bluuebunny/crossref_metadata_2025 huggingface dataset:
Familial Essential Thrombocythemia in a Child with Elevated Thrombopoietin Concentrations and Skeletal Anomalies.
Authors: Robins Edwin B. , Biguzzi Sergio
Published: April 2020

We describe a 2-year-old child with an incidental finding of persistent elevated platelet count. The platelet count at the time of referral was 900 × 109/L. His physical examination revealed bony abnormalities of the right upper extremity, and hand. (Photo1) X-Ray of the hand revealed amputation of the fingers of the right hand with rudimentary metacarpal bones, and evidence of shorter radius and ulna of the of the left forearm when compared to the right. The physical examination was otherwise normal with the exception of moderate splenomegaly. Further workup revealed normal iron stores, and no inflammatory process which could explain the thrombocytosis. Chromosomal breakage and rearrangements studies were normal and confirmed a normal male karyotype. A bone marrow aspirate and biopsy revealed megakaryocytic hyperplasia with many binucleated and trinucleated megakaryocytes. (Photo 2) The erythroid and myeloid series were well represented and of normal morphology. Flow cytometry for ALL specific and CMLspecific markers were normal. The platelet count reached 2,800 × 109/L, and serum thrombopoietin level was obtained, which returned 2119.8 pg/ml. The patient was placed on hydroxyurea and aspirin 81mg/day, and reduction in the platelet count occurred. After six months of hydroxyurea therapy the platelet count rose again to pretreatment levels. Hydroxyurea was discontinued and anagrelide started with moderate platelet reduction. Family members were also evaluated. The siblings all had normal platelet counts but the mother had an elevated platelet count 900 × 10 9/L. The bone marrow aspirate of the childs mother was also consistent with ET. The JAK2 kinase mutation was not found in the mother or the child. The childs mother was also found to have an elevated thrombopoietin level of 475.6 pg/ml and was started on hydroxyurea.

No connection has been made between our patients congenital anomalies and the thrombocytosis. To our knowledge there have been no other reports of such features in either adult or pediatric patients with ET. Because this childs mother was also subsequently diagnosed with ET, familial ET is a more likely diagnosis. The diagnosis of familial ET is further supported by the elevated TPO concentration in our patient. Sporadic cases of familial ET with elevated thrombopoietin concentrations have been reported. Although ET has been reported in patients as young a 6 weeks, our case is different than other reported pediatric cases of ET because of not only the elevated TPO but the associated skeletal anomalies of our patient. To date neither the child or the mother has suffered any thrombohemorrhagic events, which have been reported to occur at rates varying from 20% to 40%. The mother and child are being followed prospectfully with serial blood counts, serum TPO concentrations.

Figure Figure Figure Figure
A Phase II Study of Oral Interferon-Alpha (IFN-a) for Patients with Essential Thrombocythemia or Polycythemia Vera.
Authors: Quintas-Cardama Alfonso , Giles Francis , Cortes Jorge , Kantarjian Hagop , Verstovsek Srdan
Published: April 2020

Recombinant human IFN-α has been the mainstay of therapy for patients with Philadelphia chromosome-negative myeloproliferative disorders for many years. However, the response rates of IFN-α therapy have been frequently challenged by high dropout rates due to side effects and inconvenient subcutaneous dosing schedules. Accordingly, we design a phase II study to evaluate the efficacy and safety of natural human IFN-α administered by the oral mucosal route in patients (pts) with essential thrombocythemia (ET) or polycythemia vera (PV). Pts were eligible if they had failed (inability to normalize platelet count in ET, or to reduce the frequency of phlebotomy or splenomegaly by 50% in PV, after 6 months of therapy with hydroxyurea (HU) and anagrelide (AG)), were intolerant to HU and AG, or refused to receive standard cytoreductive therapy. HU or AG therapy had to be stopped at least 3 weeks prior to start of oral IFN-α. Prior exposure to IFN-α was not allowed. Oral IFN-α was administered at 150 IU three times daily as a lozenge. A total of 14 pts (8 PV, 6 ET) have been treated. Median age was 57 (range, 32 to 79), time from diagnosis to oral IFN-α therapy 2 months (range, 0 to 32), Hb 14.2 gm/dL (range, 11.2 to 15.4), WBC 9.75 ×109/L, (range, 5.6 to 17.3), platelets 812 ×109/L (range, 360 to 1389). Five patients were previously treated with HU, four with AG, and 3 with imatinib mesylate. Six pts with PV had received phlebotomies and 2 presented with marked splenomegaly. The JAK2 V617F mutation was detected in 10 of 11 evaluated pts and 2 of 14 pts (14%) had abnormal cytogenetics. All 14 pts are evaluable for response and toxicity. No responses have been observed among any of the 14 pts treated. Ten (71%) pts discontinued oral IFN-α therapy due to lack of response (n=9) or disease progression (n=1, elevation of platelets), and 4 are currently on therapy. Duration of therapy varied: 2.5 months = 2 pts, 3 months = 6 pts, 4 months = 4 pts, 6 months = 2 pts. Therapy with oral IFN-α was very well tolerated: grade 1 headache was noted in 2 pts, and grade 1 paresthesia in 1 pt. We conclude that oral IFN-α is very well tolerated but has no activity in the treatment of pts with ET or PV at the dose and schedule employed in this study.
WP1066 Inhibits Growth of Human Cells Carrying the JAK2 V617F Mutation.
Authors: Manshouri Taghi , Estrov Zeev , Quintas-Cardama Alfonso , Cortes Jorge , Giles Francis , Harris David , Priebe Waldemar , Kantarjian Hagop , Verstovsek Srdan
Published: April 2020

Myeloproliferative disorders (MPDs) are characterized by proliferation of one or more myeloid cell lineages in bone marrow and peripheral blood, with relatively preserved differentiation. Recent discovery of a dominant gain-of-function mutation in the Janus kinase 2 (JAK2) gene in patients with MPDs, involving the substitution of valine for phenylalanine at position 617 of the JAK2 protein (JAK2 V617F), represents the first acquired somatic mutation in hematopoietic stem cells described in these disorders. This discovery has opened new avenues for the development of targeted therapies for MPDs. WP1066 is a small molecule, a member of a novel class of anticancer agents whose development was based upon the backbone of AG490, a tyrphostin with activity against JAK2 V617F-expressing cell lines but limited in vivo activity. We investigated the inhibitory activity of the WP1066 against the JAK2 V617F-mutant expressing erythroid leukemia HEL cell line and peripheral blood mononuclear cells from patients with polycythemia vera (PV). WP1066 significantly inhibited the phosphorylation of JAK2 and downstream signal transduction proteins STAT3, STAT5, and ERK1/2 in a dose- and time-dependent manner. It induced a time- and dose-dependent antiproliferative and pro-apoptotic effects (activation of caspase 3, release of cytochrome c, and cleavage of PARP) in the JAK2 V617F-bearing HEL cell line in the low micromolar range. Pretreatment of cells with pan-caspase inhibitor Z-VAD abolished WP1066-induced apoptosis. The expression of apoptosis related proteins bcl-2, bax, and XIAP, however, was not changed. More important, WP1066 was effective in inhibiting cell growth in clonogenic assays of mononuclear cells harboring the JAK2 V617F mutation obtained from peripheral blood of patients with PV. We conclude that WP1066 is active both in vitro and ex vivo against cells carrying the JAK2 V617F mutation and represents a solid candidate for the treatment of JAK2 V617V-expressing MPDs.
MK-0457 Is a Novel Aurora Kinase and Janus Kinase 2 (JAK2) Inhibitor with Activity in Transformed JAK2-Positive Myeloproliferative Disease (MPD).
Authors: Giles Francis , Bergstrom Donald A. , Garcia-Manero Guillermo , Kornblau Steven , Andreeff Michael , Kantarjian Hagop , Jones Dan , Freedman Steven J. , Verstovsek Srdan
Published: April 2020

MK-0457 (VX-680) was developed as a small-molecule inhibitor of Aurora kinases A, B, and C (Ki,app = 0.66–18 nM). Recent screening for additional kinase activity shows that MK-0457 inhibits JAK-2 with an IC50 of 123 nM for wildtype JAK2 and 295 nM for the JAK2 V617F activating mutation. Based on these in vitro findings, eleven patients with documented JAK2-mutated myeloproliferative disorders (MPDs), including 2 patients with prior MPD progressing to AML, were treated with MK-0457 at either 20, 24 or 28 mg/m2/hr administered by a continuous 5-day intravenous infusion every 3 weeks. The age range was 37– 83 (median 64); 5 (45%) patients were male and performance status was 0 or 1 in 10 (91%) patients. Five (45%) patients were previously splenectomized and 2 (33%) of the non-splenectomized patients had splenomegaly present at baseline. Two (18%) patients had diploid cytogenetics, eight (73%) were pseudodiploid with varying abnormalities and one (9%) had a -17 abnormality. One patient with hepatosplenomegaly at enrollment experienced complete resolution of hepatomegaly and >50% reduction in splenomegaly with a single cycle of therapy. Of the 10 (91%) patients starting therapy with a normal or increased absolute neutrophil count (ANC), all experienced grade 3 or 4 neutropenia during the study period. Transient reductions in platelet counts of patients with normal or elevated baseline platelets were also seen. Six of 7 (86%) patients with serial JAK2 testing had gradual, yet steady reductions in percentage of PCR product with the V617F mutation. MK-0457 is worthy of further exploration at lower doses (20mg/m2/hr) which are more suitable for chronic administration in patients with less aggressive disease, in contrast to patients with AML transformation of JAK2-mutated MPD in which higher, more myelosuppressive doses may be warranted.
Outcomes after HLA-Matched Sibling Transplants or Chemotherapy in Children with Acute Lymphoblastic Leukemia in a Second Remission after an Isolated Central Nervous System Relapse.
Authors: Eapen Mary , Zhang Mei-Jie , Raetz Elizabeth , Devidas Meenakshi , Carroll William L. , Ritchey Arthur K. , Barredo Julio C. , Godder Kamar , Grupp Stephan A. , Lewis Victor A. , Davies Stella , Camitta Bruce
Published: April 2020

The optimal treatment for children with acute lymphoblastic leukemia (ALL) in second remission after an isolated central nervous system relapse is unknown. To address this question, we compared outcomes in 149 patients enrolled on Pediatric Oncology Group chemotherapy trials 9061 (n=79) and 9412 (n=70) and HLA-matched sibling transplant recipients (n=60) reported to the Center for International Blood and Marrow Transplant Research. Patients received treatment between 1990 and 2000. Median follow-up was 8 years and 9 years after chemotherapy and transplantation, respectively. Groups were similar with respect to sex and leukocyte count at diagnosis. Chemotherapy recipients were younger at diagnosis (5 vs. 7 years) and more likely to have had a longer duration of first remission (duration of first remission ≥18 months: 70% vs. 48%). All transplant recipients received bone marrow grafts and 83% received a total body irradiation containing preparatory regimen. The median time to transplantation after achieving a second remission was 2.5 months (range, <1 – 8). To adjust for time-to transplant bias, we used left-truncated Cox regression models to examine treatment outcomes. Risks of a subsequent leukemia relapse were similar in both treatment groups. As expected, risks of a subsequent leukemia relapse were significantly higher in older patients (11–17 years; relative risk [RR] 2.63, p=0.004) and those with a short duration of first remission (<18 months; RR 4.22, p<0.001) regardless of type of treatment. Relative to chemotherapy recipients, risks of treatment-related mortality (RR 4.29, p=0.001), treatment failure (RR 2.37, p=0.003) and overall mortality (RR 2.68, p=0.002) were significantly higher within the first 2 years after achieving a second remission in recipients of HLA-matched sibling transplants. Among transplant recipients who survived the first two years, subsequent mortality and treatment failure risks did not differ by treatment group. In both treatment groups, recurrent leukemia was the commonest cause of death (60% and 56% after chemotherapy and transplantation, respectively). The 8-year probabilities of leukemia-free survival (adjusted for age and duration of first remission) were 66% and 58% after chemotherapy and transplantation, respectively. These data support use of chemotherapy alone for patients with ALL and isolated central nervous system relapse who achieve a second remission regardless of duration of first remission.
Diagnostic Usefulness of JAK2 Mutation in Manistest or Latent Polycythemia Vera and Secondary Polycythemia.
Authors: Bang Soo-Mee , Nam Eun Mi , Ahn Jeong Yeal , Lee Jae-Hoon , Kim Bong-Seog , Nam Seung-Hyun , Chi Hyun-Sook , Cho Young Uk , Kim Hyo Jung , Zang Dae Young , Park Moo-Rim , Shim Hyeok , Lee Jong Seok , Won Jong-Ho , Oh Doyeun ,   Korean MPD Working Party
Published: April 2020

Backgrounds: The discovery of Janus kinase 2 (JAK2) mutation make it easy to diagnose polycythemia vera (PV) in which the prevalence of this mutation is reported up to 96%. The latent PV is defined in patients who show the lower hemoglobin than WHO criteria at the time of diagnosis without any cause of secondary erythrocytosis; 17~18.5g/dL in male and 15 ~16.5g/dL in female. We investigated the mutational status of JAK2 in polycythemia vera either of latent or manifest type and secondary polycythemia.

Methods : We reviewed the clinical records of 182 patients from 8 centers, who underwent bone marrow (BM) examination with a suspicion of non-BCR/ABL myeloproliferative diseases (nMPD). JAK2 mutation was examined by allele-specific PCR in 110 patients.

Results : The positive rate of JAK2 mutation was 69% of 48 patients with PV, 54% in 41 patients with essential thrombocythemia (ET), 25% of 8 patients with chronic idiopathic myelofibrosis and 100% of 1 patient with unclassifiable MPD. JAK2 mutation was not detected in 7 patients with secondary polycythemia and 5 with reactive thrombocytosis. The number of patients with latent PV was eleven (23%) among the 48 PV patients. Nine of these 11 patients were positive for JAK2 mutation. Finally, sensitivity of JAK2 mutation was 69% (33/48) in all PV, 82% (9/11) in latent PV, and 65% (24/37) in manifest PV. The specificity of JAK2 mutation was 100% for secondary polycythemia. Among the 41 patients with ET, four patient had high hemoglobin met the latent PV criteria. Only one patient among them showed JAK2 mutation. The presence of a JAK2 mutation was closely correlated with the diagnosis of PV (p=0.006), leukocytosis (p=0.000), increased megakaryocytes (p=0.013), and decreased serum EPO (p=0.034) in 98 nMPD patients. In these patients, vascular events before or after the diagnosis and survival were not affected according to JAK2 status during the median follow-up of 25 months (range; 0~211).

Conclusions : The JAK2 mutation help diagnose PV with higher sensitivity in latent PV than manifest PV. This finding can be adopted in the diagnosis of PV in newly visited polycythemic patient whose hemoglobin were under the criteria and condition could be excluded for secondary erythrocytosis. Therefore, JAK2 mutation should be incorporated into the work-up of nMPD including PV for accurate diagnosis. In the meeting, we can present the additional JAK2 data of untested 72 patients.
A Phase II Study of Nilotinib, a Novel Tyrosine Kinase Inhibitor Administered to Patients with Hypereosiniphilic Syndrome (HES).
Authors: le Coutre Philipp , Hochhaus Andreas , Heim Dominik , Rafferty Teresa , Weitzman Aaron , Zheng Ming , Baskaynak Gökben , Verhoef Gregor
Published: April 2020

Idiopathic hypereosinophilic syndrome (HES) is a rare myeloproliferative disorder characterized by sustained overproduction of eosinophils and organ involvement that can lead to chronic eosinophilic leukemia. HES is a clonal disorder characterized in some patients by constitutive activation of FIP1L1-PDGFRA. There is currently no approved therapy for HES. Nilotinib is a novel aminopyrimidine which potently inhibits Bcr-Abl, as well as the PDGF-R and c-Kit kinases. This Phase II study was designed to evaluate the safety and efficacy of nilotinib administered at an oral dose of 400 mg twice daily to patients with specific HES disease related criteria and with a clinical indication for treatment. Preliminary data are available for 11 patients. The median age is 63 (range 25–77) years. There were 10 males and 1 female. Extramedullary involvement was present in 6 (55%) patients at baseline. Treatment is ongoing for 4 (36%) patients and 7 (64%) have discontinued; 2 (18%) for adverse events; 3 (27%) for disease progression, and 1 (9%) for an administrative reason. There was one patient death due to neutropenic sepsis and endocarditis. Overall, 5 (45%) patients had investigator documented stable disease, and 1 (9%) patient a 51 year old male had a CR. Grade 3 or 4 adverse events included one patient each with rash, decreased hemoglobin, myalgia, arthralgia, pruritis, and diabetes mellitus. In summary, these data suggest that nilotinib has clinical activity and an acceptable safety and tolerability profile in patients with HES.
PTK787/ZK 222584 (PTK/ZK), an Oral Tyrosine Kinase Inhibitor, Has Activity in Patients with Agnogenic Myeloid Metaplasia (AMM).
Authors: Giles Francis J. , Carroll Michael , List Alan , Ferrajoli Alessandra , Thomas Deborah , Cortes Jorge , Feldman Eric , Roboz Gail
Published: April 2020

Increased angiogenesis and vascular endothelial growth factor (VEGF) levels are prognostic in AMM. Agents with anti-VEGF activity including thalidomide (Thomas. Cancer 2006) and lenalidomide (Tefferi. Blood 2006) have activity in AMM. PTK/ZK, a novel oral aminophthalazine, is a small molecule inhibitor of VEGFR-2, PDGFR, c-kit and c-fms. Twenty-nine patients with angogenic myeloid metaplasia (AMM) were enrolled between June 2002 and November 2003 on a phase I/II study of PTK/ZK, administered orally on a continuous twice daily (BID) dosing schedule as follows: two (7%) patients received 750 mg BID and 27 (93%) patients received 500 mg BID. The age range was 42 to 78 (median 62), seventeen (59%) patients were male, and Karnofsky performance status was ≥ 70% in all (100%) patients. In twelve (41%) previously treated patients, the median number of prior regimens was 2.5 (range 1–6). Nine (31%) of the 29 patients had abnormal cytogenetics at the time of enrollment. The most commonly reported potentially PTK/ZK-related adverse events (AEs) were nausea, vomiting, dizziness, fatigue, thrombocytopenia and anorexia. One or more of these AEs occurred in > 15% of patients. Five (17 %) patients achieved clinical improvement (CI), as determined by the International Working Group (IWG) consensus criteria for treatment response in myelofibrosis with myeloid metaplasia. Four CIs had improvement in splenomegaly and the fifth had improvement in hemoglobin. As had similarily been seen in patients with AML treated with PTK/ZK monotherapy, PTK/ZK exposure, both AUC and Cmin, showed decreases after multiple doses (Roboz.Leukemia.2006). PTK/ZK is an active, well tolerated agent in the treatment of AMM and warrants further investigation within combination regimens.
Atiprimod Inhibits the Growth of JAK2 Tyrosine Kinase Over-Expressing Cells “In Vitro” and “Ex Vivo”.
Authors: Nussenzveig Roberto , Manshouri Taghi , Kantarjian Hagop , Giles Francis , Cortes Jorge , Gaikwad Amos , Priebe Waldemar , Estrov Zeev , Verstovsek Srdan
Published: April 2020

Background:

A novel somatic single point non-synonymous mutation (1849 G to T) in the tyrosine kinase JAK2, rendering the enzyme constitutively active, has been reported in classical myeloproliferative disorders (MPD), specifically, in greater than 90% of patients with polycythemia vera (PV), and 50% of patients with essential thrombocythemia (ET) and myelofibrosis with myeloid metaplasia (MMM). Consequently, JAK2 tyrosine kinase has become a significant target for the development of specific inhibitors of its activity with possible therapeutic benefits for patients with classical MPD. We report here the results of a study using atiprimod, an anti-inflammatory and anti-cancer compound belonging to the azaspirane class of cationic amphiphilic drugs in JAK2 over-expressing cell lines and patient samples. In contrast to TKI inhibitors, this drug is causing downregulation of JAK2 protein by facilitating its degradation, possibly through the ubiquitin-proteosome pathway (Faderl, S. et al, Leukemia Research 2006 in press).

Methods and Results:

We have examined the growth inhibitory effect of atiprimod against two mouse FDCP cell lines transfected with erythropoietin receptor (Epo-R) and, either wild-type JAK2 (JAK2WT) or mutant JAK2 (JAK2V617F). Atiprimod growth inhibition, measured using 72 hours MTS assay, was significant against both mutant JAK2V617F cells (IC50 0.42μM) and wild-type JAK2WT cells (IC50 0.65μM). Atiprimod was found to induce apoptosis in cell lines as evidenced by an increase in the mitochondrial membrane potential and augmented annexin V positivity by flow cytometry. Mononuclear cells, isolated from peripheral blood of PV patients (JAK2V617F positive), were cultured in cytokine supplemented serum free media for expansion of erythroid progenitors. Expansion spanned a three week, three step sequential treatment of the erythroid progenitor cells with specific combinations of cytokines. Finally, treatment of expanded erythroid progenitor cells with atiprimod for 24 and 48 hours resulted in increased annexin V positive cells, in both, a dose and time dependent manner.

Conclusion:

Our preliminary pre-clinical results suggest that atiprimod might be a beneficial medication for MPD patients carrying the JAK2V617F mutation. Atiprimod is currently being tested in a phase I clinical study.
Study of the Expression of Angiogenic Factors and Dopaminergic Receptors: Correlation with ZAP70, CD38 and the Mutational State in Chronic Lymphocytic Leukaemia (CLL).
Authors: Eroles Pilar , Benet Isabel , Marugan Isabel , Teruel Ana Belen , Solano Carlos , Terol Maria Jose
Published: April 2020

The angiogenesis is a well known process implicate in the progression of CLL. In this disease has been described an increase of angiogenic and pro-angiogenic factors in serum and angiogenic receptors in B cells, mainly in advances stages. Recently had been known that the agonist of the dopaminergic receptor D2 can inhibit the tumour growth and a possible mechanism mediator is the internalization of VEGFR-2. The aim of this work had been to analyze the expression of the dopaminergic receptors (D1, D2, D3, D4 and D5), the angiogenic receptors and its correlation with the main biological factor implicated in the illness (ZAP-70, CD38 and the mutational status of IgVH/BCL-6). We had determinate the levels of the receptors VEGFR-1, VEGFR-2, VEGFR-3 and c-kit, the angiogenic factor bFGF and the dopaminergic receptors D1, D2, D3, D4 and D5 in B lymphocytes from peripheral blood of 29 patients with CLL in A stage (using positive selection and quantitative PCR), and we had analyzed the possible correlation with the values of CD38 and ZAP70 and the mutational status of IgVH/BCL-6 (using flow cytometry and direct sequenciation).

Results: The lymphocytes B-CLL show a significant increment of the expression of the receptors VEGFR-1, VEGFR-2 and c-kit versus normal lymphocytes B. The patients with positive ZAP70 (> 20%) show a higher expression of all the factors studied except D1. The difference is statistically significant for D5, VEGFR-2 and c-kit (3 ×), D2 and bFGF (2 ×). The patients with positive CD38 show an increment in the expression of D2 (1,4x) and bFGF (1,9x) versus patients with negative CD38. The patients with both ZAP70 and CD38 positive present a higher expression of D2 and bFGF (2,2x and 1,9x) versus negatives ZAP70 and CD38, which show higher expression of D1 (4,8x). We had not found significant correlations between the studied factor and the mutational status of IgVH and BCL-6, only D1 shows a higher expression (3,6x) in patients with mutations IgVH versus unmutated independently of the BCL-6 status.

Conclusion: The lymphocytes B-CLL in stage A of Binet show high levels of expression of the angiogenic receptors VEGFR-1, VEGFR-2 and c-kit, the dopaminergic receptors D1 and D2, and the angiogenic factor bFGF that are significant higher that in normal lymphocytes B. The biological factor of bad prognostic (ZAP-70 and CD38) in patients in the initial A stage are associated with a higher expression of the receptors VEGF-R, c-kit, and D2 and the factor bFGF. Oppositely, negatives ZAP-70 and CD38 have correlation with the elevated expression of the receptor D1. This patron of differential expression can contribute to the tumoral progression of the patients in the initial stage of the illness.
Autoimmune Thrombocytopenic Purpura Complicating Chronic Lymphocytic Leukemia: Analysis of 60 Patients.
Authors: Visco Carlo , Stasi Roberto , Ruggeri Marco , Ambrosetti Achille , Fortuna Stefania , Frattini Francesco , Giaretta Ilaria , Zanotti Roberta , Madeo Domenico , Pizzolo Giovanni , Rodeghiero Francesco
Published: April 2020

Autoimmune thrombocytopenic purpura (AITP) represents the autoimmune condition most frequently associated with chronic lymphocytic leukemia (CLL) after autoimmune haemolytic anemia. However, the main characteristics and outcome of AITP in the course of CLL, as well as the impact of this complication in the natural history of the tumor remain unknown.

We identified 60 consecutive patients with CLL who developed AITP, representing 3,5% of CLL patients diagnosed in the three participating centers between 1995 and 2004. To be included in this study patients had to experience at least one episode of AITP, which was defined as the occurrence of acute and severe thrombocytopenia in the presence of normal or augmented number of megakariocytes in the bone marrow, without extensive lymphoid marrow infiltration, splenomegaly or recent cytotoxic treatment. A complete response (CR) to AITP treatment was defined by a platelet count > or = 150×10(9)/L, and a partial response (PR) by a platelet count > 50×10(9)/L or by an increase of at least twofold the initial level. Remaining patients were considered as no responders (NR).

Median age of our 60 patients was 65 years (range 48–83) and 40 were males. At CLL presentation RAI stage was 0 to 2 in 88%, time to CLL treatment was 13,8 months (range 0–120), while first line treatment for CLL consisted of Chlorambucile alone (Chl) in 73% of patients with 18% of patients that received no treatment for their malignancy. Median overall survival was 57 months. AITP occurred concomitantly to CLL diagnosis in 13 patients (22%), while median time to AITP for remaining 47 patients was 30 months (range 2–147). The median platelet count at AITP diagnosis was 23 × 10(9)/L(range, 1–81). Twenty-five patients (42%) presented with moderate bleeding signs at AITP diagnosis, while 4 patients (7%) experienced severe hemorrhagic episodes, requiring hospitalization and blood transfusions. Fifty-two of the 60 patients (87%) received at least one treatment for AITP: 32 patients received i.v.Ig alone or in combination with steroids, leading to a short-term NR in 66% (CR 19%, PR 15%); nine patients underwent splenectomy and 7 (78%) experienced a durable CR; patients who were treated with chemotherapy (Chl, COP, CVP) +/− steroids had at least a PR in 73% of cases. With a median follow-up from AITP onset of 35 months, 17 of the 52 treated patients are still NR (33%) and 13 of them are on treatment.

In our series of patients with CLL and AITP we observed an unexpectedly short survival regardless of a large prevalence of low RAI stages at diagnosis. Treatment of AITP with i.v. Ig +/− steroids leaded to a low rate of short-term responses, while splenectomy and chemotherapy seemed sufficiently adequate therapeutic approaches.
Involvement of the Tis11b/Berg36 Family in the Regulation of B-CLL Surivival and Differentiation.
Authors: Baou Maria , Murphy John , Yong Kwee L. , Carr Robert , Jewell Andrew P.
Published: April 2020

Tis11b/Berg36 is a member of a family of proteins involved in post-transcriptional gene regulation. Members of this family bind to AU rich elements in certain mRNA species and increase mRNA stability. mRNA that contain AU rich elements and can be regulated in this way include molecules involved in the regulation of apoptosis eg bcl-2 and several cytokines eg TNF, IL-2. We have recently shown that Tis11b/Berg36 is involved in the regulation of apoptosis following Rituximab treatment of CLL cells suggesting a pro-apoptotic role of this gene in CLL cells. Thus B-CLL were stimulated with IL-4, anti-CD40, anti-CD40+IL-4 and PMA. It was found that IL-4, CD40 and their combination significantly inhibited spontaneous apoptosis while PMA was able to inhibit spontaneous apoptosis in some but not all patients tested and the overall effect did not reached statistical significance. Unexpectedly Tis11b/Berg36 mRNA remained unchanged following IL-4 treatment, but expression was induced following anti-CD40 or PMA treatment. Because it was found that regulation of Tis11b/Berg36 mRNA following the latter two treatments was under the control of NF-κB pathway and not p38 (as found for Rituximab treatment) it was hypothesized that this gene may be involved in the regulation of cell cycle or differentiation of B-CLL cells. Indeed it was found that stimulation of B-CLL cells with either PMA or anti-CD40 resulted in increase in sIgM as a marker of B-CLL differentiation. CLL cells were also found to express high basal levels of two other members of this family, Tis11 and Tis11d. From these stimuli, IL-4 or anti-CD40 were found to downregulate the basal expression of Tis11 mRNA. These data suggest that members of the Tis11b/Berg36 family are involved in the regulation of apoptosis and differentiation in B-CLL cells.
Influence of MDM2 Single Nucleotide Polymorphism SNP309 on Disease Onset and Course in CLL.
Authors: Zenz Thorsten , Häbe Sonja , Bühler Andreas , Kröber Alexander , Denzel Tina , Döhner Hartmut , Stilgenbauer Stephan
Published: April 2020

Background: A SNP in the MDM2 promotor region (SNP309) has been shown to directly influence MDM2 transcript and protein levels with the subsequent attenuation of the p53 pathway. In addition, the GG and GT genotype have been linked to earlier age of onset in patients with different cancers. Because of the well documented prognostic role of p53 in patients with CLL and the potential involvement of MDM2 particularly in patients with trisomy 12, we studied the MDM2 SNP309 in patients with CLL.

Methods: We developed a Denaturing High-Performance Liquid Chromatography (DHPLC) assay as a high-throughput approach to efficiently type this polymorphism.

Results: We analysed 627 CLL patients and found a similar genotype distribution as described for a large German control population (CLL: GG 12,75%; GT 48.8%; TT 38.4% vs. control: GG 14.4%; GT 44.1%; TT 41.8%). There was an equal genotype distribution among women and men. We compared the median age at diagnosis of the different genotype groups in CLL patients and did not find differences (female: GG 62.3; GT 59.8 TT 60; male: GG 58; GT 56.2; TT 58) (figure 1). However as a hormone dependent difference for pre- and postmenopausal female patients with cancer has been described, we separated the patients according to age and found a overrepresentation of the GG phenotype in young female CLL patients (<51 years) (25% GG vs 11% GG; p=0.04). In contrast, for older, female patients with the GG genotype there was a trend towards delayed onset of CLL (Figure1).

The overall survival among a subgroup of 483 patients was not influenced by the presence of a particular genotype (Median survival (months): GG 132.6; GT 134.1; TT 112.8). The mRNA expression levels of MDM2 and p53 were analysed by quantitative RT-PCR and correlated with the different genotypes.

Conclusion: The approach described can be used to rapidly type the MDM2-SNP309. Based on this study a role for the MDM2 polymorphism on disease predisposition of CLL is unlikely. The role of hormone dependent differences according to genotype in female patients warrants further investigation.

Figure 1 Figure 1.
Clinical Significance of Free Circulating CD3 and CD5 in Patients with Chronic Lymphocytic Leukemia.
Authors: Hodge Menna , O’Brien Susan , Abdool Adam , Keating Michael , Jilani Iman , Kantarjian Hagop , Ferrajoli Alessandra , Estrov Zeev , Giles Francis , Albitar Maher
Published: April 2020

CD5, a transmembrane protein expressed in T-cells, few B-cells, and chronic lymphocytic leukemia (CLL) B-cells, is the ligand for CD72 and may play a role in B-cell-T-cell communication. CD5 is part of the T-cell receptor (TCR)-CD3 complex in T-cells as well as the B-cell receptor (BCR) complex and serves as substrate for induction of tyrosine kinase activity. Since leukemic cells have high turnover and pour their protein, RNA, and DNA into the circulation, we speculated that free circulating CD3 (cCD3) and CD5 (cCD5) could be detected in the plasma of patients with CLL. We have developed a bead-based sandwich immunoassay to measure cCD3 and cCD5 in the plasma. Using this assay, we assessed the value of cCD5 measurement, alone and after normalization to cCD3 levels, as a tumor marker in CLL. Plasma levels of cCD3 and cCD5 were measured in 85 patients with CLL and 51 normal control subjects. cCD3 and cCD5 levels were significantly higher in patients with CLL (median, 7,465 and 55,806 U/μl, respectively) than in normal control subjects (median, 830 and 1,671 U/μl, respectively). Patients with CLL had significantly higher cCD5:cCD3 ratios (median, 5.28; range, 0–161) than did normal controls (median, 1.70; range, 0–8.06) (P <0.0001). Levels of cCD5, but not cCD3, correlated positively with WBC count, β2-microglobulin level, splenomegaly, and Rai stage (all P <0.01). The cCD5:cCD3 ratio also correlated with Rai stage (P = 0.04) and β2-microglobulin level (P = 0.03). cCD5 levels and the cCD5:cCD3 ratio both correlated with survival (P = 0.03). These findings confirm that free circulating surface markers can be detected in the circulation of patients with CLL, most likely reflect the tumor load, and can be used as tumor markers. The biological and therapeutic relevance of these free circulating proteins should be considered in pharmacokinetic and pharmacodynamic studies.
Valproic Acid Induces Apoptosis in Chronic Lymphocytic Leukemia Cells through Activation of the Death Receptor Pathway and Potentiates TRAIL Response.
Authors: Lagneaux Laurence , Gillet Nicolas , Delforge Alain , Dejeneffe Marielle , Stamatopoulos Basile , Massy Martine , Meuleman Nathalie , Kentos Alain , Willems Luc , Bron Dominique
Published: April 2020

Background: The anti-leukemic in vitro activity of valproic acid (VPA), a commonly used antiepileptic agent, was tested on lymphocytes derived from 40 patients with chronic lymphocytic leukemia (CLL) (Binet stage A=34, B=3, C=3). These patients had not been previously treated or remained untreated for the previous 6 months. Combined analysis of ZAP-70, CD38 and IgVH mutational status was performed for each patient.

Methods: Mononuclear cells were incubated with VPA at 1, 5 and 10 mM for 24 hours. Cell viability was assessed by trypan blue exclusion assay, apoptosis by annexin V/propidium iodide(PI) labelling and PI staining after cell permeabilisation. Caspase activation was studied by flow cytometry analysis after cell treatment with selective caspase inhibitors.

Results: Exposure of CLL cells to VPA resulted in dose-dependent cytotoxicity and apoptosis in all CLL patients tested. VPA-treatment induced apoptotic changes in CLL cells including phosphatidylserine (PS) externalisation and DNA fragmentation. The mean apoptotic rate was similar between IgVH mutated and unmutated patients or ZAP-70+/ZAP-70- cases. VPA induced apoptosis by the extrinsic pathway involving engagement of the caspase-8 dependent cascade. Although CLL cells are commonly resistant to death receptor-induced apoptosis, VPA increased significantly the sensitivity of leukemic cells to TRAIL (tumor necrosis factor α-related apoptosis-inducing ligand). In addition, VPA overcomed the prosurvival effects of bone marrow stromal cells.

Conclusions: These data indicate that VPA, at the pharmacological concentration of 1 mM, is a potent inducer of apoptosis in CLL and should be further explored as a single agent. Also the combination of VPA and TRAIL may be a promising approach in the treatment of CLL.
Transcriptosome and Serum Cytokine Profiling of B-CLL Identifies Oncogenic Pathways in Pathogenesis and Survival.
Authors: Choi James , Garewal Harinder , Riley Christopher , Cooke Laurence , Klinkhammer Thomas , Maddipati Sirisha , Sud Rohit , Spier Catherine , Mahadevan Daruka
Published: April 2020

Background: B-cell chronic lymphocytic leukemia (B-CLL) the most common leukemia in the Western world accounts for 25% of all newly diagnosed leukemias. Despite new therapeutic advances, B-CLL is currently not curable. Potential oncogenic signaling pathways in B-CLL require elucidation. We identified ROR-1 receptor tyrosine kinase (RTK) in the WNT/Planar Cell Polarity non-canonical pathway as a possible mechanism of oncogenesis by gene expression profiling (GEP).

Methods: Mononuclear cells from 8 low stage CLL patients were obtained through an IRB approved protocol and analyzed utilizing the HG-U133A 2.0 Affymetrix array (~18,400 transcripts, 22,000 probe sets) after isolating and purifying total RNA (Qiagen, RNAeasy). The control RNA samples were isolated from 8 normal volunteer peripheral blood (PB) B-cells (AllCell, CA) and a normal reactive lymph node. Tumor lineage was confirmed by immunohistochemistry (IHC). Quantitative real time RT-PCR was performed on 20 selected genes to validate the microarray GEP. Twenty one B-CLL patients (includes the 8 patients from the GEP) were evaluated by RT-PCR for several key components of oncogenic signaling pathways based on the GEP with gene specific probes. A serum cytokine profile (120 cytokines) on 12 CLL patients (8 for which GEP is available and 4 additional patients) and 4 normal volunteers were performed for identification of a signature for diagnostic and prognostic value.

Results: Data are represented as “robust” increases or decreases of relative gene expression common to 8 patients. ROR-2, a close member of ROR-1, has been shown to bind Wnt-5A via the cysteine rich domain (CRD) and to activate the JNK signaling pathway. Our findings identify over-expression of members of the non-canonical WNT/PCP- ROR-1- signaling pathway genes that were validated by RT-PCR. A serum cytokine profile of 12 patients provides a signature that may be useful in CLL diagnosis and prognosis.

Conclusions: GEP identified WNT/PCP-ROR-1 as key components of an autocrine pathway that helps B-CLL avoid apoptosis. Several serum cytokines are elevated and require validation as potential diagnostic and prognostic markers. GEP of B-CLL in combination with quantitative real time RT-PCR has identified several novel targets for therapy. The identification of ROR-1 RTK has led to the development of a molecular target for future therapeutic application. Several lead compounds have been identified and are being evaluated as potential therapies in B-CLL.
Ubiquitously Expressed Micro RNA miR-16 Is Not Mutated and Not Differentially Expressed in Patients with B-CLL.
Authors: Landgraf Pablo , Fuchs Uta , Novosel Astrid , Fulci Valerio , Schoch Claudia , Haferlach Torsten , Buhmann Raymund , Tuschl Thomas , Borkhardt Arndt
Published: April 2020

MicroRNAs (miRNAs) are a recent discovered class of conserved 21–23 nucleotide short non-coding RNAs regulating protein levels by translational inhibition and mRNA destabilization and are involved in a variety of processes including cell differentiation and apoptosis. About 68% of miRNAs are tissue specifically expressed and great attention was paid to this group of miRNAs. To assess which miRNAs are ubiquitously expressed we investigated 170 human, 68 mouse and 16 rat libraries from 26 different organ systems and cell types by cloning and sequencing a total of 332,070 small RNA sequences comprising 220,147 miRNA sequences. 39 human miRNA clusters are transcribed in at least 70% of tissues and cell types. Interestingly the only miRNA cluster detected in nearly all libraries including those of embryonic origin and 8 B-CLL patient samples are the mir-16 clusters comprising mir-16-1 with mir-15a on chr.13q14 and mir-16-2 with mir-15b on chr.3q26. Earlier reports linked a lower abundance of the mature miR-16 to loss of heterozygosity (LOH) of chr.13q14 and mutations in the precursor were implicated in B-CLL in those reports. We therefore extended our investigation of B-CLL samples. We did not find any mutation in the miR-15a-miR-16-1 gene in all patients examined (64 patients). There were also no mutations in 35 healthy controls.

Furthermore 37 of 39 patients had detectable levels of miR-16 in quantitative Northern blot analysis. Only one of the two patients with undetectable miR-16 had a positive LOH status of the 13q14 locus in 90% of cells examined by FISH while the other one had no chromosomal aberrations or deletions. Additionally, the expression level of miR-16 is not correlated with the del13q14 LOH status in our study (P = 0.43 in double sided Fisher’s exact test). We also investigated the stability and processing of a reported construct bearing a C->T mutation 7 nt 3′ of the mature miRNA in 293 cell culture. This mutation has been reported in two B-CLL patients. We could not detect any difference in miR-16 expression compared to the wild type construct. Another ubiquitously expressed miRNA miR-21 is less stably expressed and is not detectable in 7 out of 39 patient samples by quantitative Northern blotting. We conclude that the absence of miR-16 in patients with B-CLL is rare and is not due to mutations in the miR-15a-miR-16-1 gene cluster in the patients examined. Furthermore miR-16 seems not to be affected by gene dosis effects like other genes involved in the LOH of 13q14 maybe because of compensatory up regulation of the other miR-16 cluster.
Dipeptidyl Peptidase 2 - A Pro-Survival Molecule in Chronic Lymphocytic Leukemia.
Authors: Danilov Alexey V. , Danilova Olga V. , Klein Andreas K. , Huber Brigitte T.
Published: April 2020

Chronic lymphocytic leukemia (CLL) is a unique malignancy characterized by persistent accumulation of quiescent B-cells. Disruption of survival pathways leads to apoptosis and renders CLL cells sensitive to chemotherapeutic agents. As clinical outcomes in CLL are heterogeneous, it is important to better predict prognosis in each individual case of the disease. In this respect, detection of somatic mutations in the immunoglobulin variable heavy chain (IgVH) genes has become the gold standard. Overexpression of ZAP-70 (Zeta-chain associated protein kinase - 70 kDa) by CLL cells correlates with unmutated IgVH genes and predicts poor outcome in B-CLL. Dipeptidyl Peptidase 2 (DPP2) is a newly discovered serine protease which maintains lymphocytes in a quiescent state (G0). In this study we investigated whether DPP2 is involved in cell cycle control in B-CLL. 38 patients with B-CLL and 20 healthy controls were included in the study. Median age of CLL patients was 67 years. Median time from diagnosis to enrollment in the study was 102 months. 21 patients (55.3%) received treatment before enrollment in the study. Standard Ficoll-Hypaque techniques were used to isolate peripheral blood mononuclear cells (PBMC) from healthy donors and CLL patients. CLL cells were isolated to >98% purity by means of a MoFlo using CD19−specific antibodies. Cells were treated with Val-boro-Pro, a pan-inhibitor of DPP, or with DPP2-specific inhibitor (DSI), incubated for 16 hours and stained with propidium iodide and Annexin V. Expression of CD38 was assessed by flow cytometry, DPP2 and ZAP-70 - by real-time reverse-transcription polymerase chain reaction, Bcl-2 and p27 - by western blot analysis.

We report that CLL B-cells expressed higher levels of DPP2 mRNA than normal B-cells. Inhibition of DPP2 in PBMC with VbP or DSI resulted in caspase-dependent apoptosis. In individuals with CLL, death of B-lymphocytes (and rescue by caspase inhibitors) was observed in 22 cases (57.9%). In the remaining 16 cases (42.1%) malignant B-cells did not undergo apoptosis upon inhibition of DPP2 with either VbP or DSI. We found that CLL cells resistant to DPP2 inhibition-induced apoptosis expressed higher levels of ZAP-70. Meanwhile, protein levels of p27 (cell cycle inhibitor) were decreased in resistant CLL. These patients exhibited worse disease prognosis such as shorter treatment-free period (p<0.001), frequent episodes of treatment failure and faster disease progression. Between the two groups, we identified no differences in expression of Bcl-2. CLL B-cells express higher levels of DPP2, a serine protease involved in maintenance of G0 which may serve as a survival factor in CLL B-cells. Resistance vs. susceptibility to DPP2 inhibition-induced apoptosis can be employed as a prognostic factor in CLL.
Therapy with Fludarabine, Cyclophosphamide and Rituximab (FCR) for Relapsed or Untreated Progressive Chronic Lymphocytic Leukemia (CLL). A Single Center Experience.
Authors: Pavlovsky Carolina , Pavlovsky Santiago , Pardo Maria L. , Sapia Sandra , Monreal Mariela , Corrado Claudia , Pavlovsky Miguel A. , Fernandez Isolda , Juni Mariana
Published: April 2020

Purpose: To evaluate the efficacy of FCR in improving complete remission (CR), disease-free survival (DFS) and overall survival (OS) rates in patients previously treated with chlorambucil-prednisolone and untreated CLL patients.

Patients and methods: A total of 45 CLL patients started FCR. Forty-one patients completed treatment: 16 following previous relapse and 25 previously untreated with progressive disease. Four patients are still receiving treatment. Median patient age was 63 years (range 34–88 years). Binet’s stage were: A: 8%, B: 34% and C: 58%. CD38 expression was positive (> 7% of cells) in 56% of patients and negative in 44%. FCR consisted of: fludarabine (25 mg/m2/day × 3); cyclophosphamide (250 mg/m2/day × 3) and rituximab (375 mg/m2/day × 1), all given intravenously, every 4 weeks for 4–6 cycles. CR was defined by CLL/NCI-WG criteria. Minimal residual disease (MRD) negativity was < 1% CD19, CD5 positive cells in peripheral blood and bone marrow. Results: The results of this study are detailed in Table 1. To summarise: CR: 69%; nodular partial remission (PR): 15%; PR: 7% and stable disease: 2%. Grade 3–4 neutropenia occurred in 33% of patients and 34% required hospitalization due to infections.

Table 1: Patient details following FCR treatment Patients % CR % MRD negative % DFS 36 mo P % OS 36 mo P Untreated 25 87 96 90 95 Treated 16 53 87 75 0.10 89 0.90 Total 41 74 92 83 93 CD38 Positive 23 71 90 71 0.02 87 0.27 CD38 Negative 18 76 94 100 100

Conclusions: FCR induces a high CR (74%) and DFS (83%) rate and increases MRD negativity (92%). Significantly higher DFS rates at 36 months were observed in patients who were CD38 negative vs. CD38 positive (100 % vs. 71% P=0.02).