bluuebunny/crossref_metadata_2025 huggingface dataset:
Duffy Antigen/Receptor for Chemokines (DARC): Is There a Role in Prostate Cancer?.
Authors: Anyona Samuel B. , Zheng Shiyan , Wheeler Thomas , Ittmann Michael , Hayes Teresa G. , Mims Martha P.
Published: October 2019

Introduction: The Duffy blood group antigens function as chemokine receptors and as receptors for several species of malarial parasites. Approximately 70% of African Americans are Duffy negative as a result of a single nucleotide polymorphism in the promoter region of the Duffy antigen/receptor for chemokines (DARC) gene in a consensus-binding site for GATA 1. This mutation abolishes the promoter activity in erythrocytes, impairing the expression of DARC on red blood cells. Other than resistance to certain species of malaria, the functional consequence of Duffy negativity is unclear; however, it has recently been proposed that the Duffy antigen acts as a biological ‘sink’ to clear pro-inflammatory chemokines from tissue microcirculation. Further it has been suggested that since the incidence of prostate cancer in African Americans is 60% higher than in Caucasians, absence of the Duffy antigen might predispose African Americans to prostate cancer by impairing downregulation of proinflammatory cytokines. We tested the hypothesis that lack of expression of DARC on erythrocytes predisposes African American men to develop prostate cancer.

Methods: We conducted a case control study of 89 African American men with confirmed prostate cancer and 51 age matched healthy African American men. The samples were genotyped for the promoter polymorphism using an allele specific real time PCR assay (Taqman assay) developed for this study.

Results: The frequency of the Duffy negative allele was 75.8% among the patients and 81.4% among the controls (odds ratio = 0.72, p value = 0.28). The distribution of heterozygous and homozygous Duffy negative patients did not differ between the cases and controls.

Conclusion: It has been suggested that individuals lacking erythrocyte expression of DARC have higher prostate levels of angiogenic chemokines that might promote more rapid development of prostate cancer. The data presented here found no difference in the frequency of the Duffy negative allele between African American men with prostate cancer and healthy age matched African American controls. Since several studies suggest that polymorphisms associated with differential production of IL-8, IL-10 and VEGF are risk factors for prostate cancer, it remains possible that a polymorphism associated with differential cytokine production in combination with Duffy negativity increases the risk for prostate cancer.
The Tyrosine Kinase src Is Highly Activated in AML and Plays a Crucial Role in Leukemic Cell Proliferation.
Authors: Schuch Gunter , Schafer Ellen , Eggert Katharina , Loges Sonja , Jucker Manfred , Hossfeld Dieter K. , Bokemeyer Carsten , Fiedler Walter
Published: October 2019

The tyrosine kinase pp60src is involved in several signal transduction pathways such as signalling of hematopoetic growth factors and cytokines. The viral form v-src was the first oncogene described and mutations of regulatory tyrosine residues in cellular src (c-src) have been linked to malignant transformation. However, no mutations in the gene of c-src have been described in leukemia so far, although some data of src mutations in solid tumors have been reported. The current study was undertaken to examine the role of src in acute myeloid leukemia (AML).

Blood and bone marrow specimen of patients with newly diagnosed or recurrent AML treated at our institution were sampled. AML cell lines or CD34 positive cells of healthy donors served as positive and negative controls, respectively. RNA was isolated, and RT-PCR was performed using 4 different primer pairs spanning the coding region of c-src. Protein expression and phosphorylation was studied after protein extraction and Western blot analyses using src and phospho-src specific antibodies. The effect of src inhibitors PP1 and PP2 on leukemic cell proliferation was studied in human and murine cell lines. Mutational analyses of the coding region were performed using SSCP/heteroduplex and bi-directionally sequencing.

In all 60 patients analyzed expression of c-src mRNA was detected by RT-PCR. Western blot analyses confirmed strong expression of src on the protein level and revealed a robust activation of the protein as determined by tyrosine phosphorylation. Inhibition of src phosphorylation by src-specific inhibitors PP1 and PP2 was detected by Western blot using an antibody specific for phospho-src. Incubation of leukemic cells with PP1 and PP2 caused significant inhibition of proliferation in a dose dependent manner. Mutational analyses as performed by SSCP/heteroduplex and bi-directionally sequencing revealed wildtype sequence in all cell lines and 60 clinical samples.

In summary, pp60src is highly expressed and activated in cell lines and clinical samples of human AML. Moreover, phosphorylation of src is essential for leukemic cell proliferation. Mutations in the coding sequence of c-src causing constitutive activation could be excluded by mutational analyses of primary AML samples. These data suggest that pp60src plays a crucial role in AML and src inhibition by targeted therapy might offer a useful new approach in the treatment of AML.
X-Ray Cristallography Characterization of Human CD44 Epitopes Releasing Differentiation Blockage in Acute Myeloid Leukemia Blasts.
Authors: Lecomte Nicolas , Durand Laetitia , Roseto Alberto , Chomienne Christine , Kadouche Jean , Smadja-Joffe Florence
Published: October 2019

CD44 is a highly glycosylated type-1 transmembrane protein with multiple isoforms, including the standard one, CD44s (80kDa), which is the most abundant on hematopoietic cells, and alternatively spliced variant isoforms (CD44v). CD44 is strongly expressed on Acute Myeloid Leukemia (AML) blasts, which are immature myeloid cells which terminal differentiation is blocked at various stages, defining the distinct AML subtypes. In our laboratory, we have previously shown that several specific monoclonal antibodies (mAbs) directed to the CD44s isoform, can release the differentiation blockage of primary AML blasts (Charrad et al., Nature Medicine 1999), in all AML subtypes. These results indicated the possibility of using such mAbs to develop a CD44-targeted differentiation therapy that would be efficient in all AML subtypes. Our present aim is to characterize the molecular structure of the CD44 epitopes involved in the release of AML differentiation blockage, by using X-ray crystallography. In order to obtain high levels of soluble CD44 protein and to preserve its native conformational properties, we have constructed distinct fusion proteins comprising the glycosylated CD44s extracellular domain plus the human IgG1 Fc fragment (CD44-Fc). We have also added a polyhistidine tag, which allows to easily purify them by using Ni2+- and protein G affinity, and a TEV specific recognition sequence to cleave off the fusion partner. Eight distinct CD44-Fc constructions have been prepared, transiently expressed in COS-7 cells and the one displaying both the highest affinity for the anti-CD44 mAbs and the highest expression level, has been transfected into CHO-K1 cells. Using a stable cell clone, we have succeeded to produce large amounts of CD44-Fc (100 to 200 mg/L), in serum-free culture medium, using CELLine bioreactor flasks. This optimization of the CD44 production combined with an efficient three-step purification process (successive specific binding to G protein, to Ni2+, concluded by highly resolutive gel filtration), has allowed us to produce, for the first time, a sufficient quantity of highly pure and glycosylated CD44s extracellular domain for structural studies. SPR (Surface Plasmon Resonance) analysis have shown that the 2 differentiation-inducing anti-CD44 mAbs analyzed so far, differ in their dynamics of interaction with the CD44 protein. The molecular structure of the epitopes specifically mapped by differentiation-inducing anti-CD44 mAbs, as revealed by X-ray crystallography, will be presented and discussed, with the perspective of developing an efficient CD44-targeted differentiation therapy in AML.
Bcl-2 siRNA Induces Apoptosis and Enhances the Sensitivity to Arsenic Trioxide in NB4 Cells.
Authors: He Dongmei , Zhang Yuan , Liu Gexiu
Published: October 2019

Bcl-2 is the prominent member of a family of proteins responsible for dysregulation of apoptosis, prevention of cancer-cell death, and resistance to chemotherapy and radiotherapy. Overexpression of Bcl-2 protein was shown to result in resistance to a variety of apoptosis-inducing signals in acute leukemia. Arsenic trioxide (As203)induces clinical remission in acute promyelocytic leukemia with minimal toxicity and apoptosis in APL-derived NB4 cells at low (0.5 to 2 μmol/L) concentration. Short interfering RNA (siRNA) has been evaluated as an attractive and effective tool for suppressing a target protein by specifically digesting its mRNA. In this study, we studied the effect of transfection with siRNA targeting Bcl-2 in NB4 cells in terms of proliferation and induction of apoptosis. Further,we investigated whether the Bcl-2 siRNA may enhance the sensitivity of NB4 cells to arsenic trioxide. Synthetic siRNA was transferred against Bcl-2 into NB4 cells using Oligofectamine transfection protocol in the presence or absence of arsenic trioxide. RT-PCR and Western blotting analysis of Bcl-2 expression in NB4 cells was performed after transfection. The inhibition of cell growth was assessed by a modified MTT assay, counting cell number. Apoptosis was determined by morphological observation and flow cytometric analysis. When synthetic Bcl-2 siRNA was delivered into NB4 cells, no Bcl-2 transcripts were detected at 24h, and Bcl-2 protein levels were reduced by approximately 72% for 48h after siRNA transfer. Bcl-2 siRNA treatment for 72h led to 36% apoptosis of cells,as compared to control siRNA treatment. Control siRNA had no significant effect on growth and apoptosis of cells. Simultaneous administration of As203 and Bcl-2 siRNA was able to reduce the mRNA and protein expression of Bcl-2 significantly compared to Bcl-2 siRNA alone demonstrating a synergistic effect of combined As203 and siRNA treatment (P<0.05). Proliferation in NB4 cells was strongly inhibited (about 86%) following combination treatment for 72h as opposed to only 40% after administration of 0.5μmol/L As203 alone. When NB4 cells were incubated with 0.5μmol/L As203 for 72h after siRNA transfer, the percentage of apoptotic cells significantly increased (P<0.05), compared with combined As203 and control siRNA treatment or As203 treatment alone, respectively. No significant difference on apoptosis was seen in cells treated with As203 alone or treated with As203 and control siRNA. These results show that Bcl-2 siRNA could induce apoptosis and enhance the sensitivity to arsenic trioxide in NB4 cells. Our results suggest that the combination of arsenic trioxide and Bcl-2 siRNA might be an effective anti-leukemia protocol.
Comparative Effect of the Oral Iron Chelators ICL670A and CP20 on Cell Proliferation, Apoptosis and Polyamine Metabolism in Rat and Human Liver Cell Cultures.
Authors: Gaboriau François , Chantrel-Groussard Karine , Pasdeloup Nicole , Nick Hans Peter , Brissot Pierre , Lescoat Gérard
Published: October 2019

Iron overload increases the mitotic index in rat hepatocyte cultures stimulated by EGF. Iron, implicated in tumoral cell proliferation, promotes the development of the hepatocellular carcinoma. Conversely, iron chelation decreases cell proliferation. Polyamines play also an important role in cell proliferation. The purpose of the present work was to compare the effect of the two oral iron chelators ICL670A and CP20 on cell proliferation, apoptosis and polyamine metabolism in rat and human liver cell cultures.

We used three experimental models: the rat hepatoma cell line FAO, the rat liver epithelial cell line RLEC and the human hepatoma cell line HUH7. Chelator cell uptake was analyzed by mass spectrometry. DNA replication and cell viability were measured by thymidine incorporation and mitochondrial succinate dehydrogenase activity. Cytotoxicity was evaluated by extracellular LDH activity. Cell cycle analysis was performed by flow cytometry. Apoptosis was quantified by DNA fragmentation and caspase 3 activity. Polyamine metabolism was analyzed by measuring their intracellular concentrations by mass spectrometry as well as the ornithine decarboxylase (ODC) and spermine-spermidine acetyl transferase (SSAT) mRNAs levels. ICL670A entered the cells four times better than CP20. We observed that ICL670A (20 μM) decreased DNA replication (−75%) and cell viability (−40%) while a comparable CP20 stoechiometric concentration (30 μM) had no significant effect. ICL670A exhibited also a higher cytotoxicity than CP20. The decrease in DNA replication induced by ICL670A was correlated to a cell cycle block in S phase; CP20 (30 μM) increased slightly the cell number in G2/M phase while a higher CP20 concentration (150 μM) blocked clearly cell cycle in G2/M phase. By measuring DNA fragmentation and the activity of the executioner caspase 3, we demonstrated a high apoptotic effect of ICL670A (20 μM) compared to CP20 (30 μM). In the HUH7 human cell line, we observed that ICL670A (20 μM) decreased the intracellular levels of polyamines which was correlated to a drop in ODC and SSAT mRNAs levels. Conversely, an increase in SSAT mRNA level was observed in the presence of CP20 (150 μM) leading to an enhancement of putrescine and spermidine levels.

In conclusion, ICL670A and CP20 inhibit cell proliferation by two distinct ways. ICL670A compared to CP20:

enters better the cells; exerts a higher antiproliferative and apoptotic effect; modulates polyamine metabolism by inhibiting ODC and SSAT enzyme activities.
Arsenic Trioxide Enhances STI571-Induced Apoptosis of K562 Cells through Downregulating Bcl-XL and Bcr-Abl.
Authors: Zhang Ri , Zhang Xuhui , Zhi YaJun , Zhu ZiLing , Wu De Pei
Published: October 2019

We aimed to evaluate the effects of arsenic trioxide (ATO, Trisenox) on STI571 (Gleevec, imatinib mesylate)-induced apoptosis of a chronic myelogenous leukemia (CML) cell line, K562 cells. Cell prolifration and colony-forming assays were performed to determine the cytotoxicity of ATO alone and in combination with STI571. Apoptosis was analyzed by morphological changes, apoptosis rate and cell cycles. An Elisa assay was used to detect the levels of cytosolic cytochrome c (cyt c) and caspase-3 in K562 cells exposed to ATO and STI571 at graded concentrations. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to assay the transcriptional levels of Bcl-XL and Bcr-Abl genes in K562 cells. Results showed that both the colony-forming assay and cell proliferation assay demonstrated additive to synergistic effects of ATO on STI571-induced apoptosis in K562 cells, a Bcr-Abl positive cell line. Caspase-3 was activated during apoptosis and there was an increase in cytosolic accumulation of cytochrome c. Treatment of K562 cells with STI571 alone led to down-regulation of transcriptional levels of Bcl-XL at 12 hours and Bcr-Abl at 96 hours after drug administration. Treatment with ATO alone only led to reduce the mRNA levels of Bcl-XL, but not Bcr-Abl. Combined treatment with ATO and STI571 down regulated the transcripts of Bcl-XL at 12 hours and Bcr-Abl 72 hours after drug administration. We conclude that ATO enhanced cytotoxic and proapoptotic actions of STI571 could be mediated by the down-regulation of Bcr-Abl and Bcl-XL genes in K562 cells. Therefore ATO in combination with STI571 could be a promising therapy for CML.
A Phase I Study of Temozolomide (Temodar®) in Pediatric Patients with Relapsed or Refractory Leukemia—A Children’s Oncology Group Study.
Authors: Horton Terzah M. , Dolan Eileen , Hegde Madhuri , Berg Stacey L. , Adamson Peter , Blaney Susan M.
Published: October 2019

A phase I study of oral temozolomide, administered daily for 5 days every 28 days, was performed in children with relapsed or primary refractory leukemias. The starting dose was 200 mg/m2/day with subsequent dose escalation to 260 mg/m2/day. Ten patients (5 male, 5 female), median age 9 years (range: 1–18 years), with leukemia (6 with acute lymphocytic leukemia [ALL], 4 with acute myelogenous leukemia [AML]) received a total of 14 cycles of temozolomide. Serum pharmacokinetic (PK) samples were obtained in 6 patients using a limited pharmacokinetic sampling strategy. Samples were also obtained from 8 patients for correlative biology studies, including analysis of methyl-guanine methyl transferase (MGMT) activity and microsatellite instability (MSI). MGMT activity was measured in 7 patients; all 3 patients with ALL tested for MGMT had elevated MGMT activity, with a median MGMT activity of 1240 fmol/mg protein (range: 356–1756). In contrast, 3 out of 4 patients with AML had MGMT activity below the limits of detection. Eight patients were tested for microsatellite instability by multiplex PCR using a panel of 13 MSI loci. Two patients (one with ALL and one with AML) demonstrated probable MSI instability. Temozolomide has been well tolerated, with no patients developing dose-limiting toxicity to date. One patient with AML, undetectable MGMT activity and no microsatellite instability had a partial response (PR) lasting 4 cycles (<10% myeloblasts in bone marrow). Further analysis is underway to determine the frequency of elevated MGMT activity in ALL and AML patients at diagnosis and following relapse.
HuMax CD20 Fully Human Monoclonal Antibody in Chronic Lymphocytic Leuchemia. Early Results from an Ongoing Phase I/II Clinical Trial.
Authors: Coiffier Bertrand , Tilly H. , Pedersen L. M. , Plesner T. , Frederiksen H. , van Oers M.H.J. , Wooldridge J. , Kloczko J. , Holowiecki J. , Hellmann A. , Walewski J. J. , Flensburg M.F. , Petersen J. , Robak T.
Published: October 2019

The fully human monoclonal IgG1 antibody HuMax-CD20 targets a novel epitope of the CD20 molecule on B-cells. HuMax-CD20 stops growth of engrafted B-cell tumors in SCID mice more efficiently than Rituximab and i.v. infusion of HuMax-CD20 in cynomolgus monkeys leads to profound, long lasting, dose-dependent B-cell depletion. Data are presented from an open label, dose-escalation, multicenter phase I/II clinical trial. 3 cohorts of 3 (A), 3 (B) and an extended cohort of 27 (C) patients with relapsed or refractory chronic lymphocytic leukemia (B-CLL) received 4 weekly i.v. infusions of HuMax-CD20 and will be followed for 12 months. The first infusion administered was 100 mg, 300 mg and 500 mg in cohort A, B and C. The following 3 infusions were of 500, 1000 and 2000 mg, respectively. Patients were premedicated with oral acetaminophen and i.v antihistamine and received i.v. glucocorticoids before first and second infusions. The endpoints are B-cell depletion, adverse events, objective response according to the NCI working group guidelines for CLL, time to progression, duration of response, time to next anti-CLL treatment, and pharmacokinetics. Biopsies and CT images are evaluated centrally. Median age was 61 years; median time since diagnosis was 6.3 years. Maximum tolerated dose was not reached. Adverse events were predominantly observed on days of infusion and as expected most frequently symptoms of cytokine release e.g. rash, increased sweating, fatigue, rigors, pyrexia, and headache. 5 serious adverse events assessed as related to HuMax-CD20 treatment have been reported: hepatic cytolysis, herpes zoster, neutropenia (2 patients) and one death from pneumonia at week 4. In cohorts A and B, markedly reduced CD19+CD5+ cell counts were observed in 3 of 6 patients one week after final treatment and the depletion was sustained in one of these patients. In cohort C a pronounced CD19+CD5+ reduction was demonstrated by all patients; in fact the lymphocyte counts were ≤4.0 x 109/L in 21 patients. At week 11, ≥50% reduction in the product of diameters of lymph nodes was observed in 12 of 20 evaluable patients with enlarged lymph nodes. A response rate of 52% (11 of 21 evaluable patients in cohort C) was observed at week 11: 4 clinical CR (bone marrow and CT pending), 7 PR, 3 SD and 7 PD. In conclusion, this preliminary analysis of data from the first 33 CLL patients treated with HuMax-CD20 demonstrated significant depletion of CD19+CD5+ cells, a favorable safety profile and an indication of clinical efficacy. An updated report for all patients at week 19 will be presented.
Clinical and Interphase-FISH Studies of Deletion of Derivative Chromosome 9 in Patients with Chronic Myelogenous Leukemia.
Authors: Wu Wei , Xue Yong-quan , Wu Ya-fang , Pan Jin-lan , Shen Juan
Published: October 2019

Objective: To determine the frequency of the deletion of derivative 9 [der(9)] among chronic myeloid leukemia (CML) patients with classic Ph translocation and variant Ph translocation, and to assess the association between this deletion and clinical prognosis.

Methods: Cytogenetical analysis of bone marrow cells was performed by direct method and /or 24h culture method. RHG banding was used for karyotype analysis. Dual-color and dual-fusion DNA probe was used to perform interphase-FISH to investigate the deletion of der(9) in Ph+ CML patients and all patients were followed up.

Result: Cytogenetical studies showed typical Ph translocation in 76/105 and variant Ph translocation in 29/105. Interphase-FISH studies showed deletion of der(9) in 12 cases(15.8%) of 76 patients with classic Ph translocation and in 4 cases (13.7%) of 29 patients with variant translocation. The frequency of deletion was similar in classic and variant translocations (P>0.9). This result is contrary to previous reports which suggested that deletions are much more common in variant Ph translocation than in classic Ph translocation. When the deletion was seen in a patient, it was present in all the Ph+ metaphases and nuclei. Three patients with heterogeneous cell populations mixed with cells with single 5′-ABL or 3′-BCR deletion and with both 5′-ABL and 3′-BCR deletion. It may suggest clone evolution in the progression of deletion. Complete clinical information was available in 54 patients. There were no significant difference in peripheral leukocyte count, platelet count, hemoglobin and percentage of peripheral blood blast cells between patients with and without der(9)deletion. However, the results of following up showed that the median survival time of patients with der(9) deletion was significantly shorter than those without der(9) deletion (34 months vs 76 months; P<0.05, log-rank Test).

Conclusion: A deletion of der(9) is seen in about 1/6 Ph+ CML patients in china, with which Ph+ CML patients have shorter median survival time than those without it, indicating that it is a poor prognostic index. For evolution the prognosis of CML patients more precisely, it is best to perform cytogenetical analysis and FISH analysis for der(9) deletion simultaneously at diagnosis.
Evaluation of Molecular Response by Real-Time RT-PCR in Acute Promyelocytic Leukemia (APL): A Direct Comparison with Regular RT-PCR.
Authors: Hu Jiong , Liu Yuan-Fang , Zhu Yong-Mei , Zhang Shu-Jiang , Gu Bai-Wei , Shen Zhi-Xiang , Hu Jiong
Published: October 2019

PURPOSE: In this study, we systemically compare the molecular response by regular reverse transcript polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR in newly diagnosed patients with acute promyelocyte leukemia (APL).

PATIENTS AND METHODS: A total of 31 newly diagnosed patients with APL who entered remission by induction therapy with ATRA plus As2O3 were included in this study with median follow-up of 38 months (27–46 months). Serial bone marrow samples were obtained before and after the induction, consolidation chemotherapy and every 3 to 6 months during maintenance therapy. Regular RT-PCR and real-time RT-PCR were carried out to evaluate the molecular response. The PML-RARa fusion transcript level was calculated in two ways: normalized PML-RARa level as DoseN and log-reduction compared to the pre-treatment level.

RESULTS: A total of 29 patients remained disease free for a median of 27 to 46 months. Comparing two PCR assays, the sensitivity limit for real-time RT-PCR was 5.7 log DoseN reduction while the limit for regular RT-PCR was about 3 log. Furthermore, we established criteria for evaluation of molecular response by real-time RT-PCR: ≥3.0 log reduction of PML-RARa transcript level as minor molecular response; ≥5.0 log reduction as major molecular response and undetectable level of PML-RARa transcript as complete molecular responses. Minor molecular response by real-time RT-PCR was achieved in 22.6% and 96.8% of patients after induction and consolidation chemotherapy, which was equivalent to molecular response by regular RT-PCR (35.5% and 96.8% respectively). The major molecular response rate was 12.9% and 90.3% after induction and consolidation whereas the complete response was at 3.23% and 77.4% respectively. All patients achieved major and complete molecular remission during and after maintenance therapy. Loss of either major or complete molecular response occurred in two patients while both patients experienced subsequent loss of minor molecular response, which coincided with CNS leukemia and bone marrow relapse.

CONCLUSION: Real-time RT-PCR was more sensitive than regular RT-PCR and the evaluation criteria for real-time RT-PCR was established and proved to be clinically relevant.
Serum Tryptase Levels in Acute Leukemia at Diagnosis: Correlation with CBF AML.
Authors: Cairoli Roberto , Ripamonti Carla B. , Granata Simonetta , Folli Daniele , Brasca Paola , Grillo Giovanni , Bertani Gianbattista , Intropido Liliana , Marenco Paola , Nosari Annamaria , Pezzetti Laura , Morra Enrica
Published: October 2019

Α and β tryptase genes cluster on the short arm of human chromosome 16 and encode lineage-associated serine proteases that are abundantly expressed in mast cells and, in trace amounts, in basophils. Under physiologic conditions no other myeloid cells express tryptases. However, in several myeloid leukemia cell lines and in AML blasts, the level of tryptase is elevated. In vitro, AML blasts are capable to produce and release the α-protryptase constitutively. Recently, elevated levels of serum tryptase have been detected in certain FAB subtypes of AML, particularly in AML-M4eo. In an attempt at correlating the levels of tryptase with FAB classification and cytogenetics, we have analyzed serum samples collected at diagnosis, from 103 AML and 57 ALL patients referred to our Institution. The total serum concentration was determined by UniCAP 100 and UniCAP Tryptase Fluorenzyme Immunoassay Kit (Pharmacia-Upjohn, Uppsala, Sweden). The median value of tryptase level in the control group (50 healthy people; mean age 35 y, range 20–50; M/F= 26/24) amounted to less than 5 ng/ml. We found elevated tryptase levels (more than 15 ng/ml) in 46 out of 103 AML-patients (44.6%) and in 1 out of 57 ALL-patients (1.75%) (p = <0.0001). In AML, the patients showing elevated tryptase levels resulted equally distributed among the FAB subtypes (M0, M1, M2, M3, M4, M4Eo, M5) (p = 0.269). In the group of 18 AML with t(8;21) or inv(16), we recorded the highest frequency (89%) of cases with tryptase more than 15 ng/ml (p =< 0.001) and the highest median serum tryptase levels (>89.3 ng/ml) (p = 0.0001), in comparison with other cytogenetic groups (diploid, n= 43; t(15;17), n=10; other, n=29). Our data suggest that elevated serum tryptase levels at diagnosis may play a role as a marker for CBF AML subtypes.
Complete Cytogenetic Response in a Case of T-PLL with a Complex Karyotype after Treatment with Alemtuzumab.
Authors: Marinato André F. , de Oliveira Fábio Morato , Jácomo Rafael H. , Rizzatti Edgar G. , Falcão Roberto P. , Rego Eduardo M. , Tone Luiz G. , Simões Belinda P.
Published: October 2019

T-prolymphocytic leukemia (T-PLL) is an aggressive T-cell leukemia characterized by the proliferation of prolymphocytes with a mature postthymic T-cell phenotype and commonly involves the blood, bone marrow, lymph nodes, liver, spleen, and skin. Leukemic cells appear as small to medium-sized prolymphocytes or small variant cells, occasionally with cerebriform nuclei. T prolymphocytes are CD2+, CD3+, and CD7+ and may express CD4+CD8−, CD4+CD8+, or CD4−CD8+ markers. The most frequent chromosomal abnormalities are t(14;14)(q11;q32), inv(14)(q11q32), t(X;14)(q28;q11), i(8)(q10), and t(8;8)(p12;q11). Although patients treated with conventional chemotherapy generally have a poor prognosis, alemtuzumab (Campath®) has been associated with good clinical responses in patients with T-PLL. Routine cytogenetic analysis to determine the response to alemtuzumab has not been performed in patients with T-PLL. This study reports the complete cytogenetic remission achieved following alemtuzumab treatment in a single patient with T-PLL. The 41-year old male patient presented with weight loss, night sweats, diffuse lymphadenopathy and hepatosplenomegaly, in the absence of any skin rash. Peripheral blood counts were: platelets 128 x 109/L and leukocytes 93.6 x 109/L, and hemoglobin values were 12.5 g/dL. The lymphocytes were mostly medium-sized with round to irregular nuclei; some cerebriform cells were also observed, with intermediate chromatin, single evident nucleoli, and a basophilic cytoplasm. Human T-cell lymphotrophic virus-1 serology was negative. Immunophenotypic studies showed CD2+, CD3+, CD5+, CD7+, CD4−, CD8+, CD1−, terminal deoxynucleotidyl transferase-negative (TdT-), and T-cell receptor a/b+. Classic cytogenetics and spectral karyotyping (SKY) were performed and an abnormal karyotype was observed in 18 metaphases analyzed: 46, t(X;14)(q28;q11), t(Y;14)(q12;q11), r[i(8)(q10)]. These abnormalities have not been previously reported in cases of T-PLL. The patient then underwent chemotherapy with CHOP, and subsequent therapy with fludarabine. The patient was then treated with alemtuzumab, 30 mg twice a week, for a total of 7 doses. The patient achieved complete clinical, hematological, and cytogenetic remission, characterized by lack of symptoms, reduction of lymph nodes, liver, and spleen size to normal volumes, and the disappearance of prolymphocytes from peripheral blood. Cytogenetic studies performed 7 months after treatment by classic cytogenetics and SKY revealed a karyotype of 46,XY[20]. This study is the first to demonstrate a complete cytogenetic remission following treatment with alemtuzumab in a patient with T-PLL that was refractory to standard chemotherapy. This is especially impressive in a patient with a complex karyotype and with different cytogenetic alterations not previously described.
Acquired Extramedullary Resistance to Dasatinib Due to Selection of Philadelphia-Positive Lymphoblast Clone Harboring a T315I BCR-ABL Gene Mutation: Reversal by Dose Escalation and Hydroxyurea.
Authors: Lundan Tuija , Gruber Franz , Hoglund Martin , Simonsson Bengt , Knuutila Sakari , Porkka Kimmo
Published: October 2019

Most patients with advanced Philadelphia-positive (Ph+) hematologic malignancies develop resistance to imatinib. Acquired resistance to imatinib is commonly a result of selection for subclones bearing point-mutations in the catalytic kinase domain of BCR-ABL. Dasatinib (BMS-354825), a dual-specific SRC/ABL kinase inhibitor, has shown activity in imatinib-resistant Ph+ diseases both in vitro and in vivo. Preliminary data also indicate efficacy in patients. Based on laboratory evidence, dasatinib appears to inhibit all known BCR-ABL mutant clones, with the exception of T315I, a gatekeeper mutation conferring resistance to several kinase inhibitors. Here we describe a Ph+ ALL patient, who initially developed imatinib resistance (hematologic) possibly due to BCR-ABL amplification (FISH). His disease relapsed as extensive extramedullary tumors bearing wild-type BCR-ABL. He received dasatinib 70 mg BID as part of the BMS CA180–015 study and achieved a very good partial remission. After 5 months of therapy, the disease relapsed as a solitary axillary tumor and several small palmar skin lesions. He also had blasts in the CSF indicative of neuroleukemia. Bone marrow remained in cytogenetic remission. FISH analysis of the tumor revealed 2–3 copies of BCR-ABL as previously. A highly sensitive, quantitative, mutation-specific PCR (Gruber F, ASH 2004) showed the presence of the T315I mutation, which was confirmed by sequencing. A very low level of T315I transcript was also detected in the blood. Dasatinib dose was escalated to 100 mg BID, and low-dose hydroxyurea 500 mg BID was initiated to putatively enhance the access of dasatinib in the CSF sanctuary. He also received two doses of i.t. therapy (methotrexate, cytarabine). Patient’s symptoms (confusion, headache) related to neuroleukemia resolved rapidly, skin lesions disappeared and axillary tumor decreased in size. He is currently symptom-free and has no signs of active ALL. The favorable response to dasatinib dose escalation and low-dose hydroxyurea was unexpected. Preclinical data on T315I mutant cell lines would argue against a significant concentration dependence in kinase inhibition by dasatinib. Putatively, targets other than BCR-ABL may be of importance in particular in Ph+ ALL (e.g. Src, Lyn), and this effect may account for the response. Similar off-target activity of hydroxyurea is utilized in clinical trials to overcome resistance to multidrug HIV therapy - a setting resembling current treatment of Ph+ malignancies with kinase inhibitors.
Assembling the Players for Evaluation of Anti-Leukemic CTL Activity in NOD-SCID Mice.
Authors: Hsu Andy K.W. , Kerr Beverley M. , Lock Richard B. , Hart Derek N.J. , Rice Alison M.
Published: October 2019

Acute Lymphocytic Leukemia (ALL) patients, who relapse after hematopoietic stem cell transplantation, have a poor prognosis with few therapeutic options. An immunotherapeutic approach that enhances the graft versus leukemia effect post transplant may improve the survival of relapsed patients. We postulate that dendritic cells (DC) derived from cord blood CD34+ stem cells will be ideal for this. Further we propose the use of RNA as a source of antigen to induce T cell responses to poorly immunogenic tumours, such as ALL. We have shown that CD34+ cells can expand and differentiate into DC and are functional antigen presenting cells. In addition, we regularly achieve >90% transfection efficiency with >90% cell viability when CD34+-DC are electroporated with eGFP mRNA. Electroporation of CD34+-DC with flu mRNA results in processing, translation and presentation of epitopes by the DC, which are recognised by flu-specific cytotoxic T lymphocytes (CTL). We have induced anti-leukaemic CTL with leukaemic total RNA from the HLA-A2+ NALM-6 cell line. We have established a NOD-SCID mouse model of primary human ALL to test the efficacy of the anti-leukaemic CTL in vivo. Mice engraft within 4–6 weeks of transplantation. Mice transplanted with ALL3 had 93.9±0.8% (BM) and 84.3±4.7% (spleen) CD45+cells at sacrifice. Mice transplanted with ALL7 had 79.1±5.5% and 89.9±1.2% CD45+ cells in the BM and spleen respectively. Cells from the BM and spleen have been cryopreserved and total RNA extracted for later CTL generation. Experiments aimed at replicating the minimal leukaemia burden post transplant, show that the kinetics of appearance of human cells in the blood and the time of palpable splenic engraftment relates to the cell dose. This suggests that while the leukaemic burden is low, the kinetics of growth are identical and thus, by varying the inocula we model emerging relapse. Thus, we have “assembled the players” and are ready to test the efficacy of anti-leukaemic CTL generated by stimulation with RNA loaded CD34+-DC to control in vivo leukaemic growth.
Granulocytic Sarcoma (GS) as First Manifestation of AML.
Authors: Khalil Maged , Malik Tamer , Mousa Albeir , Kalavar Madhumati , Fogler Richard , Stephen Lichter
Published: October 2019

Granulocytic sarcoma (GS), or chloroma is an extramedullary tumor of immature myeloid cells which occurs throughout the body, often associated with acute myeloid leukemia (AML) or in the accelerated phase of chronic myelogenous leukemia, but rarely as the first manifestation of AML, preceding the onset in marrow and blood by months or years[1, 2] Herein, we report GS of lymph nodes presenting as first manifestation of AML

Case Report

A 53 years old male presented with recurrent episodes of epigastric abdominal pain without history of trauma or infections, Physical examination was normal. Laboratory examination showed WBC 5.2, ANC 4.4, Hemoglobin 13.6 gm/dl, PLT.306, 000 and Retic 1.8. Computerized Tomography (CT) of abdomen and pelvis showed extensive abdominal and pelvic lymphadenopathy, gastric wall thickening and prominence of the pancreatic head versus peripancreatic lymphadenopathy. Subsequently Esophagogastroduodenscopy and colonoscopy were both negative for malignancy. Peripheral smear showed normal RBC, and no myeloblasts. Laparoscopic retroperitoneal lymph node biopsy revealed granulocytic sarcoma, with immunohistochemistry positive for CD43, CD117, CD68 and MPO

Bone marrow biopsy and aspirate were performed, showing increased M: E ratio, increased blasts (29%), with flow cytometry reporting positivity for CD13, CD33, CD34, CD117, and HLA-DR supporting the diagnosis of AML. Chromosomal analysis including Fluorescence in situ hybridization studies (FISH) showed no evidence of chromosomal abnormalities. Patient continued to have epigastric pain radiating to the back without fever, night sweats or weight loss.

He was treated with standard induction chemotherapy with Ara-c (7 days) and Idarubicin (3days), resulting in complete remission with resolution of the abdominal and pelvic lymphadenopathy.

Patient is currently receiving consolidation therapy with high dose Ara-c

Conclusion

Granulocytic sarcoma is a rare disease. The overall prognosis is poor. However, AML-type chemotherapy appears to improve survival.
Novel Therapeutic Approach Yields Excellent Results in Minimally Differentiated Acute Myeloid Leukemia (AML-M0): Time To Think out of the Box.
Authors: Banavali Shripad , Goyal Lovenish , Padhye Bhavana , Arora Brijesh , Bhagwat Roshni , Gujral Sumit , Nair Chandrika , Parikh Purvish
Published: October 2019

Background: AML M0 has dismal complete remission (CR) rates (<50% in most series) and poor long term survival with conventional combination chemotherapy (CT). Thus it is considered a candidate for allogenic bone marrow transplantation (BMT) in first remission and also for other novel therapeutic approaches. There is increasing recognition of angiogenesis as a pathogenic and prognostic factor in AML. The objective of our study was to evaluate the efficacy of low-dose oral metronomic CT in patients with AML-M0.

Materials and methods: Eleven patients fulfilling the diagnostic criteria for AML-M0 and ineligible for standard CT and/or BMT were accrued in this pilot protocol between June 1998 and Feb 2005. The treatment consisted of Prednisolone 40 mg/m2/d, Etoposide 50 mg/m2/d and 6-TG 40 mg/m2/d (PET), given orally on out-patient basis continuously for 21 days every month. Post-induction high dose Ara-C (HD-Ara-C) was given whenever possible.

Results: The median age was 17 yrs (range 1.5 to 52); 9 were males and 2 females. The median leukocyte count at presentation was 15.4 x 109/L (range 0.9 to 142) and platelet count was 62 x 109/L (range 21 to 442). CD56 and CD7 were positive in 3/5 and 8/11 of those evaluated. Cytogenetic studies were done in 7 patients. Ten of the eleven patients received at least 1 cycle of PET CT of which 9 (90%) achieved CR. The eleventh patient received only 1 week of CT before being lost to follow up, however by then she had already cleared all blasts (57%) in the peripheral blood. Importantly the whole induction was given on out patient basis with only 2 patients requiring admission. There was minimal to nil blood, platelet and antibiotic support required during the entire Induction therapy. Four of them received HD-Ara-C after achieving CR; two of them died in remission at 6 and 11 months. The other two are alive in CR at 36 months and 14 months. Others who could not get HD-Ara-C because of socio-economic reasons were continued on PET or ET chemotherapy for up to maximum of 12 cycles. The median overall survival was 20 months for those who continued treatment. So far no relapses are noted in those who received PET x 2 followed by HD-Ara-C.

Conclusions: The dramatic remission rates, excellent tolerability, minimal cost and morbidity with this protocol provide proof of principle that metronomic approach may be a superior alternative to conventional induction chemotherapy in the treatment of AML-M0. Additionally, incorporation of HD-Ara-C consolidation and metronomic maintenance may improve the long term outcome of this disease with dismal prognosis.
Prognostic Significance of Early Peripheral Blood Blasts Clearance Following Induction Chemotherapy for Adult Acute Myeloid Leukemia.
Authors: Pakkala Suchita , Winton Elliott , Langston Amelia , Heffner Leonard T. , Khoury Hanna J.
Published: October 2019

Early bone marrow blast clearance, defined by a remission marrow obtained on day 14 post-induction, is an independent prognostic indicator of CR, OS, EFS and RFS in adult AML. The prognostic significance of early peripheral blood blast clearance is unknown. We sought to evaluate the relationship between time to peripheral blood blast clearance post-induction and results of the day+14 bone marrow in a cohort of 100 adult AML patients homogeneously-treated at Emory University, between 1997 and 2004. Patients with acute promyelocytic leukemia were excluded from this analysis. In this cohort, median time to peripheral blood blast clearance was 5 days (range, 1–18). Patients were divided into 2 groups: early blast clearance (< 5 days; n=43; EBC) and delayed blasts clearance (≥ 5 days; n=57; DBC). Patient characteristics are summarized in the Table. EBC patients presented with a lower WBC (8.4 vs. 32.4) and LDH (99 vs. 389), but had comparable cytogenetic risks. Remission day+14 marrow, defined as a hypocellular marrow (<20%) with <5% blasts morphologically, was attained in 74% and 79% in the EBC and DBC groups, respectively. However, a trend for improved relapse-free and overall survival was observed in the EBC group. In conclusion, our analysis shows that early peripheral blood blasts clearance post-induction chemotherapy does not correlate with a remission day 14 marrow but may predict improved outcomes.

Patient Characteristics EBC DBC Number of patients 43 57 Gender, M/F 20/23 34/23 Median age (range), yrs 49 (23–86) 56 (18–79) Median WBC (range), μLx103 8.4 (0.4–278) 32.4 (0.4–763) Median LDH (range), U/L 99 (84–2700) 389 (59–2042) Cytogenetic Risk Groups Favorable 12% 18% Intermediate 59% 51% Unfavorable 26% 28% Unknown 5% 3% Remission day 14 Bone marrow 74% 79% Median DFS, months 22 18 Median OS, months 23 19
SNS-595, a Novel S-Phase Active Cytotoxic, Demonstrates Pharmacologic Properties Appropriate for the Treatment of Advanced Hematologic Malignancies.
Authors: Lawrence Chris E. , Arbitrario Jen P. , Kumer Jeff L.
Published: October 2019

Despite advances in the treatment of leukemia patients, response rates with intensive chemotherapy regimens remain less than 30% for relapsed or refractory acute leukemias and advanced chronic myelogenous leukemias. SNS-595, is a novel naphthyridine analog small molecule, a class of drugs not previously used in cancer treatment. SNS-595 acts specifically during the S-phase of the cell cycle to induce rapid apoptosis of cells that are actively synthesizing DNA, and a subsequent cell cycle arrest in the G2 phase of the cell cycle. Sunesis Pharmaceuticals, Inc., is developing SNS-595 for the treatment of both solid and hematologic malignancies. SNS-595 was previously evaluated in two models of hematologic cancer in nu/nu mice, causing a dose-dependent tumor growth inhibition (97% to 99%, n=6 mice per treatment group) in a LM-3 Jck (human acute lymphoma) hematologic xenograft. All 6 mice treated at 30 mg/kg had a major reduction in tumor volume, with 2 mice showing a complete response. SNS-595 also significantly inhibited tumor growth in mice bearing CCRF-CEM (human acute lymphoblastic leukemia) xenograft tumors. In phase I clinical trials in solid tumors the dose-limiting toxicity was neutropenia, and the incidence of gastrointestinal effects, nausea, and vomiting was low. No mucositis or peripheral neuropathies were observed. In the present study, CD1 mice (n=4 per treatment group) were administered either SNS-595 15 mg/kg or 20 mg/kg, by IV bolus on days 0 and 4, or the S-phase acting drug, cytarabine (Ara-C) 150 mg/kg, BID, by IP injection on days 0 and 4. SNS-595 was well tolerated, with no body weight loss observed through to day 16. Bone marrow isolated from the femurs showed a dose-dependent reduction in cellularity on day 6, 48hr after the second of the two SNS-595 administrations; at 15 and 20 mg/kg, cellularity was reduced to 15% (±2.9, n=4) and 7.5% (±1.4, n=4) respectively. SNS-595, 20 mg/kg, reduced absolute neutrophils to a nadir of 51 ±24 cells/μL blood (n=4) on day 8, from 1244 ±55 cells/μL at day 0 (n=4), but subsequently rebounded to normal levels by day 16. Total WBCs also reached a nadir on day 8, returning to normal by day 16. In the same timeframe cytarabine caused bone marrow ablation, with absolute neutrophil counts substantially reduced at day 8 and returning to normal by day 16. In conclusion SNS-595 has significant anti-tumor activity in hematologic xenografts, as well as significant ability to reversibly ablate murine bone marrow cells. These properties, combined with the good clinical safety profile observed in phase I patients, suggest that SNS-595 has the potential to demonstrate anti-tumor activity in patients with hematologic malignancies.