bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunophenotypic Diagnosis of Lymphoproliferative Disorders Using the Abbott CELL-DYN Sapphire Hematology Analyzer.
Authors: Settegrana Catherine , Maloum Karim , Souni Fadila , Septier Isabelle , Vinet Sandrine , Bonnemye Patrick , Merle-Beral Helene
Published: April 2020

The CELL-DYN Sapphire (Abbott) is a routine hematology analyzer which uses integrated optical and fluorescence measurements, with FL1 (FITC) and FL2 (PE) detectors being configured for fluorescence analysis. Enumeration of CD3/CD4 and CD8 lymphocytes as well as platelets quantification using an anti-CD61 monoclonal antibody (moAb) are now included in the automated process of the analyzer. Recently, Johannessen et al. (Clin.Lab.Haematol.,2006) demonstrated that the CELL-DYN Sapphire analyzer allows a possible complete automated leukocyte immunodifferential count, using a panel of different moAbs (Johannessen et al., 2006).

Aim To test the ability of this blood analyzer to rapidly detect and quantify abnormal lymphoid cells by using a restricted panel of moAbs.

Patients and Methods Seventy blood samples were analyzed by using the following moAb combinations: CD3FITC/CD4PE, CD3FITC/CD8PE in an automated assay and CD3FITC/CD19PE and CD5FITC/CD19PE by using a manual procedure which only consists of mixing EDTA blood and antibodies. Results were obtained in 4 and 15 minutes for the automated assay and for the manual procedure, respectively. The results were compared with an extensive immunophenotype performed by using the FacsCanto cytometer (Becton Dickinson).

Results Sixty out of 70 samples were evaluated:

the quantification of CD3/CD4 and CD3/CD8 lymphocytes was well correlated with classical flow cytometry (FC), as previously demonstrated (R2 = .94 for CD3, .93 for CD3/CD4 and .95 for CD3/CD8). Interestingly, two T cell lymphomas (one with double positive T cells and one double negative T cells) were clearly recognized by the CELL DYN Sapphire and confirmed by classical FC and molecular biology. B-cells immunophenotyping: 25 samples with B cell lymphoproliferative disorders and with circulating abnormal lymphocytes (20 CLL, 1 HCL, 1 B-PLL and 3 MZL) were studied. In all cases, a very good correlation between the Abbott analyzer and classical FC was observed: CD19+/CD5+ (R2 = .99), CD19 (R2 = .91), CD3 (R2 = .91). Twenty controls without abnormal circulating B lymphocytes were studied and results were similar to those obtained with classical FC (R2 = .92 for CD3, .94 for CD19 and CD5). Of note in 10/70 samples, immunophenotyping using the Abbott analyzer was not possible because of the interference of lytic resistant red blood cells and/or platelets aggregates.

Conclusion The CELL-DYN Sapphire Abbott hematology analyzer provides a simple and fast immunophenotyping method useful for a preliminary screening in routine laboratory practice to detect and quantify abnormal circulating lymphoid cells simultaneously with the blood cell count and leukocyte differential.
Analysis of In Vitro Effects of Bortezomib (Velcade) on T Cell Prolymphocytic Leukemia (T-PLL).
Authors: Ozpuyan Fulya , Meyer Paul N. , Al-Masri Hytham , Ni Hongyu , Alkan Serhan
Published: April 2020

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.
Clinical Significance of Alterations of the NOTCH1, FLT3 and p53 Genes in Pediatric T-Cell Acute Lymphoblastic Leukemia.
Authors: Park Myoung-ja , Taki Tomohiko , Shimada Akira , Sotomatsu Manabu , Hanada Ryoji , Hayashi Yasuhide
Published: April 2020

Activating mutations of the NOTCH1 gene have been reported in about 50% of T cell acute lymphoblastic leukemia (T-ALL). We performed mutation analysis of the NOTCH1, FLT3 and p53 genes by polymerase chain reaction followed by direct sequence. Mutations of the NOTCH1 were identified in 24 (30%) of 80 fresh samples and 10 (71.4%) of 14 T-ALL cell lines. Six missense mutations and 2 insertion in HD domain, 2 nonsense mutations and 6 insertions in PEST domains were found in 14 cell lines. Eight missense mutations, 9 insertions and one deletion in HD domains, 5 missense mutations, 3 nonsense mutations and 3 deletions in PEST domain were found in 80 fresh samples. The incidence of the NOTCH1 mutations is less frequent than that of previous reports. We observed about 95% of single nucleotide polymorphisms (5097 C/T) in HD domain, which is more frequent than 30% of previous report, possibly due to the racial difference. FLT3 internal tandem duplication, which is known as the poor prognostic factor in acute myeloid leukemia, were not identified in any T-ALL cell lines or fresh samples. Mutations of the p53 gene were found in 5 of 8 cell lines and 5 of 50 fresh samples. Mutations of both NOTCH1 and p53 genes were identified 3 of 8 cell lines and one of 50 fresh samples. In our study NOTCH1 mutations were not significantly associated with high WBC count and prognosis. Further studies are needed to find the association between novel genes and clinical features of pediatric T-ALL.
JNK/c-Jun Is Required for HMC-1 Cell Proliferation.
Authors: Wang Bin , Tsukada Junichi , Higashi Takehiro , Mizobe Takamitsu , Matsuura Ai , Mouri Fumihiko , Sawamukai Norifumi , Ra Chisei , Tanaka Yoshiya
Published: April 2020

Activation of c-jun N-terminal kinase (JNK) through c-kit-mediated phosphatidylinositol 3 (PI3) and Src kinase pathways plays an important role in cell proliferation and survival in mast cells. Gain-of-function mutations in c-kit are found in several human neoplasms. Constitutive activation of c-kit has been observed in human mastocytosis, acute myeloid leukemia, lymphoma, germ tumor and gastrointestinal stromal tumor. In the present study, we demonstrate that an anthrapyrazole SP600125, a reversible ATP-competitive inhibitor of JNK inhibits proliferation of human HMC-1 mast cells expressing constitutively activated c-kit mutant. We found that JNK/c-Jun was constitutively activated in HMC-1 cells without stimulation. When spontaneous activation of JNK/c-Jun was inhibited by treatment with SP600125, cell proliferation was suppressed. The concentration which effectively inhibited JNK/c-Jun activity in our experiment had no effect on SCF-induced phosphorylation of Akt or Erk, suggesting that SP600125 specifically inhibited JNK/c-Jun activity in HMC-1 cells. Moreover, we demonstrated that SP600125 induced HMC-1 cell apoptosis in dose- and time-dependent manner. Caspase-3 and PARP were cleaved as early as 12 h after treatment with SP600125, but caspase-9 was not. Also, cell cycle arrest in G1 phase was observed in SP600125 treated cells. Thus, the inhibitory effect of SP600125 on cell proliferation was associated with cell cycle arrest at the G1 phase and apoptosis accompanied by cleavage of caspase-3 and PARP. Caspase-3 inhibitor Z-DEVD-FMK almost completely inhibited SP600125-induced apoptosis of HMC-1 cells. In contrast, caspase-9 inhibitor Z-LEHD-FMK failed to block SP600125-induced apoptosis, suggesting that apoptosis induced by SP600125 was caspase-3 dependent. Following SP600125 treatment, down-regulation of cyclin D3 protein expression, but not p53 was also observed. Take together, JNK/c-Jun is essential for proliferation and survival of HMC-1 cells. The results obtained from the present study suggest the possibility that JNK/c-Jun may be a therapeutic target in diseases associated with c-kit mutant.
Late Appearance of t(9;22) at Relapse, in an Adolescent with an Homozygous 9p Deletion T-ALL.
Authors: Papadakis Vassilios , Papadhimitriou Stefanos , Paisiou Anna , Belesi Chrysoula , Parcharidou Agapi , Tourkantoni Natalia , Sambani Constantina , Paterakis George , Polychronopoulou Sophia
Published: April 2020

T-ALL has increasing incidence during adolescence and it is rarely associated with Philadelphia chromosome positivity. Moreover, extremely rare is the event of Philadelphia chromosome negativity at diagnosis and leukemia clone evolution with t(9;22) at relapse. Such a patient is being described and relevant issues are being raised. A 14 year old boy was diagnosed with leukemia due to malaise and ecchymoses. CBC revealed: WBC 170 k/uL (80% blasts), Hb 13.1 gr/dL, Ht 37.7% and PLT of 26 k/uL. Bone marrow confirmed the diagnosis of ALL (L2 morphology). Bone marrow flow cytometry depicted a T II ALL by EGIL classification (Tdt 54%, cCD3 99%, CD7 96%, CD2 96%, CD4 0%, CD8 2%, CD5 75%, TCRαβ 0%, CD1α 0%, sCD3 31%). Karyotype with G-banding, technically failed. By PCR, blasts were negative for TEL-AML1, E2A-PBXand MLL-AF4 while RNA coding for proteins p190 and p210 was also negative. Additionally, the 9 chromosome was evaluated by FISH technique (centromere and 9p21 areas/genes p16/p14 and p15- Vysis) and an homozygous deletion of 9p16 area was detected in the great majority of the blasts tested. The patient was treated according to the ALL-BFM-95 protocol and proved to be a poor prednisone responder, with residual disease detected on day +15 bone marrow and remission on day +33. He proceeded to the High Risk arm of the protocol. Thirteen months from diagnosis and 5 months on maintenance treatment, the patient suffered a bone marrow relapse. CBC revealed: WBC 30 k/uL (25% blasts), Hb 13.8 gr/dL, Ht 40.9 % and PLT of 201 k/uL. Bone marrow confirmed the relapse (L2 blast morphology) with a more mature T-cell type (cCD/TdT coexpression, with CD3 87% and TCRαβ 53.3% expression). Five of 25 metaphases revealed the following complex karyotype: 46,XY,del(6)(q21),?del(7)(q36), -9, t(9;22)(q34;q11),+mar1. By FISH , monosomy of 9 chromosome was not proved , while homozygous deletion of 9p16 area was again documented and t(9;22) was clearly evident. By PCR, there was no detection of RNA coding for proteins p190 and p210 with standard commercially available probes. The patient is treated with a fludarabine based regimen and imatinib, with the prospective of stem cell transplantation.

In conclusion, at diagnosis, T-ALL without t(9;22) but with an homozygous deletion of chromosome 9, was documented. At relapse, there is clone evolution with

a more mature T-origin blast detection of a complex karyotype with t(9;22). Ph+ T-ALL is rare, as well as rare is the event of ALL clone evolution, with emergence of Ph+ at relapse.

This patient’s data are being completed and there is prospective of further illuminating the underlying pathogenesis. Chromosome 9 deletions are under investigation for contributing in genetic instability. Homozygous 9p16 deletion of ALL blasts at diagnosis and relapse might play a key role in these events.
Changes in Thymic Recent Output Function in Patients with B-Cell Lymphocytic Malignancy.
Authors: Li Yangqiu , Yang Lijian , Chen Shaohua , Geng Suxia , Przybylski Grzegorz , Schmidt Christian A.
Published: April 2020

Since 1998 the use of signal joint T cell receptor excision circles (sjTRECs) to study changes in the frequency of recent thymic emigrants was reported, this study drew attention to the fact that the level of sjTRECs can be used to estimate the recent thymic output function. Therefore, sjTRECs was used as a new marker for analysis of thymic output function in different immunodeficiency diseases, immune reconstitution in patients after stem cell transplantation. Defects of cellular immunity, however, may also play a role in hematologic malignancies. Little is known about the feature of T-cell immune state in B-cell lymphocytic malignancy. In order to identify number of naïve T-cells in B-cell lymphocytic malignancy, sjTRECs-content was determined in the mononuclear cell fraction and the sjTRECs-number was related to the number of T-cells (according to the number of CD3-positive cells). Quantitative analysis of sjTRECs in DNA of peripheral blood T cells from 45 cases with B-cell lymphocytic malignancy (including 17 cases with ALL, 4 cases with CLL, 15 cases with B-NHL and 6 cases with MM) were preformed by real-time PCR and TaqMan analysis, and 14 normal individuals as controls. The results showed a dramatic reduction of sjTRECs values in patients. Some cases no sjTRECs copies could be detected in 40000 T cells. The mean value of sjTRECs was 0.39±0.75 copies/1000 PBMCs in ALL, 0.11±0.15 copies/1000 PBMCs in B-CLL, 0.67±1.39 copies/1000 PBMCs in B-NHL, 0.91±1.16 copies/1000 PBMCs in MM patients, as compared with 4.1±3.65 copies/1000 PBMCs in normal individuals, the sjTRECs level in all goups of B-cells lymphocytic malignancy were significant decrease (p=0.0001, p=0.048, p=0.002) except MM group (p=0.053). However, when comparison of the sjTRECs value in CD3+ compartments, it was not statistically different from the sjTRECs counts between patients with ALL (3.91±7.20 copies/1000 CD3+cells) and normal individuals (6.36±5.28 copies/1000 CD3+cells) (p=0.2139), suggesting that the reduction of sjTRECs level in patients wit ALL may due to lower numbers of T cells in peripheral blood. In conclusions, the results suggest that some thymic dysfunction in T cell generation was detected at least in most patient with B-cell lymphocytic malignancy. However, whether this is due to clonal expansion of T-cells to antigens, or reflects impairment of immune function associated to the malignancy, remains an open question. In a prospective study sjTRECs levels will be determined in different T- cell compartments including CD45RA+, CD4+ and CD8+ cells and so on.
Effect of Roline Rich Domain of an RNA-Binding Protein Sam68 in B Lymphocyte Leukemia Cell Line Growth Process, Death and B Cell Signal Transduction.
Authors: Pang Tianxiang , Li Qinghua , Wang Jianxiang , Yuan Wensu , Zhang Lei , Han Zhongchao
Published: April 2020

Sam68 plays an important role as a multiple functional RNA binding nuclear protein in cell cycle progress, RNA usage, signal transduction regulation of cell growth and proliferation, and tyrosine phosphorylation by Src during mitosis. However, its precise impact on these essential cellular functions remains unclear. The purpose of this study is to further elucidate Sam68 functions in B lymphocyte leukemia DT40 cell line. By using gene targeting method, we isolated a mutation of deleted proline rich domain from Sam68 in DT40 cells and described its effect on cell growth process and signal transduction. The results have demonstrated that mutant cells grew at a slower rate (2.1 hours growth elongation). Cell cycle pattern was observed in mutant cells by flow-cytometric analysis, the retardation of cell growth was mainly due to the elongation of S phase (1.7 hours elongation). When the cell cycle was analyzed at different serum concentration conditions, we found that the effect of Sam68 mutant on the S phase was further supported by the serum depletion experiment. In light of evidence that RNA binding activity is essential for functions, Sam68 is implicated in cell growth control probably by modulating the function of mRNAs in S phase or earlier. Larger amounts of dead cells was observed in the culture of mutant cells, and serum depletion enhanced the ratio of dead cells in both culture conditions. Since the putative control of cell cycle by Sam68 appeared to be related to signal transduction, we next assessed the involvement of Sam68 in B cell signal transduction pathway. After cross-linking of B cell receptor on the DT40 cells by IgM antibody, phosphorylation of cellular proteins was analyzed by protein blotting with anti-phosphotyrosine antibody. The result demonstrated that the overall pattern of B cell receptor in Sam68 mutant cells was similar to that in the wild type DT40 cells. However, the maximal level of tyrosine phosphorylation on BLNK, a prominent signal molecule in B cell, has been observed no significantly changes in the mutant cells. Since the expression levels of B cell receptor on the Sam68 mutation clones were essentially same as that of parental DT40 cells, it was suggested that deletion of partial of proline region in Sam68 resulted in the decrease in B cell signal transduction. We have concluded that the proline rich domain of Sam68 is involved in cell growth control by modulating the function of mRNAs in S phase or earlier and the functions as an adaptor molecule in B cell signal transduction pathways.
Syndrome of Inappropriate Secretion of Anti-Diuretic Hormone in Children with Acute Lymphoblastic Leukemia.
Authors: Borker Anupama S. , Hutchins Sabrina , Grant Robin , Hijiya Nobuko , Sandlund John T. , Metzger Monika L. , Jeha Sima , Razzouk Bassem I. , Ribeiro Raul C. , Rubnitz Jeffrey E. , Hudson Melissa M. , Relling Mary V. , Pui Ching-Hon , Howard Scott
Published: April 2020

Background: The syndrome of inappropriate secretion of anti diuretic hormone (SIADH) is a well known complication of vincristine. Available literature on SIADH in pediatric acute lymphoblastic leukemia (ALL) focuses on patients with severe manifestations. We performed this study to determine the clinical consequences of SIADH that occurred during ALL therapy.

Patients and Methods: We studied all episodes of hyponatremia in patients with ALL diagnosed from December 1991 to October 2002 at St Jude Children’s Research Hospital who had not relapsed or undergone stem cell transplantation. SIADH was defined as sodium ≤ 130 meq/L in a euvolemic patient with increased urinary sodium excretion, decreased serum osmolarity and increased urine osmolarity with creatinine normal for sex and age. For each episode, we reviewed demographic features, treatment phase, predisposing factors, presenting features, duration, severity, treatment and outcome.

Results: Of 605 patients studied, 68 patients had 79 episodes of SIADH. Fifty-nine patients (87%) had only 1 episode while 9 patients (13%) had recurrences. The mean lowest serum sodium was 128.3 mEq/L (range, 121–130 mEq/L) and the mean duration of hyponatremia was 1.9 days (range, 1–14 days). Ninety-six percent of episodes were associated with the administration of 1 (n=29) or 2 (n=43) doses of vincristine in the preceding 2 weeks. Episodes were associated with infection in 51 cases and central nervous system pathology in 3 cases. Only 6 (8 %) patients had serum sodium 125 mEq/L or lower; 50% of these had altered mental status, compared to 7% whose sodium never fell below 125 mEq/L. Treatment included fluid restriction (46%), change or cessation of intravenous fluids (6%), diuretics (2%), or observation (46%). All symptomatic patients had complete neurological recovery.

Conclusions: SIADH is common during treatment for ALL but is rarely severe or symptomatic. The risk is greatest within 14 days of vincristine administration. Most episodes are self limited and need neither extensive investigation nor aggressive therapy.
Impact of mTOR Inhibition on FoxP3+ Regulatory T Cells as Compared to Conventional T Cells after Allogeneic Bone Marrow Transplantation.
Authors: Zeiser Robert , Leveson-Gower Dennis B. , Zambricki Elizabeth A. , Hou Jing-Zhou , Negrin Robert
Published: April 2020

FoxP3+CD4+CD25+ regulatory T-cells (Treg) have been shown to effectively reduce the severity of experimental acute graft-versus-host disease (aGvHD) while sparing graft-versus-leukemia activity. These findings, in concert with the observation that human and murine Treg share functional characteristics, have fueled interest in clinical trials to control aGvHD. Recent data indicates that the immunosuppressant rapamycin (RAPA) in contrast to cyclosporine A does not interfere with in vivo function of Treg and could enhance Treg expansion in vitro by a yet unknown mechanism.

To investigate the impact of mTOR inhibition on proliferating Treg and Tconv, both cell types were exposed to CD3/CD28 Mabs in the presence of different RAPA concentrations in vitro. Phosphorylation of mTOR downstream products p70S6K1 and 4E-BP1 were assessed by western blot and flow cytometry. Inhibition of the phosphorylation of p70S6K1 and 4E-BP1 was observed in both populations in the presence of RAPA. Interestingly, Treg were more resistant to mTOR inhibition as compared to Tconv and displayed significantly higher phosphorylated products in the presence of RAPA at 10 nM (MFI Treg vs Tconv, p<0.001) and at 100nM (MFI Treg vs Tconv, p<0.001).

To investigate whether Treg and RAPA protect from aGvHD in a synergistic manner, BALB/c recipients were transplanted with H-2 disparate BM and 1.6x10e6 T-cells (FVB/N) after lethal irradiation (8 Gy). aGvHD lethality was only slightly reduced when suboptimal Tconv:Treg ratios were employed (4:1, 8:1), or when recipients were treated with a non-protective RAPA dose (0.5 mg/kg bodyweight). Combining a suboptimal Tconv:Treg ratio with a non-protective RAPA dose reduced expansion of luciferase expressing (luc+) Tconv and pro-inflamatory cytokines and improved survival indicative for an additive in vivo effect of RAPA and Treg.

To evaluate the impact of RAPA on in vivo T cell expansion, either luc+ Tconv or luc+ Treg were adoptively transferred. In vivo bioluminescence imaging demonstrated that RAPA had a more potent inhibitory effect on proliferation of Tconv as compared to Treg (p<0.05 vs. NS). We did not observe RAPA to increase FoxP3+ Treg numbers in vivo, or to enhance GITR or CTLA-4 expression.

Thus, increased Treg numbers observed in RAPA containing expansion cultures are likely due to a lower susceptibility of this cell population to mTOR inhibition. This could explain the observed synergistic effect of RAPA and Treg in aGvHD protection which has relevance for clinical trials utilizing Treg to prevent aGvHD.
Proteinase-Activated Receptor 1 Expression in Human Leukemia HL-60 Cells and Its Significance.
Authors: Xie Wan-Zhuo , Lin Mao-Fang , Huang He , Cai Zhen
Published: April 2020

The coagulation system is regularly activated in patients with acute leukemia, especially with acute promyelocytic leukemia. Up to 30% of these patients suffer from thromboembolic complications. Obviously, activation of thrombin occurs in the natural course of cancer. During tumor progression, many patients develop coagulation disorders; treatment is complicated by increased resistance of tumor cells to chemotherapy. Thrombin-mediated activation of cellular receptor(s), such as proteinase-activated receptor-1(PAR-1), plays an important role in tumor growth, local progression, and distant metastasis. Our hypothesis is, that leukemia cells that express PAR-1 have a survival benefit if they are activated by thrombin.We harvested HL-60 cells in vitro. We detected PAR-1 expression using reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence activated cell sorting (FACS) scan.We found that HL-60 cells expressed PAR-1 receptor. Furthermore,we employed a series of different concentrations of thrombin into the HL-60 cell culture system.With certain interval of low level of thrombin, HL-60 cell growth was enhanced,in contrast, we found that HL-60 cell apoptosis was observed under higher concentration of thrombin.In summary, we found that human leukemia HL-60 cell exhibits a positive PAR-1 expression. Also, thrombin could influence the growth of HL-60 mediated by PAR-1 possibly. Further experiments will show if these cell-biological mechanisms aggravate cellular proliferation/apoptosis in leukemia cells. Therapeutic consequences, such as therapeutic thrombin inhibition in addition to chemotherapy are discussed.
CD44-Ligation Triggers IL-1, GM-CSF and IL-8 Driven Differentiation Pathways in Human Monoblastic Leukemia Cells.
Authors: Smadja-Joffe Florence , Delaunay Jacques , Qi Junyuan , Gadhoum Zeineb , Ingeneer Laetitia Durand , Chomienne Christine
Published: April 2020

The CD44 surface antigen is highly expressed on AML cells. We previously reported that its ligation with specific monoclonal antibodies (mAbs) triggers terminal differentiation of leukemic blasts in all AML subtypes. This raised the perspective of developing a CD44-targeted therapy that would be efficient in all AML subtypes, including the monoblastic one (AML-M5) in which, in the absence of specific genetic abnormalities, no targeted therapy is being developed. Here we investigated the molecular mechanism responsible for the CD44-induced differentiation in primary AML-M5 blast samples (n=13). Firstly, by using cDNA arrays, we show that CD44 ligation:activates a burst of cytokine and chemokine genes, through the p38MAPK pathway, indicating that normal CD44 pathways are functional in AML-M5 blasts; andmodulates the gene expression of enzymes, surface proteins and transcription factors as in normal monopoiesis.Secondly, by using functional inhibitors, we demonstrate that the differentiation of CD44-ligated AML-M5 blasts is mediated by IL-1b, GM-CSF and IL-8. These proteins act locally, through an autocrine/paracrine pathway, suggesting that most of the drawbacks often provoked by their systemic injection should be very attenuated. Together, these results provide new support for the development of a CD44-targeted differentiation therapy in acute monoblastic leukemia.
Clinical Significance of Free Circulating CD34 in Patients with Acute Myeloid Leukemia and Myelodysplastic Syndrome.
Authors: Hodge Menna , Giles Francis , Abdool Adam , O’Brien Susan , Keating Michael , Jilani Iman , Estrov Zeev , Estey Elihu , Kantarjian Hagop , Albitar Maher
Published: April 2020

CD34 is an approximately 116-kd glycophosphoprotein expressed in hematopoietic progenitor cells, endothelial cells, and some mesenchymal and neural cells. CD34 is a typical adhesion molecule capable of inducing the cell signal transduction pathway leading to adhesion and differentiation. We used a newly developed bead-based assay to measure cell-free circulating CD34 (cCD34) in the plasma of patients with acute myeloid leukemia (AML; n = 98) and myelodysplastic syndrome (MDS; n = 50). Levels of cCD34 were significantly higher in AML (median 10983, range: 844–100,4191 U/10 μl than in MDS (median: 8749, range:102–791,350 U/10 μl) patients (P<0.01). cCD34 levels were higher among patients with high-risk cytogenetic abnormalities in AML (P = 0.01) but not MDS (P = 0.92). When grouped together, AML and MDS patients with cCD34 levels higher than the median (10,845 U/μl) had significantly shorter survival than those with lower levels (P = 0.01). This association was independent of cytogenetic grouping, age, and performance status. cCD34 levels did not correlate with percent of blasts or CD34+ cells but did correlate with WBC count (R = 0.36) in patients with AML, suggesting that cCD34 reflects the overall leukemia load. Although further study is needed for confirmation, cCD34 appears to result from turnover of leukemic cells and may affect the activation of certain pathways, therefore influencing survival and clinical outcome.

Figure Figure
Stromal Cell Line Enhances Survival of Myeloid Leukemia Cells by Regulating the Expression of bcl-2 Family Genes and Caspase-3.
Authors: Xu Zhenshu , Liu Yongjun , Han Zhibo , Lv Lulu , Yang Ting , He Rui , Chen Zhizhe , Han Zhongchao
Published: April 2020

Approximately 20–30% acute myeloid leukemia (AML) patients are not cured by traditional chemotherapy. The possible explanation is that residual leukemia cells are harbored in association with stromal cells in the bone marrow, which causes late relapse of leukemia. This study was designed to investigate interactions between marrow stromal microenvironment and leukemia cells. AML cells were cultured in different conditions with or without serum, Ara-c, the monolayers or the conditioned medium of a stromal cell line HESS-5. Apoptosis frequencies, cell viability, cell cycle distribution, DNA fragment, and bcl-2/bcl-xl gene as well as caspase-3 expression were determined by Typan blue exclusion, MTT assay, flow cytometry analysis, PCR and Western blot respectively. Direct contact of HL-60 cells with HESS-5 significantly inhibited serum-deprivation- or Ara-c-induced apoptosis of HL-60 cells, and resulted in a significant increase of short-term proliferation and viability. Soluble factors had negligible effect. The interaction of HL-60 with fibronectin did not confer a significant resistance to serum-deprivation and Ara-c agent. HESS-5 cells were also able to maintain cell cycle progression of HL-60 during chemotherapy exposure. HESS-5 cells-mediated apoptosis inhibition was associated with increased bcl-2/bcl-xl expression and down-regulation of caspase-3 in primary myeloid leukemia cells. These results indicate that stromal cells enhance the survival of myeloid leukemia cells by regulating bcl-2 family genes as well as caspase-3 protein and suggest that specific interactions between stromal cells and leukemia cells may enhance the resistance of leukemia cells to chemotherapy.
Analysis of NPM1 Gene Mutations in Chinese Adults with Acute Myeloid Leukemia.
Authors: Wu De Pei , Yan Lingzhi , Chen Suning , Liang Jianying , Feng Yufeng
Published: April 2020

Many European groups have described that mutations at exon-12 of the nucleophosmin (NPM1) gene are the most frequent genetic lesion in acute myeloid leukemia (AML) (45.7%~61.7% of all adult AMLs with normal karyotype). This outstanding discovery provides a promising minimal residual disease (MRD) marker for AMLs with normal karyotype. To clarify the prevalence and the clinical profile of NPM1 mutations in Chinese patients with AML, we analyzed a cohort of 156 newly diagnosed adult AMLs for this mutation. Genomic DNAs were prepared from bone marrow samples of these patients. NPM1 exon 12 mutations were detected using direct sequencing or fragment analysis of DNA-PCR products. NPM1 mutations were present in 28.2% of the overall population, including 1/1(100%) of M0, 11/27(40.7%) of M1, 11/46(23.9%) of M2, 0/29(0%) of M3, 2/9(22.2%) of M4, 18/39(46.2%) of M5 and 1/5(20.0%) of M6. NPM1 gene mutations were more prevalent in patients with a normal karyotype (37 of 90; 41.1%), when compared with patients with karyotypic abnormalities (7 of 66; 10.6%; P<0.001). Sequence analysis of 25 NPM1 mutated cases revealed known mutations (type A, B, NM, and PM) as well as 1 novel sequence variation (here named as type S). All mutational types were heterozygous and showed a 4 bp insertion between position nucleotide 960 and 961 (Genebank accession number: NM-002500). NPM1 mutations were significantly associated with old age (P<0.05), high peripheral white cell count (P<0.05) and FAB-M1/M5, but negatively associated with expression of CD34 (P<0.05) and CD117 (P<0.05). In conclusion, NPM1 mutations also represent a common genetic abnormality in Chinese adults with AML, especially in the presence of a normal karyotype. The occurrence of NPM1 mutations indicates an age-dependent characteristic. NPM1 mutated cases show a special clinical subtype of AML. Further studies are urgently needed to confirm the role of NPM1 mutations in leukemogenesis. The altered nucleo-cytoplasmic transport of NPM1 mutated protein is probably a potential therapic target for AML with NPM1 mutations.
Gain-of-Function WASP Mutations in Pediatric and Adult Patients with Myelodysplasia or AML.
Authors: Beel Karolien , Schollen Els , Uyttebroeck An , Verhoef Gregor , Demuynck Hilde , Devriendt Koen , Vandenberghe Peter
Published: April 2020

X-linked neutropenia (XLN, OMIM #300299)) was first described in 2001 in a three-generation Belgian family with five affected members and with a L270P gain-of-function mutation in the GTPase binding domain (GBD) of the Wiskott-Aldrich-syndrome protein (WASP) (Nat Genet2001,27,313). Although all five L270P cases originally presented with neutropenia, two of them eventually developed a myeloid malignancy while under G-CSF, with monosomy 7 in the malignant cells. One male (II.3) developed a myelodysplastic syndrome (MDS-RAEB) at age 65, achieved stable remission after three courses of decitabine, but died three years later due to an unrelated cause. A second patient (III.6) succumbed to refractory MDS RAEB, rapidly evolving to MDS-RAEBt at age 38. Since our original report, two more cases with XLN have been reported, one with a I294T and one with a S272P WASP mutation. (Ancliff et al. Blood online 2006). The I294T case, 4 years old, had myelodysplastic features at presentation. These observations have prompted us to investigate whether inherited gain-of-function WASP mutations are associated with paediatric MDS or AML. In addition we have explored whether acquired WASP mutations occur in adult myeloid malignancies. 207 cases were examined. Male/female ratio was 1.35. 34 cases were between 2 months and 20 years old and 173 cases were between 21 and 89 years old. There were 48 cases with myelodysplasia, 157 had acute myeloid leukaemia and 2 had myelofibrosis. 29% had monosomy 7. There were two brothers with monosomy 7, one with AML M5 and the other with secondary ALL after MDS. Exons 7-10, encoding the GTPase Binding Domain (residues 230-381), were amplified and screened for mutations using dHPLC. In these 207 samples from patients with MDS or AML, no mutations were found in the exons 7-9 of the WAS gene. One mutation was found in intron 6 in a male patient with AML and monosomy 7. The mutation did not influence splicing of the exon and was thus considered irrelevant. Four patients had a known T1029C (V332A) single nucleotide polymorphism in exon 10. One patient with AML (age 50y) had a C975T (P314S) mutation in exon 10, which has not been reported previously. Since this mutation occurs in less than 1%, it does not qualify as a polymorphism. If relevant for the pathogenesis of AML, the mutation is very rare whatsoever. Although the eventual risk of malignant myeloid transformation appears to be increased in L270P XLN, we could not, with one exception, identify mutations in exons 7-10 of the WAS gene in this series of paediatric and adult MDS/AML. Therefore, inherited or acquired WAS mutations do not seem to be commonly associated with pediatric or adult MDS or AML. Our data therefore do not support screening for exon 7-10 mutations of WAS in the context of paediatric or adult MDS and AML.
Co-Operativity between Antibody-Dependent Cellular Cytotoxicity (ADCC) and Immunotoxin-Mediated Killing of Human T-ALL Cells in a SCID Mouse Model: Simultaneous Delivery of Saporin Via CD7 and ADCC Via CD38 Is No Better Than the Simultaneous Delivery of Each Against Only CD7.
Authors: Flavell David J. , Flavell Sopsamorn U.
Published: April 2020

We have previously shown that the anti-CD7 saporin-based immunotoxin (IT) HB2-SAPORIN and host antibody-dependent cellular cytotoxicity (ADCC) co-operate in the killing of human T-ALL a SCID mouse model of this disease (Flavell et al 1998 Cancer Res 58, 5787). In this described model system it is the same anti-CD7 IT that is responsible for delivering both the ribosome inactivating protein saporin and eliciting ADCC by recruiting NK cells via binding of their FcγRIIIA to the Fc domain of target cell bound IT. There is however a conflict here in that for the IT to be effective it needs to internalize whilst for maximally effective ADCC, the IT needs to remain on the cell surface in order to recruit cytotoxic effector cells. Over 90% of HB2-SAPORIN IT is internalized by CD7+ HSB-2 target cells within 12h following bivalent ligation by this IT and yet the ten percent or less of CD7 remaining on the target cell surface appears sufficient to elicit adequate ADCC to achieve the co-operative effect in vivo. The question is if ADCC is performing sub-optimally due to the reduced amount of target cell expressed CD7 doe this mean that the subsequent co-operative effect is also sub-optimal and could this be improved upon to achieve a greater and more effective target cell kill?

To attempt to answer this question we have targeted saporin to the HSB-2 target cell via CD7 using a F(ab)2 IT (HB2-F(ab)2−-SAPORIN) that is incapable of eliciting ADCC whilst simultaneously delivering ADCC via an intact IgG1 anti-CD38 antibody (OKT10) that also binds to the target cell surface. The expression level of CD38 on HSB-2 cells is approximately ten-fold higher than CD7 and importantly the observed CD38 internalization rate and extent is considerably reduced compared with CD7 when ligated by cognate antibody. We therefore predicted that increased ADCC would be deliverable to HSB-2 target cells with OKT10 antibody and an in vitro chromium release assay confirmed this. However, the in vivo treatment of SCID-HSB-2 mice with a combination of HB2-F(ab)2-SAPORIN and intact OKT10 antibody was not significantly better than treatment with HB2-SAPORIN intact IT alone. These preliminary data indicate that even though additional ADCC is apparently deliverable with the OKT10 antibody this does not result in a further enhancement of the co-operative effect in vivo suggesting that the optimum co-operativity threshold has already been reached in the HB2-SAPORIN mono-therapy system.
Changes in the Immune Functions during ALL Induction Therapy - Prospective Immune Monitoring in 23 Children.
Authors: Wiegering Verena A. , Eyrich Matthias , Schlegel Paul G. , Winkler Beate
Published: April 2020

Introduction: Inadequate lymphocyte functions are an important clinical problem complicating chemotherapy by severe bacterial and fungal infections. During an ongoing immune response, cytokines produced by T-cells are critical to the effectiveness of that response. To understand what happens with the immune system in the beginning of therapy, we investigated lymphocyte subpopulations and functioning (cytokines, immunoglobulins).

Patients: 23 children (median age 5y;range 2m-14y;15 female, 8 male) with B precursor ALL were treated according to the ALL-BFM 2000 protocol. Blood samples were drawn at diagnosis and on days 8, 15, 33 and 64 and before beginning of protocol M, protocol 2 and during maintenance therapy.

Methods: We analyzed lymphocyte subpopulations using flow cytometry. Intracellular cytokines (IFNg, IL2, TNFa, IL4, IL5, and IL10) were determined by FACS after in vitro stimulation with PMA, ionomycin and brefeldin for 24h. Additionally, we measured various cytokines in unstimulated sera by ELISA. Furthermore, we studied TRECs and Spectratype values and immunoglobulins.

Results: As to lymphocyte subpopulations, B-cells decreased rapidly from a median value of 301+120/mm_ before chemotherapy to 85+138/mm_ during chemotherapy (“day 33”) and did not reappear until the end of therapy.

T cells decreased at the beginning of chemotherapy, but there was a partial recovery proportional to the intensity of chemotherapy. Also we found a shift towards CD8+ cells. NK cells did not show significant changes.

As to cytokines in CD3+ T-cells, we detected an increase of IFNg (diagnosis:11+9%, day 33: 22+7%). We could detect a correlation between gd-TCR and IFNg production (FACS) as well as IFNg values (ELISA) and high memory cell count. Also CD45RA+cells correlate with CD4IL2+cells. Finally TGFb in unstimulated sera correlated strongly (p<0,01) with CD19+cells. TGFb was also produced by blasts. We detected difference in cytokine profile between conditioning with dexamethasone vs. prednisone, especially the IFNg secretion was much higher in the prednisone branch (p<0,01). TREC values were higher in the dexamethason branch, but this might be due to the age, because younger children had significantly higher TREC values. Analyses of TCR repertoire show a slightly higher complexity in the prednisone branch. Until d15 the repertoire complexities were reduced and increase slowly continuingly during therapy. We could detect Vb gene families with high complexity during the whole therapy (for example BV22, BV23) and with low complexity (BV13b, BV6a). The complexity correlated positively with the number of naïve T-cells and with the age (p<0,01).

Summary: We detected a shift towards Th1-cytokines during ALL-induction-therapy. B cells were erased during chemotherapy. This might be of clinical importance of the possible necessity of prophylactic immunoglobulins to prevent serious infection because of the lack of B-cells.
Multi-Agent Phase I Trials in Childhood Acute Lymphoblastic Leukemia (ALL).
Authors: Sposto Richard , Raetz Elizabeth A. , Reynolds Charles P. , Gaynon Paul S.
Published: April 2020

Background: Single agent phase I trials with conventional methodology may not be suitable for children with relapsed leukemia. Accrual of children with ALL in relapse to single-agent phase I trials is poor due to clinical urgency and a > 30% likelihood of complete response (CR) with a variety conventional agents combinations (Br J Haematol.2005; 131(5): 579) with the option of hematopoietic stem cell transplant in remission. As most drugs are ultimately used in combination, a Phase I trial testing a new agent in combination with conventional agents would seem most useful and might increase accrual. However, with conventional phase I methodologies determination of a maximum tolerated dose is complicated by the toxicities of the accompanying conventional agents and by the background morbidity of relapsed leukemia.

Methods: The Children’s Oncology Group (COG) study, AALL01P2, employed vincristine, prednisone, doxorubicin, and pegylated asparaginase for children with ALL in first marrow relapse. We determined the incidence of conventional non-hematologic dose limiting toxicities (DLT’s) and modeled the impact on a hypothetical phase I trial of a candidate agent with no additional toxicity.

Results: Among 111 patients on AALL01P2, 19% had conventional non-hematologic DLT’s. Induction therapy was judged clinically acceptable. With a traditional Phase I escalation scheme that accepts 0/3 and 1/6 DLT’s at a dose-level and rejects 2/3 and 2/6 DLT’s, an agent that adds no morbidity would be rejected as too toxic at any dose 30% of the time.

Conclusion: Background morbidity confounds identification of an acceptable dose of a non-toxic new agent tested in combination with conventional drugs for recurrent ALL. We propose a modification to the traditional Phase I design that increases the DLT thresholds to 1/3 and 2/6, which effectively compensates for background toxicity and reduces the chance of falsely rejecting an acceptable agent.
A Phase I Study of Oral 6-Thioguanine, Followed by Continuous Infusion Cytarabine, Followed by Intramuscular PEG-Asparaginase (“TGAP”) in Children with Relapsed or Refractory Acute Leukemia or Non-Hodgkin’s Lymphoma.
Authors: Fu Cecilia H. , Franklin Janet L. , Gaynon Paul , McCarthy Colleen , Panosyan Edward , Siegel Stuart E. , Avramis Vassilios I.
Published: April 2020

Background: We found enhanced cytotoxicity against human leukemia cell lines resistant to cytarabine with the sequence specific three-drug combination, 6-thioguanine (6TG) + cytarabine + PEG-asparaginase (“TGAP”) [Fu et al, Cancer Chemother Pharmacol 48:123–133, 2001] and developed a Phase I study to evaluate TGAP’s feasibility in children with relapsed/refractory leukemia or non-Hodgkin’s lymphoma with marrow involvement.

Patients and Methods: Three days of twice daily 6TG was followed by cytarabine @ 487 mg/m2 loading dose and 126 mg/m2/hr by 72-hour continuous infusion. Cytarabine was followed by pegylated asparaginase @ 2,500 units/m2. Complete response (CR) required fewer than 5% marrow blasts with recovery of neutrophils and platelets. Partial response required marrow blasts between 5% and 25% with no peripheral blasts.

Results: Between April 1999-January 2006, 11 patients, age 1 to 18 years, were enrolled after local IRB approval and individual or parental informed consent. Six patients had relapsed/refractory ALL, 4 patients had relapsed ANLL (2/4 had Down syndrome) and 1 patient had relapsed Burkitt’s lymphoma/leukemia. Six patients were enrolled at dose level #1 (6TG @ 100mg/m2/dose) and 5 patients were enrolled at dose level #2 (6-TG @ 150mg/m2/dose). The last patient enrolled was later found ineligible, but was included for toxicity. Chemotherapy was well tolerated in 9 patients. All patients developed grade 3–4 neutropenic fevers or bacteremia. Other grade 3–4 toxicities included transient elevations in transaminases. Asparaginase related pancreatitis occurred in two patients, in one with symptoms. Dose limiting acute vascular leak syndrome occurred in two patients at dose level #2 during the cytarabine infusion and just after the completion of therapy. Both required mechanical ventilation but recovered fully. Complete responses (CR) occurred in 2 of 10 evaluable patients and partial response (PR) occurred in 2 for total response rate (CR + PR) of 40%. All four responders tolerated a second course of TGAP. Both CR patients subsequently underwent matched unrelated BMT with no veno-occlusive disease or other excessive toxicity. In one CR patient, ara-CTP incorporation in the patients leukemic blasts ex vivo studied at baseline, pre chemotherapy, and repeated after 6TG and a 24-hour infusion of cytarabine showed a four-fold increase in intracellular ara-CTP, confirming our previous work in cell lines.

Conclusions: TGAP is a feasible combination for patients with relapsed leukemias. A phase II study is currently under development using the maximum tolerated dose for 6TG of 100mg/m2/dose in combination with CI ara-C and IM PEG-asparaginase.