bluuebunny/crossref_metadata_2025_split huggingface dataset:
WT1 Expression in Childhood Acute Leukemias.
Authors: Spanaki Adriani , Linardakis Emmanouela , Perdikogianni Chryssoula , Stiakaki Eftichia , Cilloni Daniela , Morotti Alessandro , Kalmanti Maria 
Published: October 2019

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.