bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel Stilbene-Based Antileukemic Agents Active in P-Glycoprotein Expressing and Apoptosis-Resistant Acute Leukaemia Cell Lines.
Authors: Grimaudo Stefania , Di Cristina Antonietta , Abbadessa Vincenzo , Daniele Simoni , Roberti Marinella , Tolomeo Manlio
Published: October 2019

The stilbene scaffold is a basic element for a number of biologically active natural and synthetic compounds and in accordance with Evans’ definition it can be considered as a privileged structure. One of the most relevant and studied stilbenes is Resveratrol, a phytoalexin present in grapes, endowed with chemopreventive and chemotherapeutic properties and able to induce apoptosis in different cancer cell lines. Since reduced apoptosis has been implicated in the development and progression of malignant tumors and in the occurrence of chemoresistant phenotypes, resveratrol-induced apoptosis might therefore contribute to its antitumor activity. However, resveratrol is a not potent cytotoxic compound if compared with others chemotherapeutic drugs and it is scarcely active in P-glycoprotein expressing (MDR) and Bcr-Abl expressing leukaemia cells. With the aim to find new stilbene compounds active in resistant leukaemia cells we synthesized a small library of resveratrol analogs, bearing the 3,5-dimethoxy motif at the A phenyl ring and amino, methoxy and hydroxy moieties at the 3′-and/or 4′-positions. Moreover, we synthesized analogues which incorporate a phenyl ring as bioisosteric substitution of the alkenyl bridge. Among these new stilbenes we identified two compounds endowed with interesting antileukemic properties: a) a methoxylated cis derivative active at nanomolar concentrations in P-glycoprotein expressing HL60-R and CEM VBL100 acute leukaemia cell lines and in P-glycoprotein and Bcr-Abl expressing K562-ADR cell line which is resistant to apoptosis induced by most common anticancer agents, and b) a terphenyl derivative active in MDR and Bcr-Abl expressing cell lines. Both compounds induced apoptosis prevalently through the mitochondrial pathway. Differently from resveratrol and other stilbenes, the therphenyl derivative induced a block of cells in G0-G1 phase of cell cycle which was associated to the shift of the phosphorylation state of pRb from hyperphosphorylated to hypophosphorylated. Morover, low concentrations of this compound were able to induced a potent granulocytic and monocytic differentiation of HL60 cells.
Switching Death Pathways in AML; Induction of Necrosis by Arsenic, Ascorbic Acid and Antabuse (AAA).
Authors: Nichols Gwen L. , Wagner Michael J. , Dinnen Richard , Fine Robert L.
Published: October 2019

Virtually all chemotherapy drugs induce apoptosis. Cancer cells have multiple mechanisms of resistance to this form of cell death. Necrosis may have fewer mechanisms of resistance, providing a rationale to discover ways to switch cell death pathways from apoptosis to necrosis. Arsenic trioxide (ATO) is clinically effective against acute promyelocytic leukemia and studies have suggested efficacy in treating other malignancies, including other leukemias. ATO increases the intracellular concentration of reactive oxygen species and inhibits glutathione (GSH) reduction resulting in redox changes which activate caspases and induce apoptosis. Ascorbic acid (AA) and other agents which reduce glutathione concentrations and increase hydrogen peroxide concentrations have been shown to enhance the antitumor activity of ATO in vitro. This combination of agents is currently in clinical trials. In previous experiments using pancreatic cancer cell lines, disulfiram (DSF), in clinical use as an inhibitor of aldehyde dehydrogenasel, was added to the ATO/AA combination. Synergistic induction of cell death occurred. We sought to test this combination in myeloid cell lines. We tested the ATO/AA/DSF (AAA) combination ([ATO], 1uM; [AA], 100uM; [DSF], 0.125 uM) on two myeloid leukemia cell lines, KG1 and K562. After treatment for 48 hours in AAA, annexin and propidium iodide staining revealed an increase in annexin(+) PI(+) cells (necrosis) in the KG1 cell line that was similar to the effect in pancreatic cancer cells. Nearly five fold more KG1 cells underwent necrosis after exposure to AAA than with arsenic alone. Arsenic combined with ascorbic acid or disulfiram yielded an approximately two fold increase in necrosis. We propose that the mechanism of action of disulfiram is through quenching peroxide (H2O2) radicals generated by ATO and AA. The reducion of DSF leads to oxidation of GSH and NADH which decrease [ATP]i, shifting the cells from apoptosis to necrosis. K562 cells were largely resistant to the triple combination, remaining in a state of cytostasis after exposure to AAA. BCR-ABL positive cell lines, such as K562, have a relative increase in intracellular ROS. The lack of necrosis in K562 was surprising, and suggests that the activated BCR-ABL kinase may override this proposed mechanism. Imatinib has been shown to induce necrosis, and combinations including imatinib will be tested in BCR-ABL positive cell lines. To study the potential safety of this combination for human trials, AAA was tested on peripheral blood stem cells from normal donors (obtained after informed consent) and did not appear to have synergistic effects on colony formation as compared to the drugs applied individually. These results suggest a new mechanism of cell death, switching from apoptosis to necrosis, that can be targeted to treat leukemias and other malignancies. More studies to confirm the mechanism of this cell death are underway.
The Role of Bcl-2 in Apoptosis Induced by khat (Catha Edulis) in Acute Myeloid Leukemia Cell Lines.
Authors: Bredholt Therese , Dimba Elisabeth A. , Johannessen Anne C. , Costea Daniela , Francis G. W. , Vintermyr Olav K. , Gjertsen5 Bjorn T.
Published: October 2019

The herbal drug khat (Catha edulis Forsk.) is habitually used in Eastern Africa and the Middle East. The pharmacological activity of khat is due to its alkaloid fraction which initiates a rapid release of monoamine neurotransmitters from nerve terminals when ingested. Extracts from khat have been shown to induce cell death caspase-dependent apoptosis in human leukaemia cells. In this study, apoptotic sensitivity is correlated with endogenous protein expression of the anti-apoptogen Bcl-2, as well as transfection enforced over expression of Bcl-2. Exposure of a panel of human leukaemia cell lines to khat extract resulted in the development of similar apoptotic morphotypes. The sensitivity of the cell lines to khat induced apoptosis revealed a trend of inverse correlation with endogenous levels of Bcl-2. Stable or transient transfection of Bcl-2 inhibited khat-induced apoptosis, and the threshold for khat-induced apoptosis was lowered by blocking Bcl-2 synthesis with small interfering RNA. Together, these data suggest Bcl-2 modulation as a possible mechanistic pathway through which khat-induced apoptosis may occur.
IgH and TCRgamma Gene Rearrangements, Cyclin A1 and HOXA9 Gene Expression in Biphenotypic Acute Leukemias.
Authors: Golemovic Mirna , Sucic Mirna , Zadro Renata , Mrsic Sanja Davidovic , Mikulic Mirta , Rajic Ljubica , Labar Boris , Batinic Drago
Published: October 2019

Biphenotypic acute leukemia (BAL) represents a rare type of acute leukemia (AL) with the reported incidence of 4.0–8.5% and poor response to treatment. In adult BAL, poor prognosis is related to unfavorable cytogenetics and P-glycoprotein over-expression (Legrand et al, Br J Haematol1998;100:147–55). It is presumed that better knowledge of its biological features could improve the classification and clinical management of this rare type of acute leukemia. Due to paucity of data regarding biological parameters of BAL, in this study we have analyzed the rearrangements of immunoglobulin heavy chain (IgH) and T-cell receptor g (TCRg) genes, expression of cyclin A1 (CycA1) and HOXA9 genes as well as in vitro growth of 10 de novo BALs and compared them with 15 AML and 15 ALL, respectively. The diagnosis of BAL was established according to EGIL criteria. BAL represented 4.3% of adult and 3.0% of pediatric patients with de novo AL referred to our institution during the 1999–2003 period. Of 10 BALs studied, lymphoid BAL (L2) was identified in 3 children, myeloid BAL (M1 and M5) was identified in 3 adults and one child, BAL of undifferentiated cytomorphology (AUL) in 2 adult patients, whereas one 5-month-old child presented with two separate blast cell populations (L1+M5b, bilineal BAL). With regard to immunophenotype, the group of BAL patients was also heterogeneous: 8/10 expressed the combination of myeloid and B-lymphoid antigens and 2/10 expressed myeloid and T-lymphoid antigens. The majority of BAL expressed CD34 (89%). Cytogenetic analysis was obtained in 7 BAL patients, of whom 3 showed chromosome 11 aberrations and 1 had Ph+ chromosome. Our results indicate that IgH and TCRγ gene rearrangements correlated well with lymphoid BAL morphology. Interestingly, the majority of lymphoid BAL showed oligoclonal amplification of antigen-receptor genes, indicating the coexistence of several malignant clones at the time of presentation. The expression of CycA1 correlated well with myeloid and undifferentiated BAL morphology. In contrast, the expression of HOXA9, a marker associated with myeloid cell lineage, showed no strong correlation with BAL morphology. BAL showed almost equal in vitro growth in three different serum-free culture conditions (no addition of GF, addition of IL-7 and FL or GM-CSF and FL), indicating that their growth was less dependent on the addition of exogenous GFs. Finally, in vitro growth of blasts during a 7-day culture showed autonomous cell growth in 3/10 AML and 3/8 myeloid BAL samples tested, but not in any of the AL with lymphoid features. Finally, further studies are needed to confirm these findings and to extend research to a broader spectrum of cell markers aiming to give a better definition of this rare group of AL.
Risk Factors in Childhood Acute Lymphoblastic Leukemia: A Single Center Experience in a Developing Country.
Authors: Anak Sema S. , Agaoglu Leyla , Akcay Arzu , Saribeyoglu Ebru T. , Atay Didem Y. , Garipardic Mesut , Unuvar Aysegul , Karakas Zeynep , Devecioglu Omer , Ozturk Gulyuz , Gedikloglu Gunduz
Published: October 2019

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, with the survival rates up to 80–90%, but in high-risk patients the survival rate is still unsatisfactory. The aim of this study is to analyze the pediatric ALL data of a single pediatric university center between 1987–2005 retrospectively to identify risk factors effecting the event free survival (EFS). In order to determine the risk factors possibly effecting the survival, we analyzed gender, age, physical examination findings, blood cell count, FAB morphology, immunophenotyping results, translocations and extramedullary involvement. During the same period, chemotherapy regimens used and response to these protocols were also analyzed. A total of 372 cases [220 male (60%) and 151 female (41%)] were diagnosed and treated in our center between 1987–2005. The age distribution was as follows: 7% patients under 2 years, 68% between 2–10 years, 25% above 10 years of age. At diagnosis, 76% patients had a hemoglobin level <10gr/dl, 56% WBC >10.000/mm3, 74% platelet <100.000/mm3. Primary CNS involvement was positive in 2.5%, mediastinal mass in 8% of all patients. The morphological subtypes were as follows: L1 64%, L2 32%, L3 4%. Immunophenotypic results revealed T ALL in 22%, mature B ALL in 8% and B ALL (common, pre B, proB) in 70%. All patients received CCG modified BFM protocol (80% of all patients) until 1999. Since then high-risk patients were treated with the augmented BFM protocol and L3 patients BFM NHL 95 Protocol, while standard risk patients continued to get the CCG modified BFM protocol. The remission rate at day 33 was 97.8%. Eighty-one patients relapsed (68% patients isolated bone marrow, 16% bone marrow + extramedullary, 9% CNS, 7% testes). According to the univariate analysis of our patient population, the factors negatively effecting the EFS were age <1 year, hepatomegaly >2cm., white blood cell >50.000/mm3 and platelet count <20.000/mm3, L3 FAB morphology, ≥M2 bone marrow status at day 14; CD3, HLADR, CD45 and Tdt negativity. According to the multivariate analysis the most important negative risk factors effecting the EFS were age <1 year, hepatomegaly >2cm. and ≥M2 bone marrow status at day 14 and CD 45 negativity. After a follow up of 72±59 months (1–300 months) 58% of patients are alive & well, 28% were lost to follow up and 14% patients succumbed to death. Overall survival (OS) for 60&120 months follow up were 83%, 83% and event free survival (EFS) 72%, 70% respectively. In conclusion, in ALL patients risk assessment is very important to conduct appropriate therapy. Identifying such factors and implementing risk adapted therapy will improve our treatment results decreasing the toxicity rates in pediatric ALL. Therefore treatment of all ALL patients still remains a challenge.
Peculiar Morphological, Cytochemical, Biochemical and Immunophenotyping Features in One Case of Acute Myeloid Leukaemia at the Onset.
Authors: Zini Gina , Rossi Elena , Garzia Mariagrazia , Rumi Carlo , Laurenti Luca , Di Mario Antonella , Sica Simona
Published: October 2019

On July 28th 2005, a 53-year old woman was admitted to our Hematology Dpt. with fever (37.5 C), fatigue, marked gingiva hypertrophy, mild hepatomegaly and absence of lymphoadenopaty and splenomegaly. Laboratory tests showed high leukocyte count of 182 x109/L, moderately increased platelet count (438 x109/L), normocytic anemia (10,9 g/dl, MCV 84 fl). Biochemical parameters, including lysozyme, were in the normal range except for high level of lactate dehydrogenase (2524 UI/l). Peripheral blood (PB) smear showed the presence of 94% of blasts, without evident granules. At the cytochemical myeloperoxidase staining blast cells revealed the presence of single or multiple Auer rods, while cytoplasmic granules were positive to the α-naphtyl butyrate esterase. In the bone marrow (BM) aspirate blasts were 80% showing same morphological and cytochemical features as in peripheral blood, while the granulocytic maturating series was 14%. No evidence of morphologically identifiable monocytic series in PB neither in BM. Megakariocytes were slightly increased and there was trilineage dysplasia. Blast immunophenotyping in PB and in BM was as follow: CD45, CD33, CD13, CD7 and HLA-Dr positive, 36% MPO positive and 48% co-express CD11c an CD117. Moreover blasts were CD14, CD19 and CD56 negative. FAB diagnosis was AML-M4. Interphase Fluorescence in situ Hybridization (FISH) for detection of AML1/ETO and bcr/abl gene rearrangements was negative. After treatment with 2 g hydroxyurea and leucapheresis, the WBC count was still high (140 x109/L) while after 1g of aracytin, WBC count decreased to 31000/mm3. Patient is presently enrolled into the GIMEMA protocol AML-12.

This case shows at the onset following abnormalities:

- an increased platelet count is a rare evenience in an AML at the onset; - negativity for lysozyme; - absence of CD14; - presence of Auer rods in quite all the blast population, which is a very rare evenience associated with monocytic subtypes of acute myeloblastic leukemia (AML); - atypical homogenous cytochemical pattern of blasts, showing Auer rods MPO positive and granules ANBE positive; - absence of any morphological differentiation of monocytic series. S4479
Reliability of Computer Algorithm-Based Binding Predictions for the Identification of Leukemia Antigens.
Authors: Gomez-Nunez Marta , Pinilla-Ibarz Javier , Dao Tao , Korontsvit Tatyana , Zakhaleva Victoriya , May Rena J. , Scheinberg David A.
Published: October 2019

Major Histocompatibility Complex class I (MHC-I) molecules present antigenic peptides to T cells on the cell surface as a prerequisite for stimulating cytotoxic T cell response. Thus, the ability to reliably identify the peptides that can bind to MHC molecules is of practical importance for rapid vaccine development. Several computer-based prediction methods have been applied to study the interaction of MHC class I/peptide binding. Here we have compared three of the most commonly used predictive algorithms BIMAS, SYFPEITHI and Rankpep with actual binding of HLA-A*0201 peptides in vitro. Forty six HLA-A*0201 peptides were selected from several target oncoproteins: Wilms’ tumor (WT1), native and imatinib- mutated bcr-abl p210 and JAK2 protein. Experimental peptide binding to HLA-A*0201 was assessed using a MHC stabilization assay on T2, TAP deficient cells. Peptides were considered to show positive in vitro binding if the mean fluorescence was at least 50 % of the binding of a high affinity reference peptide. Peptides qualified as positive in vitro if the BIMAS score was ≥ 100, the SYFPEITHI score ranked ≥ 24 or the Rankpep was ≥ 50. Results are summarized below:

BIMAS SYFPEITHI RANKPEP Sensitivity 84 % 72 % 60 % Specificity 76 % 71 % 81 % Positive Predictive Value 84 % 72 % 60 % Negative Predictive Value 80 % 68 % 63 %

Combining two or more computer methods did not appear to improve the predictive value. In conclusion, of the three predictive algorithms, the best correspondence with the actual MHC binding was demonstrated with the BIMAS algorithm. Predictive computer algorithms are important for preselection of potential T-cell epitope candidates for the application in vaccine design.
Redirection of CMV Specific CTL towards B-CLL Via CD20 Targeted HLA/CMV Complexes.
Authors: Mous Rogier , Savage Philip Savage , Remmerswaal Ester BM , van Lier Rene A.W. , Eldering Eric , Van Oers Marinus H.J.
Published: October 2019

Because B cell chronic lymphocytic leukemia (B-CLL) can not be cured with current therapies, but in general has a slow progression and rather long median survival, it is considered an attractive candidate for active T cell mediated immunotherapy. However B-CLL cells have poor antigen presenting capacity because they express low levels of co-stimulatory molecules. Moreover, most immunotherapeutic strategies require knowledge of the eliciting tumor antigen and/or ex vivo manipulation of patient cells. To circumvent these drawbacks we aim to redirect existing viral immunity towards B-CLL. Previously, we have shown that in patients with B-CLL considerably expanded numbers of cytomegalovirus (CMV)-specific CD45RA+CD27 CD8+ cytotoxic T cells are present (W. Mackus et al. Blood2003; 102:1057). These cells are potent cytotoxic effector cells when directed against B-CLL cells loaded with CMV peptide (A.Kater et al. Br.J. Haematol.2004; 126:512). In the current study, we apply a novel bridging reagent to redirect CMV-specific CTL to specifically target B-CLL. The targeting complex is composed of a streptavidin fused anti-CD20 single chain variable fragment (scFv) in combination with biotinylated MHC class I molecules containing CMV pp65 peptide (HLA/CMV). We demonstrate that this complex is stable on the cell surface for ≥24 hours, and that B-CLL cells coated with this CD20-HLA/CMV complex can be lysed by autologous CMV-specific CD8+ CTL with similar efficiency as B-CLL cells directly loaded with CMV-peptide. Killing occurs at scFv CD20 concentrations of ≥100 ng ml−1 and HLA/CMV concentrations of ≥20 ng ml−1. Lysis of CD20-HLA/CMV complex coated CLL cells could not be blocked by anti-LFA1 antibodies, in contrast to B-CLL cells directly loaded with CMV-peptide, indicating a different immunological synapse. HLA-A2 positive B-CLL cells coated with HLA-B7 /CMV complexes were only lysed by HLA-B7 positive CMV-specific CTL, whereas HLA-A2/CMV complex targeted HLA-A2 positive B-CLL cells were unaffected by HLA-B7 positive CMV specific CTL, proving HLA restriction of the killing.. Furthermore, CD20-HLA/CMV complex coated B-CLL cells induce both proliferation and cytokine production (interferon γ, tumor necrosis factor α, and macrophage inflammatory protein-1 β) in CMV-specific CD8+ T cells. Thus, CD20-HLA/CMV complexes elicit both immune activation and direct cytotoxicity towards B-CLL cells. The findings of our study constitute a necessary step towards possible application of CD20-HLA/CMV complexes for immunotherapy of B cell malignancies. It is obvious that this recently recognized capacity to redirect existing antiviral immunity towards tumor cells has a utility in cancer immunotherapy far beyond CMV and B-CLL.
The Results of Molecular Relapses Treatment in Acute Promyelocytic Leukemia (APL) Patients.
Authors: Shuravina Elena N. , Parovitchnikova Elena N. , Demidova Irina A. , Olshanskaia Yulia V. , Misiurin Andrei V. , Savchenko Valeri G.
Published: October 2019

Nearly all de novo APL patients (pts) undergo complete remission (CR) when treated with all- trans-retinoid acid (ATRA) plus chemotherapy. Nevertheless a significant portion (20%) of these pts ultimately relapse during the first 2 years of therapy. Minimal residual disease (MRD) monitoring using RT-PCR is widely recognized method for detection of molecular relapses (MR) in APL pts during hematological remission. However, the precise MR definition and significance of therapy changing when MR is detected are still controversial. We report the results of molecular monitoring and MR treatment in 40 pts (17 male, 23 female, median age = 33) who were included in the APL studies from 1997 to 2005. The induction and consolidation consisted of four 7+3+ATRA courses or four courses of 5 days Daunozome + ATRA or AIDA-protocol. Maintenance (MT) was performed for 2 years of CR with chemotherapy (5+2 courses) or chemotherapy combined with ATRA or monotherapy with ATRA+INF-alfa after Daunozome. RT-PCR for PML/RARα was performed on fresh marrow aspirates in all pts before treatment and periodically (2–3month) during 2 years after CR induction (2–18 investigations, median 6, for each pt). RT-PCR was performed using recommendations of BIOMED-1 Concerted Action (1999). MR was defined as probable if chimeric transcript was detected once and was not find out by second investigation and as proved when PML/RARA was detected at least twice by consecutive investigations (in 2–4 weeks). In case of proved MR we changed the type of MT. In pts with early MR (<12 mo of CR) MT was changed to Methotrexate+6-Mercaptopurine +ATRA. Pts with late MR (> 12 mo of CR) were treated with ATRA+IFN-α. After two hematological relapses (HR) in the group of probable MR we started to change the type of MT even if MRD was detected once. The Table 1 reflects the effect of MT changes on the frequency of HR in pts with MRs.

Table 1 Maintenance Probable MR Proved MR No MR/relapsed MT changed /pts relapsed MT unchanged/pts relapsed MT changed/pts relapsed MT unchanged /pts relapsed Chemotherapy (n=19) 3/0 2/1 3/0 1/1 10/1 Chemo+ATRA (n=15) - 6/1 1/0 - 8/1 ATRA+IFN- α (n =6) 1/0 1/1 1/0 - 3/2 Total (n=40) 13/3 6/1 21/4

Our data shows the equal frequency of HR in pts with and without MR: 4/19 and 4/21. The absence of difference is probably due to the treatment changes in case of MR. We also can admit that ATRA monotherapy as maintenance is ineffective approach (3 MR and 3 HR). We revealed the statistically significant difference in the probability of hetmatological relapse in pts with MR in whom MT was changed (0 from 9 relapsed) and not changed (4 from 10 relapsed) (p= 0,04). So it seems reasonable that in proved and probable MRs MT should be changed. However, more investigations should be done for definition of the best postremission treatment strategy in APL pts with MRD.
WT1 Expression in Childhood Acute Leukemias.
Authors: Spanaki Adriani , Linardakis Emmanouela , Perdikogianni Chryssoula , Stiakaki Eftichia , Cilloni Daniela , Morotti Alessandro , Kalmanti Maria
Published: October 2019

WT1 has been identified as a new molecular marker in acute leukemias, although the significance of WT1 overexpression remains unclear. The purpose of the present study was to investigate WT1 expression levels in children with acute lymphoblastic and myeloid leukemia. Twenty-one children were enrolled in the study. Thirteen patients (6 males-7 females), 11 with ALL and 2 with AML and a mean age of 8 years old (range from 4 months to 15 years old), were studied. Normal bone marrow from 8 children with non malignant hematological diseases was used as control. Total RNA was isolated either from frozen bone marrow samples using the TRIzol reagent protocol as described by the supplier or from mononuclear cells fractioned by Fycoll-Isopaque density gradient centrifugation, using the QIAamp RNA mini protocol following the manufacturer’s instructions. Qualitative PCR reactions were performed according to the protocols established by the European BIOMED 1 Concerted Action. Five of the 13 patients studied (38.5%), expressed high levels of WT1. Specifically, five of the 13 patients, three with ALL and two with AML, were positive for WT1. Four of the WT1 positive patients, who had additional adverse prognostic factors according to their treatment protocols have succumbed to their disease. One of the patients of the control group, while remaining under investigation for thrombocytopenia and leukopenia, revealed significantly high WT1 expression levels and was therefore excluded from the control group. In conclusion, though it has been reported that leukemic patients express WT1 at a higher percentage than the children in this study, our above findings are concomitant with the fact that WT1 is considered to be an adverse prognostic factor.
JAK2 V617F Tyrosine Kinase Mutation in Leukemia Cell Lines.
Authors: Quentmeier Hilmar , MacLeod Roderick A.F. , Reinhardt Julia , Zaborski Margarete , Drexler Hans G.
Published: October 2019

It has recently been shown that the majority of patients with polycythemia vera, and substantial numbers of patients with idiopathic myelofibrosis and essential thrombocythemia, carry a single nucleotide mutation in the JAK2 gene. The JAK2 V617F mutation was also found in patients with other myeloproliferative disorders, though at lower percentages. The JAK2 point mutation may contribute to the development of these diseases, and the elucidation of the cellular function of mutated JAK2 might facilitate the development of a suitable drug. In numerous cases, the effects of a mutationally altered protein on cellular functions like proliferation, differentiation or apoptosis, have been revealed using immortalized cell lines that carry the mutation in question. To find such a model system for JAK2 V617F in hematopoietic neoplasia, we tested a panel of 37 cell lines from patients with myeloproliferative disorders, myelodysplastic syndromes (MDS) and the megakaryoblastic variant of acute myeloid leukemia (AML M7) and compared these with cell lines derived from other types of AML (M1-M6), and acute lymphoblastic leukemia (ALL). Cell lines were tested for JAK2 V617F expression applying a DNA tetra-primer amplification refractory mutation system (ARMS), a method that specifically amplifies the normal and mutant sequences plus a positive control band in a single reaction. According to this method, three cell lines carried the JAK2 V167F mutation:

cell line MB-02 derived from a patient with a history of myelofibrosis and myeloid metaplasia that developed into AML M7; cell line MUTZ-8 established from a patient with AML M4 25 years after onset of MDS; cell line SET-2 from a patient at the stage of leukemic transformation of essential thrombocythemia.

We present data on these cell lines including JAK2 ARMS and sequencing results, cytogenetic analysis focusing on chromosome 9, and JAK-2 protein expression and activation. In summary, we detected three leukemia cell lines that carry the recently discovered JAK2 mutation. These cell lines promise to represent important research tools in the further analysis of this genetic alteration.
Use of Transmission Electron Microscopy in Diagnosis of Acute Leukemias: A Prospective Study of Fifty Cases.
Authors: Chatterjee Tathagata , Mahapatra Manoranjan , Pati Hara P. , Panigrahi Inusha , Kumar Rajat , Wadhwa Shashi , Saxena Renu
Published: October 2019

Ultrastructural studies have contributed substantially to the understanding of the cellular morphology of the acute leukemias. We present here 50 cases (aged 3-55 years, M: F-32: 18) of acute leukemias, which were studied for morphology, conventional cyto chemistry, immunophenotyping, and transmission electron microscopy (TEM) including ultrastructural cytochemistry using myeloperoxidase (MPO) and platelet peroxidase activity. TEM morphology using ultrastructural cytochemistry of MPO helped diagnose three cases of acute myeloid leukemia with minimal myeloid differentiation (AML M0). The blasts showed large round nuclei, 1–2 nucleoli, chromatin with peripheral condensation and abundant mitochondria. Of total of 5 cases of acute promyelocytic leukemias (APML), all had strong Sudan Black, MPO, dual esterase positivity; one case was non-specific esterase positive and sensitive to fluoride. On TEM, this unusual case was identified to be microgranular variant of APML. TEM morphology and ultrastructural cytochemistry using platelet peroxidase helped diagnose 3 cases of AML M7 (acute megakaryocytic leukemia), 2 cases of acute biphenotypic leukemias and also in differentiating one case of acute proerythroblastic leukemia (AML M6b) from 3 cases of AML M6a or acute erythroleukemia. Thus, TEM is helpful in differentiating further the subgroups of AML-M5 and AML-M6, in identifying the microgranular variant of APML, and in confirming the diagnosis of AML-M0 and biphenotypic leukemia. Also, in cases with very hypercellular marrow and with associated myelofibrosis, where the bone marrow aspiration gives low cell count, TEM and ultrastructural cytochemistry are a valuable aid to arriving at a accurate diagnosis.

Characteristics of acute leukemia cases (n=50) FAB Subtype Morphology, Conventional Cytochemistry and Immunophenotyping Transmission Electron Microscopy (?) = doubt in diagnosis AML-M0 3 (?) 3 AML-M1and M2 11 11 AML-M3 4, 1(?) 5: Hypergranular-4, hypogranular-1 AML-M4 5 5 AML-M5 5 5a-4, 5b-1 AML-M6 4 6a-3 (erythroleukemia), 6b-1 AML-M7 3 3 Biphenotypic 2 (?) 2 ALL-L1 8 8 ALL-L2 4 4
The Residual Malignant Cells Could Be Masked by Therapeutic Use of Granulocyte-Colony Stimulating Factor in the Patients with AML1/ETO+ Acute Myelogenous Leukemia.
Authors: Moon Hee Won , Kim Ho Young , Kim Young Ree , Cho Han Ik , Yoon Sung-Soo , Park Seonyang , Kim Byoung Kook , Chun Honggu , Kim Hee Chan , Lee Dong Soon
Published: October 2019

Among 200 AML cases for the past 7 years, we observed 6 cases with acute myelogenous leukemia(AML) which showed remission by morphologic criteria in BM examination, but revealed clonal changes in most cells by FISH after G-CSF administration. Remarkably, 5 of 6 cases were AML with AML1/ETO rearrangement, and FISH study revealed that most of AML1/ETO+ cells were mature neutrophils, suggesting differentiation of leukemic cells. A true remission of leukemia was probably never achieved with G-CSF alone and 4 of 6 cases have relapsed and 3 have died. Most cases of present study had infections or were suspected as infection, thus G-CSF which was administered and endogenously produced by infection seems to bring synergic effect (Table 1). To elucidate the mechanism involved in this finding, we measured the numbers of G-CSF receptor (G-CSFr) in AML1/ETO positive (Kasumi-1) and negative AML cell lines (CTV-1), and in leukemic cells from 8 patients with AML1/ETO positive and negative AML by flow cytometry. The number of G-CSFr was 2,673/cell in AML1/ETO+ Kasumi-1 cell line and 522/cell in AML1/ETO− CTV-1 cell line(Table 2). In 8 patients with AML, the number of baseline G-CSFr in AML1/ETO+ AML cells was significantly higher than that in AML1/ETO− AML cells (mean number 446.2 VS 226) (p value =0.0029). We assume that therapeutic G-CSF administration could result in differentiation and proliferation of AML1/ETO+ leukemic cells due to higher expression of G-CSF receptor. In conclusion, we strongly recommend that complete remission should be confirmed by FISH test, because malignant clone can be differentiated and masked in morphological examination or conventional cytogenetic test, especially for AML1/ETO+ AML.

Table 1. Clinical and laboratory summary of cases Case No. Initial diagnosis Follow-up* Cytogenetics FISH % Blast in BM Cytogenetics FISH Other findings Outcomes * when showing discrepancy between morphologic examination and FISH test 1 t(8;21)(q22;q22) AML1/ETO: 94.5% 0%(PB) - AML1/ETO:95.5%(PB) Pneumonia Relapse 2 t(8;21)(q22;q22) AML1/ETO: 98% 39% - AML1/ETO:99% Suspected infection, G-CSF administration Relapse, death 3 t(8;21)(q22;q22) AML1/ETO: 47% 0.5% t(8;21)(q22;q22) AML1/ETO:55% Suspected infection, G-CSF administration Alive 4 t(8;21)(q22;q22) AML1/ETO: 94.5% 3.9% t(8;21)(q22;q22) AML1/ETO:77.5% Fever Relapse, death 5 t(8;21)(q22;q22) AML1/ETO: 93.5% 0.3% Normal karyotype AML1/ETO:33.5% G-CSF administration Relapse 6 Trisomy 8 Trisomy 8: 98.5% 28.1% Trisomy 8 Trisomy 8:96.5% Candidiasis, G-CSF administration Death Table 2. Quantitation of G-CSF receptors in Kasumi-1 and CTV-1 cell line G-CSF receptor (PE molecule per cell) Baseline After G-CSF administration Kasumi-1 cell line 2,673 1,953 CTV-1 cell line 522 556
High FLT3 Transcript Level Is Associated with Unfavorable Prognosis in Pediatric Non-Promyelocytic Acute Myeloid Leukemia.
Authors: Kang Hyoung Jin , Lee Soo Hyun , Ahn Hyo Seop , Shin Hee Young
Published: October 2019

FLT3 is a receptor tyrosine kinase involved in the survival of hematopoietic stem cells, and mutations of FLT3 have been reported to be a risk factor in acute myeloid leukemia (AML), especially for pediatric patients. Recently, FLT3 expression level is proposed to have a prognostic significance in adults, but little is known for childhood AML.

FLT3 transcript levels at diagnosis were measured with real time quantitative reverse transcription polymerase chain reaction to verify the clinical significance in pediatric AML. FLT3 transcript levels were analyzed in 20 (M: 12, F: 8) pediatric patients with median 6 (0 – 16) years old received same treatment scheme (Leuk Res. 29; 617). Patients with secondary AML or RAEB-t were included, but patients with FAB M3 type were excluded. Relative gene expression levels were calculated by using standard curves made with KG-1 cell line copy and adjusted on the basis of the expression level of the GAPDH gene (106 copy). Each gene expression level was examined in triplicate, and the mean value was subjected to analysis. The copy number of FLT3 in each sample was calculated by comparing the Ct values of samples with standard curves. Mann-Whitney test was applied for comparison of means. For survival data, Kaplan-Meier life tables were constructed and the curves were compared by means of log-rank test.

The median value of adjusted FLT copy number was 58 (0.06–19,302) x 103. FLT3 copy number was not associated with gender, age and other risk factor except FLT3 internal tandem duplication (FLT3/ITD) which was present in 3 patients with significantly higher mean copy number (7,130 x 103) than others (372 x 103) (P = 0.023). The mean value of FLT3 copy number of 12 refractory or relapsed patients (2,278 x 103) was higher than that of patients in continuous remission (48 x 103), significantly (P = 0.031). The overall survival rate of 10 patients with high FLT3 copy number more than 50 x 103 (25.0%) was lower than that of others (59.3%) significantly (P = 0.0457). All patients with high copy number were refractory to induction or relapsed early before post remission transplantation except 2 patients (one received bone marrow transplant from matched sibling and another received autologous peripheral stem cell transplant).

High FLT3 transcript level was an unfavorable prognostic factor for early relapse and overall survival in pediatric AML and associated with FLT3/ITD. New therapeutic scheme such as stem cell transplantation with more intensive conditioning just after complete remission and administration of FLT3 inhibitor could be applied in pediatric non-promyelocytic AML patients with high FLT3 transcript level.
Prolonged Survival of a Patient with De Novo Acute Myeloid Leukemia (AML) and Trisomy 13 with Intensive Chemotherapy and Autologous Peripheral Blood Transplantation (PBSCT): A Case Report.
Authors: Joshi Nitin D. , Amin Alpesh , Nath Rajneesh
Published: October 2019

Trisomies are uncommon cytogenetic abnormalities in patient with de novo AML. Survival of patients with trisomy 13 ranges from 0.5 to 14.7 months. We present the treatment outcome of a 71-year-old man with de novo AML and trisomy 13 who had PBSCT in first complete remission. A 71-year Puerto Rican male was diagnosed with AML in April 2003. His CBC showed WBC count 177 K/mm3, hemoglobin 10.3 gm/dl, platelets 43 K/mm3 and blast cells 75%. Flow cytometry revealed that the leukemic blasts were CD33, CD13, CD11c and CD56 positive but negative for CD34. Cytogenetics failed to yield any metaphases. Peripheral blood FISH studies revealed trisomy 13 positivity in 300 of 325 cells analyzed. Patient received induction chemotherapy with high dose Ara-c (HiDAC) 3g/m2 QD x 5 doses and mitoxantrone 80mg/m2 on day # 2. Bone marrow done day 28 post induction chemotherapy revealed residual leukemic blasts. Cytogenetics showed that one out twenty metaphases had trisomy 13 along with translocation t (9:18) (q34; q10). 11.9% of cells had trisomy 13 by FISH analysis. The patient then received a second cycle of chemotherapy with HiDAC at 2 g/m2 Q12 x 12 doses. Bone marrow biopsy on day 35 following reinduction chemotherapy revealed normocellular-regenerating marrow in remission and FISH was negative for trisomy 13. On the third cycle of chemotherapy, patient received Etoposide 11 mg/kg. Neupogen was started on day #3 and 10.3 x 106 CD34 positive cells/kg were collected. The patient then underwent autologous PBSCT using Melphalan 160 mg/m2 as the preparative regimen. On Day +87 and Day +182 post transplant, bone marrow biopsy showed complete remission with FISH negative for trisomy 13. The patient is still alive 27 months after initial treatment and 22 months post PBSCT. Autologous PBSCT in first complete remission for AML with trisomy 13 may provide a superior survival than chemotherapy alone.
A Comprehensive Set of In Vitro Transcribed RNAs as Controls for Leukemia Molecular Diagnostics.
Authors: Hedges John , Ye Fei , Labourier Emmanuel
Published: October 2019

Introduction: Leukemia is one of several cancers in which genetic sub-types are differentially responsive to specific chemotherapeutic agents, with success rates being directly proportional to early detection and treatment. Detection of specific fusion transcripts from chromosomal translocations (sub-typing) is required for rapid differential diagnosis and initiation of treatment strategies. Since leukemia assays are often performed in small test runs in many facilities, a pooled control that minimizes kit usage and cost by supplying multiple markers in a convenient single-tube format is optimal. As a means to better assess the accuracy and robustness of leukemia assays, we have developed a simple yet flexible external control comprised of in vitro transcribed RNA fusion transcripts of leukemia-associated chromosome translocations. These transcripts include b2a2, b3a2, e1a2, E2A/PBX1, TEL/AML1, AML1/ETO, PML/RARa (Long and Short forms), MLL/AF4 (e10e4 and e9e5) and inv16 (A and D types). To accompany this positive control is translocation-negative, HL-60 cell-line RNA. It serves as a negative control to assess assay background levels.

Materials and Methods: The RNA fusion transcripts include about 150 nucleotides flanking each side of the transcript fusion point. Additionally, we fused an MS2 bacteriophage operator to the 3′ end of the transcript sequences to provide a binding site for MS2 coat protein dimers for eventual conversion into Armored RNA Quant™, a stable synthetic quantitative standard that can be utilized upstream in the workflow to run alongside patient samples at the collection and RNA extraction steps. Once all the sequences have been verified for their accuracy, the transcripts were manufactured individually and formulated into a mixture with HL-60 total RNA to mimic leukemic cell extract conditions. The HL-60 total RNA was also tested as an external negative control.

Results: We demonstrated that these controls can be used with the Signature LTx assay for simultaneous detection of twelve leukemia-related fusion transcript targets in a single well. We anticipate that these control transcripts will be compatible with current conventional RT-PCR or real-time PCR platforms as well.

Conclusion: The twelve transcripts in a single-tube pooled positive control plus the accompanying negative control will provide clinical laboratories with an invaluable tool for tracking day-to-day performance for both multiplex and simplex leukemia molecular diagnostic assays.
Pin1 in Acute Myeloid Leukemia Blast Cells.
Authors: Hangen Hanne , Reinhard Dirk , Griesinger Frank , Truemper Lorenz , Wulf Gerburg M. , Wulf Gerald G.
Published: October 2019

Pin1 is a peptidyl-prolyl-isomerase catalysing conformational changes on ser/thr-pro-sites of proteins, and has been shown to play a pivotal role for the activity of cell cycle regulating proteins in breast and prostate cancer cells. Overexpression of Pin1 has been found in many cancers, and we here present first data on its expression in blast cells of patients with acute myeloid leukaemia (AML). Having documented Pin1 expression in leukemic cell lines with highest expression levels in the promyelocytic leukemia cell line HL60, we analyzed the expression of Pin1 in bone marrow samples with high blast counts from 24 pediatric and 28 adult patients with AML by semiquantitative RT-PCR and flow cytometry. Expression levels varied considerably, ranging from undetectable in 16 of 52 cases, to very high levels of up to 13.7 units normalized on abl-expression in the RT-PCR. Pin1 levels did not correlate with patient age, nor the expression of progenitor or myeloid differentiation markers on the blast cell population. Also, Pin1 level did not segregate with FAB classification, nor the prognostic implications of cytogenetic findings. Comparing Pin1-negative to Pin1-positive leukaemia cases at a median observation time of 32 months by Kaplan-Meier analysis, we found a tendency towards better event free (p=0.01) and overall survival (p=0.058) for the Pin1-positive cases. In summary, Pin1 was found in the leukemic blast cells of most patients with AML, and further investigations on prognostic significance and function of Pin1 in AML appear warranted.
Stimulatory Auto-Antibodies to the PDGF Receptor in Patients with Chronic GVHD.
Authors: Olivieri Attilio , Svegliati Silvia , Campelli Nadia , Luchetti Michele Maria , Trappolini Silvia , Poloni Antonella , Leoni Pietro , Gabrielli Armando
Published: October 2019

Background

Experimental data are consistent with the hypothesis that activation of the PDGF receptor (PDGFR) is characteristic of scleroderma (SSc) fibroblasts and may contribute to their activation. We have recently demonstrated that fibroblasts from SSc patients contain high Ha Ras and ROS (Reactive Oxygen Species) levels and constitutive activation of ERK1/2 (Svegliati et al: JBC in press). Furthermore, SSc patients have circulating auto-antibodies against the PDGFR which induce type I collagen gene expression in normal human fibroblasts through the Ha Ras-ERK1/2- ROS pathway (Svegliati et al: Submitted). These findings suggest that anti PDGFR auto-antibodies play a pivotal role in the pathogenesis of scleroderma. Clinical chronic graft-versus-host disease (cGVHD) can show manifestations that are very similar to those of SSc. Although it is conceivable that the two diseases can share a similar pathophysiological mechanism there are no data supporting this assumption. In view of these considerations we tested the hypothesis that patients with cGVHD have serum auto-antibodies that stimulate PDGFR and activate collagen gene expression in fibroblasts.

Methods

Serum from 7 patients with extensive cGVHD showing scleroderma-like features either in the skin or in the lung was analyzed for the presence of stimulatory autoantibodies to PDGFR. Patients receiving allogeneic transplantation, but without any signs of cGVHD were used as controls. The median F-U after transplant was 23 months (range 16–36) in patients with cGVH and 42 (range 9–51) in the control group. The assay was carried by incubating purified IgG of the patients with mouse embryo fibroblasts carrying inactive copies of PDGFR α or β chains (PDGFR −/−) or the same cells expressing PDGFR α or β, respectively. Production of reactive oxygen species was assayed in the presence or absence of specific PDGFR inhibitors. The antibodies were characterized by immunoprecipitation, immunoblotting and absorption experiments in primary human fibroblasts and endothelial cells.

Result

Stimulatory antibodies to the PDGFR were selectively found in all patients with cGVHD and fibrotic lesions. The antibodies specifically recognized PDGFR, induced tyrosine phosphorylation and ROS accumulation. Their activity was completely and selectively abolished by pre-incubation with cells expressing PDGFR α or β chains or by PDGF receptor tyrosine kinase inhibitor. Anti-PDGFR antibodies induced selectively Ha-Ras-ERK1/2 and ROS cascade and stimulated the expression of type I collagen gene and myofibroblast phenotype conversion in normal human primary fibroblasts. Antibodies were absent in all controls.

Conclusions

Stimulatory auto-antibodies against PDGFR represent a specific hallmark of patients with cGVHD. Their biological activity on fibroblasts strongly argues for a causal role in the pathogenesis of the disease.
Increased Risk for Venous Thromboembolism in Patients with Acute Leukemia.
Authors: Mohren Martin , Markmann Ilka , Jentsch-Ullrich Kathleen , Koenigsmann Michael , Lutze Gerd , Franke Astrid
Published: October 2019

Introduction: Patients with solid tumors have an increased risk for venous thromboembolisms (VTE) associated with substantial morbidity and mortality, but so far there exists little data on hematologic malignancies. We have recently found a VTE rate of 7,7% in patients with malignant lymphoma. However, information on patients with acute leukemia (AL) is very limited so far.

Patients and methods: Medical records of all patients with AL treated in our institution between january 1992 and april 2005 were reviewed and data was collected and analyzed in a microsoft excel data base. P-values to show correlation of VTE with leukemia type, patient age and gender were calculated using Fisher’s exact test. All reported p-values are two-sided.

Results: Of a total of 455 patients 310 (68%) had AML, 108 (24%) had ALL and 37 (8%) had blast crisis. 248 patients (55%) were male and 207 (45%) were female, median age was 60 years. 55 patients with AL (12,1%) had at least one VTE, occuring during therapy in 82% of events. 27 patients (5,9%) had central venous catheter associated VTE, whereas 28 patients (6,2%) had deep vein thrombosis and/or pulmonary embolism. Neither leukemia lineage - myeloid versus lymphocytic - (p=1,0) nor patient gender (p=.193) had an impact on the VTE risk. However, central venous catheter associated VTE more likely occured in younger patients (< 60 years) than in patients ≥ 60 years (p=.003). There was no statistically significant difference in the incidence of non-central venous catheter associated VTE between both age groups (p=.563).

Discussion: Patients with acute leukemia have a substantial risk for VTE, half of which occur with the use of central venous catheters. A recently published study analysed the incidence of VTE in close temporal relationship to onset of disease and found a VTE rate of 2,09% with equal risk in ALL and AML. Our study included VTE occuring prior to diagnosis of AL as well as during chemotherapy and follow up, revealing a VTE rate that is 6-fold higher and showing a considerable association with central venous catheters.

Conclusions: The risk of VTE is surprisingly high in patients with acute leukemia, thus improvement of prophylactic measures, especially in regard to central venous catheter use is warranted.