and differential drug sensitivity scores (DSS), representing leukemia-specific responses, were calculated by comparing patient samples with those obtained from healthy donors. In addition, both exome and RNA sequencing was performed from T-PLL cells (patient 1). ResultsBoth patient samples showed high sensitivity to small molecule BCL2-inhibitors navitoclax (EC50 values 44nM and 10nM) and ABT-199 (EC50 23nM and 20nM) (Fig. 1 and 2). HDAC-inhibitors (quisinostat, belinostat and panobinostat) also showed high sensitivity in both patients in low nM concentrations (EC50 values 1-80nM). As AKT1/mTOR pathway is activated in most T-PLL patients due to the TCL1 oncoprotein, it was interesting to observe that neither of the patient samples showed any response to an AKT1 inhibitor (MK-2206 EC50 values >1000 nM) nor to mTOR inhibitors (temsirolimus and everolimus)(Fig. 1). Furthermore, T-PLL cells were resistant to corticosteroids such as prednisolone and methylprednisolone. [Display omitted] [Display omitted] To further elucidate the molecular mechanism behind the drug responses, exome and RNA sequencing was performed from T-PLL cells (patient 1). No deletion was found in the ATM gene, but instead a homozygous missense mutation K2413Q was detected. This particular mutation is in the region coding for the FAT domain and while it has not been described earlier in T-PLL, it is in a cancer mutation hotspot region of ATM, suggesting that it is inactivating. No mutations directly linked to the BCL2-family genes were observed. In the RNA sequencing analysis, TCL1A was overexpressed when compared to the healthy CD4+ cells as expected. Similarly, AKT1 was overexpressed. The expression of BCL-2 and BCL-XL did not differ from those observed in healthy CD4+ cells while pro-apoptotic BCL-2 family members BID and BAD were elevated compared to the healthy control. ConclusionsPrimary T-PLL cells showed sensitivity to BCL-2 and HDAC inhibitors in a systematic high-throughput ex vivo drug sensitivity testing across a range of clinical and investigational drugs. The BCL-2 inhibitor sensitivity was not related to increased BCL-2 expression or activating mutations in the BCL-2 family genes, and further studies are needed to clarify the mechanism of action. However, the results suggest that BCL-2 inhibitors could be a novel promising candidate drug for T-PLL-patients and warrant further clinical development in this group of patients. In contrast, inhibitors of AKT and mTOR, kinases known to be activated by TCL1, showed no efficacy ex vivo in this assay. Disclosures:Porkka:BMS: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau. Mustjoki:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau.

The human T-cell lymphotropic viruses types I/II are not involved in T prolymphocytic leukemia and large granular lymphocytic leukemia.

The possible involvement of the human T lymphotropic viruses type I and II (HTLV-I and -II) in lymphoproliferative disorders of mature T cells other than adult T cell leukemia/lymphoma (ATLL) has been controversial. Most studies have focused primarily on the cutaneous T cell lymphomas. However, skin involvement is a frequent feature of T prolymphocytic leukemia (T-PLL) and antibodies against HTLV-I and -II have been reported in individuals with large granular lymphocytic (LGL) leukemia. We examined 36 patients with T-PLL and 28 with LGL leukemia for evidence of HTLV-I and -II. Polymerase chain reaction (PCR) was performed on DNA from fresh peripheral blood mononuclear cells (PBMCs) and PBMCs after short-term culture (STC) using primers against all parts of the HTLV-I genome (LTR, gag, env, pol, tax/rex) and against HTLV-II pol and gag. Reverse transcriptase (RT) activity was measured on supernatants from STCs using a sensitive PCR-based technique. No HTLV-I or -II sequences were found by PCR nor RT activity detected in the 64 cases. Our findings do not provide evidence of HTLV-I or -II infection in T-PLL and LGL leukemia patients from an HTLV-I nonendemic area. Previous positive reports on these disorders may represent technical artefacts, detection of endogenous HTLV-like sequences or reflect patients from endemic areas and a variable etiology of T cell diseases.

The role of pentostatin in the treatment of T-cell malignancies: analysis of response rate in 145 patients according to disease subtype.

To assess the results of treatment with the purine analog 2'deoxycoformycin (pentostatin [DCF]) in patients with postthymic T-cell malignancies. One hundred forty-five patients with postthymic T-cell malignancies were given DCF intravenously at 4 mg/m2/wk for the first 4 weeks and then every 2 weeks until maximal response; the last 30 patients received weekly injections until maximal response. The overall response rate was 32% (complete responses [CRs] plus partial responses [PRs]), with marked variation according to diagnosis. The best responses occurred in patients with Sézary syndrome (62%) and T-prolymphocytic leukemia (T-PLL) (45%), with CRs in three of 16 Sézary syndrome and five of 55 T-PLL patients. In contrast, no responses (NRs) were documented in 13 patients with other types of cutaneous T-cell lymphoma, including five mycosis fungoides. Two of five patients with large granular lymphocyte (LGL) leukemia had a CR and two of four with Sézary cell leukaemia had a PR. A low response rate was observed in 27 patients with peripheral T-non-Hodgkin's lymphoma (T-NHL) (19%) and in 25 with adult T-cell leukemia/lymphoma (ATLL) (12%). The latter included two CRs and one PR. Toxicity was low and DCF was generally well tolerated. No significant differences were observed when results were analyzed according to previous treatment. Disease subtype was the most important factor to influence results. We conclude that DCF is effective as a single agent in T-PLL, Sézary syndrome, and LGL leukemia, but has low activity in other T-cell disorders.