bluuebunny/crossref_metadata_2025 huggingface dataset:
Bloodless Cancer Treatment Results of Patients Who Do Not Want Blood Transfusion: Single Center Experience of 77 Cases
Authors: Kim Sung Hyun , Oh Sung Yong , Kwon Hyuk-Chan , Lee Suee , Lee Dong Mee , Kim Kyeong Hee , Han Jin-Yeong , Park Joo- In , Lee Jong-Hwan , Lee Kyu-Yeol , Kim Hyo-Jin
Published: April 2020

Background: Patients may be unwilling to accept blood products for religious reasons – Jehovah’s Witnesses (JW). Because of its minority, as well as JW patients, physicians have a lot of difficulty and anxiety to treatment. Furthermore, sometimes cancer patients are needed transfusion during operation or chemotherapy. So, we analyze the clinical outcomes of JW cancer patients who had treated for identifying the risk of treatment and transfusion need.

Methods: We analyzed 77 cases of histological confirmed cancer patients (JW) from Jan. 2001 to Apr. 2008.

Results: Male to female ratio was 1:1.48. Median age of patients was 59 years (range, 8 –83 years). The most common primary site was stomach (20.8%) followed by breast (14.3%), colorectal (11.7%), liver (7.8%), lung (6.5%), and hematologic malignancy (5.2%). At diagnosis, 53 patients (68.8%) were operable early cancer patients. Operation was done in 47 patients (89%). three patients refused operation itself. Change of CBC profile after operation was observed in hemoglobin (Mean ± SD; 12.7 ± 2.1 g/dL to 10.6 ± 2.3 g/dL, P<0.001). WBC and platelet were not affected by operation (P=0.818, P=0.179). Twenty-eight patients received adjuvant chemotherapy. Of them, 21 patients (78%) had completed their planed schedule. There is no treatment related transfusion need or death during adjuvant chemotherapy. Initially, 20 patients had been presented with advanced stage and 8 patients relapsed after curative treatment. 127 cycles of palliative intravenous chemotherapy was administrated in 19 patients. G-CSF and erythropoietin was used in 45 and 20 cycles of treatment, respectively. Grade I/II anemia (38.6%) and leucopenia (42.5%) were common hematologic toxicities. Grade ≥III thrombocytopenia and anemia were observed in 3.9% and 2.4%. Only 1 patient had stopped chemotherapy due to thrombocytopenia. Reduced dose chemotherapy was administrated at 23 cycles (18.1%). The median overall survival for all the patients has not yet been determined. 3 year and 5 year survival rate was 80% and 70%, respectively. Major cause of death was disease progression, not bleeding

Conclusion: Bloodless cancer treatment was not accompanied with serious complication. A few cases of palliative chemotherapy were needed transfusion, but adequate physician’s assist - dose modification and use of G-CSF and erythropoietin - could make keep the patient’s hope and quality of life.
The Effect of Soluble P-Selectin in Coagulation
Authors: Korakas Ingrid , Guillen Darlene , Martin Erika , Tranholm Mikael , Barnett Thomas , Li Chester
Published: April 2020

P-selectin, a member of the selectin family, is a vascular cell adhesion molecule. It is expressed and stored in alpha granules of platelets and in Weibel-Palade bodies of endothelial cells, and upon activation, P-selectin is translocated/transferred to the membrane surface. A key function of the P-selectin is to mediate leukocyte, lymphocyte and platelet interactions in inflammation and possibly in the thrombus formation. A soluble variant, S-Psel, comprising the extracellular domain of P-selectin, has been identified in healthy individuals, but is markedly elevated in patient with vascular disorders. Recent work on S-Psel suggested that S-Psel may play a role in hemostasis/coagulation through the generation of procoagulant TF-bearing microparticles (MP), and therefore, has potential in treating patients with the bleeding disorders, like hemophilia. The aim of this study is to verify studies reporting that S-Psel exhibits in vitro and in vivo pro-coagulant activity. S-Psel and S-Psel-Fc (IgG) fusion were purchased commercially or prepared at Novo Nordisk Research US (NNRUS) and their biological activity was verified by P-selectin/Pselectin glycoprotein ligand-1(PSGL-1) interaction in vitro. The clotting times of human whole blood and plasma treated with S-Psel or S-Psel-Fc, or with an irrelevant human IgG control protein, were measured by thromboelastography and aggregometry respectively. After up to 8 hours of incubation with S-Psel and S-Psel-Fc at a concentration of 15ug/ml, we found no significant difference between samples treated with S-Psel, S-Psel-Fc and the IgG controls. The ability of S-Psel to generate TF-bearing microparticles in human whole blood was examined in a FXa substrate cleavage assay; however, no significant difference in cleavage was observed. Finally, we evaluated S-Psel in vivo. Hemophilia A mice were injected with recombinant mouse S-Psel-IgG or S-Psel-Fc (IgG) at the concentration of 1.2 mg/Kg body weight and human IgG was used as control. As suggested from published results, the effect of S-Psel was determined 6 h after the treatment. Contrary to previous reports, the results revealed no significant difference in bleed time and blood loss between the experimental and control group. In conclusion, we were unable to demonstrate the procoagulant activity of S-Psel in our laboratory either in vitro or in vivo.
Anti-Thrombotic Effects of Targeting Outside-in αIIbβ3 Signaling in Platelets
Authors: Ablooglu Ararat J. , Kang Jian , Petrich Brian G. , Ginsberg Mark H. , Shattil Sanford J.
Published: April 2020

Platelet activation by agonists stimulates high affinity binding of adhesive ligands, such as fibrinogen, to integrin αIIbβ3. In turn, ligand binding initiates Src kinase-dependent outside-in signaling, platelet spreading on sub-endothelial matrices, and platelet aggregation. Short-term integrin blockade by parenteral αIIbβ3 antagonists is effectively anti-thrombotic in certain clinical situations, but long-term blockade by oral αIIbβ3 antagonists has been problematic. As an alternative approach, we evaluated the feasibility of disrupting outside-in αIIbβ3 signaling in vivo. A “β3(Δ760-762)” knock-in mouse was generated that lacked the three C-terminal β3 amino acid residues (RGT) necessary for αIIbβ3-dependent c-Src signaling, but retained β3 residues necessary for agonist- and talin-dependent fibrinogen binding. β3(Δ760-762) mice were compared with wild-type β3+/+ littermates, β3+/− heterozygotes, and “β3/β1(EGK)” knock-in mice where β3 RGT was replaced by three C-terminal residues in β1 (EGK) to potentially enable outside-in integrin signaling by certain Src kinases other than c-Src. Whereas β3+/+, β3+/− and β3/β1(EGK) platelets spread and underwent tyrosine phosphorylation normally on fibrinogen, β3(Δ760-762) platelets spread poorly and exhibited reduced tyrosine phosphorylation of c-Src substrates, including the β3 cytoplasmic domain itself (Tyr-747). Furthermore, unlike the three groups of control mice, all of the β3(Δ760-762) mice tested were protected from carotid artery occlusive thrombosis following vessel injury with FeCl3. The median tail bleeding time for the β3(Δ760-762) mice was approximately two-fold longer than that of the control mice. However, β3(Δ760-762) mice did not exhibit spontaneous bleeding, excess bleeding after surgical exposure of the carotid artery, fecal blood loss or anemia. Fibrinogen binding to β3(Δ760-762) platelets was normal in response to saturating concentrations of agonists to the PAR4 thrombin receptor or the GP VI collagen receptor; however, responses to ADP were impaired. These studies establish that deletion of β3 RGT disrupts outside-in αIIbβ3 signaling and confers protection from arterial thrombosis in vivo. Currently available anti-platelet drugs either inhibit agonist-induced activation of αIIbβ3 or block fibrinogen binding to the integrin. The studies reported here suggest that targeting outside-in αIIbβ3 signaling may represent an alternative anti-thrombotic strategy.
Identification and Functional Characterization of Novel Telomerase Variant Alleles in Patients with Bone-Marrow Failure Syndromes
Authors: Carroll Kathryn A , Calado Rodrigo , Young Neal S , Danzy Shamika , Ly Hinh
Published: April 2020

As multiple studies have shown a link between mutations in telomerase components and the disease pathogenesis of bone-marrow failure syndromes (BMFS), we screened blood or marrow cells from patients with various forms of blood disorders for novel pathogenic mutations in the telomerase hTERC and hTERT genes. We identified several heterozygous mutations in these genes, which were observed only in patient samples and not in controls. As these mutations are predicted to affect proper telomerase function, we introduced these and other mutations identified previously in patients with other forms of blood disorders into telomerase-negative cells in order to reconstitute telomerase enzymatic activity and to assess the effect of the individual mutations on modulating a wild-type telomerase function. All disease-associated mutations disrupted telomerase function to various degrees and most modulated a wildtype telomerase enzymatic function by haploinsufficiency based on results obtained from the telomeric repeat amplification protocol (TRAP). We also tested several previously reported natural sequence changes in the promoter region of the hTERC gene for their effect on gene expression and telomerase function via luciferase-reporter assay, TRAP, and Northern blot. The results obtained from these studies appear to contradict those that have recently been reported for some of the naturally occurring hTERC-promoter associated mutations. This study, therefore, offers new insights into the mechanism of natural telomerase mutations in regulating telomere lengths and marrow stem cell renewal capacity in patients with hematological disorders.
Possible Selection for the Jak2 V617F Mutation in Bone Marrow Failure
Authors: Dunsmore Sara , Williams Gaynor J. , Houston Donald S
Published: April 2020

The acquired JAK2 V617F mutation has been well documented to be common in the classical myeloproliferative disorders. As it is an activating mutation in a kinase involved in proliferative signaling in hematopoietic cells, it is presumed to play an etiopathogenic role. The same acquired JAK2 V617F mutation has been described in other hematological disorders such as myelodysplastic syndrome with a prevalence which, though much lower than in myeloproliferative disorders, exceeds what could be attributed to the chance coincidence of two rare disorders. The direction of causality in this association is unclear. CASE DESCRIPTION: A 42 year old woman initially presented at the age of six with anemia and was subsequently diagnosed with congenital dyserythropoietic anemia (CDA) type I. At initial presentation her other blood counts, including platelets, were normal. When she returned to hematology clinic as an adult, her anemia persisted, but she had elevated platelet and white blood counts. Bone marrow biopsy showed hypercellularity with increased representation of the myeloid and megakaryocyte lineages. It also showed megaloblastoid dyserythropoiesis and binucleate erythroid precursors. These changes are consistent with essential thrombocythemia superimposed on CDA type I. The marrow cells were heterozygous for the JAK2 V617F mutation.

DISCUSSION: This patient has a unique pairing of congenital dyserythropoietic anemia and a myeloproliferative disorder. No archival tissue is available so it is impossible to be certain she did not have the JAK2 V617F mutation on initial presentation, although the normal platelet and white blood count at age 6 support the inference that the myeloproliferative disorder arose later. Among patients with essential thrombocythemia, those who have the JAK2 V617F mutation tend to have higher hemoglobin levels, though in her case the development of the JAK2 V617F mutation did not lead to amelioration of her anemia. We hypothesize that the hematopoietic failure state associated with CDA may, like that of myelodysplasia, create an environment in which a genetically-mediated proliferative advantage due to an acquired mutation may be especially favored, and may thus have been a factor in the development of the myeloproliferative disorder.
Electronic Cell Volume and ALDH Expression of Hematopoietic Stem Cells Obtained by Apheresis
Authors: Shariatmadar Sherry , Sharma Siddharth , Cabana Raquel , Krishan Awtar
Published: April 2020

Small cell volume or diameter (3–7μm) appears to be one of the characteristics that may be used to identify early stem cells. Hematopoietic stem cells may be characterized by the expression of cell surface markers such as CD34, CD117 and CD133. In addition, stem cells such as “SP cells” can be recognized by the efflux of dyes such as Hoechst 33342 and presence of aldehyde dehydrogenase (ALDH). In prior studies we used the Beckman Coulter® Cell Lab Quanta(™) SC MPL analyzer to determine the electronic cell volume, diameter and marker expression (CD34, CD90, CD117 and CD133) of cells from peripheral blood apheresis (HPC, Apheresis) samples (Shariatmadar et al. Cytometry B, 74B:182–188, 2008; Sharma et al. Cytometry A, 73A:160–167, 2008). In the present study, we used this analyzer to determine cell volume, diameter and marker expression of ALDHpositive stem cells from HPC, Apheresis samples of 44 patients with hematologic malignancies mobilized with granulocyte colony-stimulating factor. The mean electronic volume of the ALDHpositive cells was 286μm3 (SD±27) corresponding to a diameter of 8.2μm. The mean percentage of ALDHpositive cells analyzed was 0.51% (SD± 0.004). CD34 expression was seen in 0.13% (SD±0.001) of the ALDHpositive cells. The mean electronic volume of the ALDH and CD34 positive cells was 270μm3 (SD± 33) corresponding to a diameter of 8.0μm. CD133, CD117 and CD90 expression was seen in 0.28% (SD± 0.01), 0.17 (SD± 0.05) and 0.04% (SD± 0.03) of the ALDHpositive cells. The mean electronic volume of the ALDHpositive cells with CD133 expression was 275μm3 (SD±29) corresponding to a diameter of 8.1μm while that of the ALDHpositive cells with CD117 and CD90 expression was 284μm3 (SD± 32) and 265μm3 (SD± 35), corresponding to a diameter of 8.15 and 7.9μm.

Stem Cell Marker Electronic Volume (μm3) Diameter (μm) CD90 (Thy-1) 300μm3 (SD± 38.5) 8.3μm (95% CI=8.1–8.4) CD117 (c-Kit) 349μm 3 (SD± 47.4) 8.7μm (95% CI=8.5–8.8) CD133 (Sca-1) 322μm3 (SD± 44.5) 8.5μm (95 % CI= 8.3–8.6) ALDH+ 286μm3 (SD± 27.0) 8.2μm (95 % CI= 8.1–8.3) ALDH+CD34+ 270μm3 (SD± 33.0) 8.0μm (95 % CI= 7.8–8.2) ALDH+ CD90+ 265μm3 (SD± 35.0) 7.9μm (95 % CI= 7.8–8.1) ALDH +CD117+ 284μm 3 (SD± 32.0) 8.15μm (95 % CI= 7.9–8.2) ALDH +CD133+ 275μm3 (SD± 29.0) 8.1μm (95 % CI= 8.0–8.3)

The use of electronic cell volume in conjunction with side scatter might provide a useful method for characterization of stem/progenitor cell populations with specific marker expression. In our study, hematopoietic stem cells expressing ALDH and bearing early stem cell markers were relatively larger than those previously reported. Future studies will aim to characterize the long term repopulating capability of this fraction of stem cells in in-vivo models.
Incidence of Febrile Episode During Stem Cell Mobilization (SCM) After High Dose Ciclophosphamide Chemotherapy (HD-CTX) and G-CSF (filgrastim or lenograstim) Administration in Multiple Myeloma (MM) Patients: II Interim Evaluation
Authors: Orciuolo Enrico , Buda Gabriele , Mauro E. , Marturano E. , Pastore Domenico , Cioppa Paola Della , Pietrantuono Giuseppe , Lombardini Letizia , Di Renzo Nicola , Irrera Giuseppe , Cangialosi Clotilde , Brunori Marino , Petrini Mario
Published: April 2020

Introduction: Clinical use of G-CSF in pts with high grade chemotherapy induced neutropenia does not conduce to a reduction of the incidence of febrile episodes (FE). This paradox may be explained by the acquisition of a defective chemotaxis by neutrophils (PMN) exposed to filgrastim (Fil), due to a higher adhesivity and cytoscheletric alterations. Lenograstim (Leno), a glicosilated form of G-CSF, is able to stimulate PMN production, manteining in vitro all the functional capabilities. On these bases, we hypotized that Leno may prevent FE and reduce their lasting in pts with chemotherapy derived neutropenia.

Patients and methods: starting from April 2005, 105 MM pts achieving HD-CTX for SCM were enrolled in 12 Centers. Treatment plan consisted in: HD-CTX (3 or 4 g/sqm) on day 1, G-CSF (random: Fil or Leno) 30 MU/day from day +4 to +9, 60 MU/day from day +10 to the achievement of an optimal CD34+ cell count for staminoapheresis. Random, 1:1, was effectuated on the base of a generated random list. FE, significant if equal or higher than 38 °C for at least 2 different determinations, were recorded till day +30. Primary endpoint is the incidence of FE; secondary endpoints are the duration of FE, efficacy in the CD34+ cell mobilization, time to mobilization.

Results: 105 pts were enrolled. All pts underwent post-chemo grade 4 neutropenia and G-CSF was administred starting from day +4. FE were recorded in 23 pts, 14 in the Fil arm (53 total pts) and 9 in the Leno arm (52 total pts). The global fever incidence was 21.9%, 26.4% with Fil and 17.3% with Leno, with a 9.1% difference. Average days with fever are 4.00 with Fil and 3.67 with Leno. Related to the neutropenia grade, 8 FE are recorded with Fil and 1 FE with Leno with absolute PMN count >500/μL (grade 4); 7 episodes with Fil vs 1 with Leno when PMN are >1000/μL (grade 3–4). CD34+ SCM occurs in after an average time of 10.3 day with Fil and 9.8 day with Leno, with an higher absolute count with Leno when compared to Fil: 131.9 CD34+/μL (range 40–640) vs 111.6 (range 40–616) CD34+/μL.

Conclusions: Leno is associated with a reduced incidence (17.3% vs 26.3%) of FE in MM patients undergoing to HD-CTX and SCM when compared to Fil. FE are recorded with Fil even in presence of PMN confirming the functional block by Fil on PMN documented in vitro. CD34+ mobilization occurs shorter and with higher efficiency with Leno when compared to Fil. On these evidences, patients’ enrollment will continue to 180 to validate these results.
Detection of Genetic Abnormalities for Diffuse Large B-Cell Lymphoma by Selective Interphase Analysis with FISH Method
Authors: Jiang Shi-Ping , Bouman Derek , Hovanes Karine
Published: April 2020

Diffuse Large B-cell Lymphoma (DLBCL) accounts for approximately 30% of all lymphoid malignancies. It can be difficult to separate DLBCL from Burkitt lymphoma and plasmablastic myeloma by morphology and flow cytometric immunophenotyping alone. Fluorescent in situ hybridization (FISH) targetgene analysis can assist the differential diagnosis by detection of genetic abnormalities associated with DLBCL.

Though no consistent numeric chromosomal abnormalities or translocations are observed as a hallmark for DLBCL, most cases have rearranged immunoglobulin heavy chain (IgH), BCL2 and MYC genes. Standard interphase FISH analysis is widely utilized to detect these genetic rearrangements. However, standard interphase FISH analysis could be falsely negative if the neoplastic cells are limited or if abundant benign cells are present in the background. Thus, the sensitivity to detect genetic abnormalities is low among the cases with a low number of neoplastic cells.

Herein we present three DLBCL cases in which interphase FISH analysis was applied on selective cells that are morphologically consistent with neoplastic large lymphocytes. All three cases showed genetic changes in a high percentage of the selected cells. These three cases were originally analyzed for IgH gene rearrangement, IgH/BCL2 and IgH/MYC translocation by using standard non-selected interphase study and showed false negative results. However, by review of the H&E slide and the flow cytometry results prompted a re-evaluation of the FISH studies using selective analysis of neoplastic cells only.

Our observations indicate that selective interphase FISH analysis improves the sensitivity of detecting genetic abnormalities in DLBCL. This analysis does require a corporation between hematopathologists and technologists performing interphase FISH studies.
Rel a Is a Novel Prognostic Marker in CLL That Is Independent of VH Gene Mutation Status, CD38 Expression and ZAP-70 Expression
Authors: Hewamana Saman , Lin Thet Thet , Rowntree Clare , Karunanithi Kamaraj , Burnett Alan , Brennan Paul , Fegan Chris , Pepper Chris
Published: April 2020

We recently demonstrated that the NF-kB subunit Rel A is associated with in vitro survival and clinical disease progression in CLL. We therefore hypothesized that Rel A would have prognostic significance in this disease. Rel A DNA binding was quantified in nuclear extracts derived from 131 unselected CLL patient samples using a quantitative DNA binding ELISA-based method. We tested the ability of Rel A to predict for the requirement for treatment and survival and compared our findings with other established prognostic markers. Rel A DNA binding was strongly associated with advanced Binet stage (P<0.0001) but did not correlate with IgVH mutation status (P = 0.25), CD38 expression (P = 0.87) or ZAP-70 expression (P = 0.55). It was predictive of time to first treatment (P = 0.02) and time to subsequent treatment (P = 0.0001). In addition, Rel A was the most predictive marker of survival both from date of diagnosis (hazard ratio 9.1, P = 0.01) and date of entry into the study (hazard ratio 3.9, P = 0.05) and retained prognostic significance in multivariate analysis for both time to first treatment and overall survival in the presence of Binet stage, IgVH mutation status, CD38 and ZAP-70. Take together our data shows that Rel A is an independent prognostic marker of survival in CLL and appears to have the unique capacity to predict the duration of response to therapy. Prospective assessment of Rel A as a marker of clinical outcome and as a therapeutic target is now clearly warranted.
LPL and ADAM-29 Gene Expressions Stratify Prognosis in Clinical Stage BinetA and B Chronic Lymphocytic Leukemia Patients
Authors: Barreto Wolney Góis , Belluzzi Lorenza Oliveira , Siufi Grazziella Curado , de Andrade Silva Marçal Cavalcante , Kimura Eliza Yuriko Sugano , Vasconcelos Yuri , Arce Inara Lucia , Yamamoto Mihoko
Published: April 2020

Introduction: Chronic Lymphocytic Leukemia (CLL) is the most frequent adult leukemia in western countries. It is known to have a heterogeneous clinical course, some patients presenting an indolent disease while others have an aggressive disease requiring prompt treatment. Therefore, an individualized approach, especially in early clinical stage patients is necessary. Recent studies suggest that the biological markers LPL and ADAM-29 could be useful to predict prognosis in CLL patients: LPL being associated with an unfavorable prognosis while ADAM-29 to favorable one.

Aims: to evaluate the expression of LPL (L), ADAM-29 (A) and the L/A ratio in CLL patients in regard to Binet clinical stage and progression free survival (PFS).

Patients and Methods: thirty CLL patients followed at UNIFESP/EPM and HSPE were studied. RNA extraction was done by the TRIZOL method followed by c-DNA synthesis. c-DNA was amplified by PCR using LPL and ADAM-29 specific primers. T-Student’s exact test was used to compare the genes frequencies and Kaplan Meyer analysis to evaluate PFS. The results were considered statistically significant when p<0.05.

Results: Of the 30 CLL patients studied (16M/14F, median age of 67 years, varying from 38 to 64 years) 19 (63,3%) were early clinical stage (Binet A), 8 (26,7%) Binet B and 3 (10%) Binet C. In the Binet A group LPL was negative in 52.63% of the patients and ADAM-29 positive in 63.15% with relation LPL-/ADAM29+ (or L/A=0) in 52.63% (10/19). In contrast among Binet B and C patients 90% (10/11) presented relation LPL+/ADAM-29- (or L/A=1). Those who were LPL+ or ADAM-29- or L/A=1 had a shorter PFS when compared to those LPL-or ADAM-29+ or L/A=0 (p<0.05). Patients, even those in early clinical stages (Binet A and B), when LPL+, ADAM- and L/A=1 constitute a group with an unfavorable prognosis with a short DFPS (p<0.05).

Conclusion: Our study shows that patients with a positive expression of LPL, negative for ADAM-29 and with L/A=1 have unfavorable prognosis independent of clinical stage. Analyzing Binet A and B together, we identified a subgroup LPL+, ADAM- and L/A=1 with a poor outcome. Thus, the expression profile of these genes could be useful to identify Binet A and B patients with an unfavorable prognosis, contributing to the decision making process to begin therapy in an individualized manner. (Support–FAPESP proc n. 05/57792-0)
A Scoring System Based on Clinical, Blood Count and Immunophenotypic Data Can Predict the Risk of Disease Progression in Patients with Chronic Lymphocytic Leukemia
Authors: Alipour Sina , Leitch Heather , Vickars Linda M , Foltz Lynda M , Galbraith Paul F , Leger Chantal S , Ramadan Khaled M
Published: April 2020

There is limited access to advanced molecular and immunophenotypic techniques such as immunoglobulin heavy chain variable mutational status and ZAP-70 expression analysis in many parts of the world. To facilitate the development of a clinical model based on readily available clinical and well-standardized pathological data, we analyzed the well known prognostic factors in chronic lymphocytic leukemia (CLL) and proposed a scoring system. We searched the CLL database and among 465 patients (pts) we included 128 pts where the staging, blood counts, immunophenotypic and follow up data are complete. The baseline characteristics of this group and the significance of the proposed factors on the progression-free survival on univariate analysis are summarized in the table. Progression–free survival (PFS) was defined as the time from diagnosis to the time where treatment for CLL is indicated and if no treatment id indicated, the time of last follow up. In this 5 point scoring system we added one point for the presence of each one of the following factors: male gender, Rai stage 1 or more, lymphocyte count of 20 ×109/l or higher, lymphocyte doubling time less than 12 m, CD 38 expression of 20% or more. We further lumped these groups into 3 groups: no risk factors, one risk factor and tow or more risk factors. Thirty eight patients (30%) were found to have no risk factors and their 5 and 10 year PFS was 100% and 100% respectively. Forty nine pts (38%) have at least one risk factor and their 5 and 10 y PFS was 92% and 81% respectively. Forty pts (31%) have two or more risk factors and their 5 and 10 y PFS was 48% and 20% respectively. Those differences in PFS were significant (p<0.00001). We conclude that readily available clinical and laboratory information in patients with CLL can be utilized to predict the risk of disease progression. Pts with no risk factors can be reliably reassured and do not require regular monitoring. Correlation of these findings with other molecular prognostic factors is needed.

Table: Risk factors for disease progression

Parameter N (%) 5-y PFS 10-y PFS p value (UVA)* *Univariate analysis Sex: Male 71 (56) 67% 43% 0.01 Female 56 (44) 91% 73% Rai stage: 0 103 (80) 87% 77% <0.0001 1,2,3,4 25 (20) 49% 18% Lymphocyte count: < 20×109/l 108 (84) 85% 60% <0.0001 ≥20×109/l 20 (16) 40% 40% Lymphocyte doubling time ≥ 12m 111 (87) 84% 68% 0.001 <12 m 17 (13) 50% 22% CD38 <20% 99 (77) 83% 67% 0.006 ≥20% 29 (23) 62% 36%

Fig: Progression free survival based on the number of risk factors for disease progression (p<0.00001) Fig:. Progression free survival based on the number of risk factors for disease progression (p<0.00001)
Anti Hsp70 Antibodies in Untreated Patients with Chronic Lymphocytic Leukaemia
Authors: Piszcz Jaroslaw , Cichocka Edyta , Bolkun Lukasz , Galar Marzenna , Mazgajska-Barczyk Katarzyna , Milewski Marek , Kloczko Janusz
Published: April 2020

Background: Chronic lymphocytic leukaemia (CLL) is one of the diseases in which processes of proliferation and apoptosis are altered. It has been intensely studied in the recent years in order to understand the mechanisms of neoplastic development. Hsps or heat shock proteins are molecular chaperones involved in a number of cellular functions in stress conditions. Feng et al. (2002) have reported that heat-stressed apoptotic 12B1-D1 leukaemia cells (BCR-ABL(+)) express Hsp70 on their surface. It has also been suggested that an increased surface expression of heat shock proteins on apoptotic tumour cells results in the generation of potent antitumour T-cell responses. Furthermore, different hsps including Hsp70 have been discovered in indolent lymphoma cells. Anti Hsp70 antibodies are known to play a role in immunological and neoplastic processes. It has been well documented that this antibodies level tends to increase with age likewise an incidence of CLL. However their significance in CLL has not been clearly understood.

Aims: The aim of our study was the assessment of the anti Hsp70 antibody concentration in the patients with CLL.

Material and methods: We assessed 60 peripheral blood samples from the patients with newly diagnosed CLL. (aged 40–77), including 31 males and 29 females. A group of 20 healthy age matched subjects were used as a control group. The patients were in A-C stages of CLL according to Binet scale. Quantitative determination of anti-human Hsp70 antibodies in the serum was done using commercial test (anti Hsp70 Elisa Kits, Stressgen). The results are presented as mean ± SEM. Statistical analysis was done using Shapiro-Wilk, Mann-Whitney and Spearman’s tests.

Results: The levels of anti Hsp70 antibodies were significantly lower in the group of patients with CLL in comparison to healthy controls (105,70±16,86 ng/ml vs.243,71±60,36 ng/ml; p=0,0027). In our analysis there was no association between the levels of antibodies and the stage of the disease. There were no statistically significant correlations between the levels of anti Hsp antibodies and other parameters such as age, gender and some prognostic factors (LDH, β2-microglobulin, and lymphocyte doubling time) in the studied group.

Conclusions: The significantly lower concentrations of anti Hsp70 antibodies in CLL patients suggest that these molecules may play a role in the biology of the disease. Our study revealed lack of correlation between the level of antibodies and the disease prognostic factors. However, further studies utilizing new discovered factors are required to elucidate the role of these proteins in leukaemic biology. The encouraging results from our study suggest that it might be interesting to assess their level in CLL patient after chemotherapy.
Molecular and Functional Analysis of Stem Cell Compartment of Chronic Myelogenous Leukemia Reveals the Presence of a CD34− cell Population with Intrinsic Resistance to IMATINIB Treatment
Authors: Salvestrini Valentina , Manfredini Rossella , Fogli Miriam , Bianchi Elisa , Salati Simona , Amabile Marilina , Bertolini Francesco , Tafuri Agostino , Zini Roberta , Testoni Nicoletta , Rabascio Cristina , Rossi Lara , Martinelli Giovanni , Baccarani Michele , Ferrari Sergio , Lemoli Roberto M
Published: April 2020

We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin−CD34−) hematopoietic stem cells from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular caryotyping and quantitative analysis of BCR-ABL transcript demonstrated that about one third of CD34− cells are leukemic. CML Lin−CD34− cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures induced CD34 expression on some cells, cell cycling, acquisition of clonogenic activity and increased expression of BCR-ABL transcript. Lin−CD34− cells showed hematopoietic cell engraftment rate in immunodeficient mice similar to Lin-CD34+ cells whereas endothelial cell engraftment was significantly higher. Gene expression profiling revealed the down-regulation of cell cycle arrest genes, genes involved in antigen presentation and processing, while the expression of genes related to tumor progression, such as angiogenic factors, was strongly up-regulated when compared to normal counterparts. Flow cytometry analysis confirmed the significant down-regulation of HLA class I and II molecules in CML Lin−CD34−cells. Imatinib mesilate did not reduce fusion transcript levels, BCR-ABL kinase activity and clonogenic efficiency of CML Lin− CD34− cells in vitro. Moreover, leukemic CD34− cells survived to BCR-ABL inhibitors in vivo. Thus, we identified a novel CD34− leukemic stem cell subset in CML with peculiar molecular and functional characteristics.
A Single Nucleotide Polymorphism in Pro-Apoptotique Protein BIM Affects Chronic Myeloid Leukaemia Evolution during Imatinib Treatment
Authors: Augis Vanessa , Belloc Francis , Airiau Kelly , Chollet Claudine , Mahon Francois-Xavier
Published: April 2020

The tyrosin kinase inhibitor imatinib induces apoptosis in chronic myeloid leukaemia (CML) cells. RNA interference experiments have demonstrated that the pro-apoptotic protein BIM (Bcl-2 interacting mediator of cell death) was absolutely essential to this apoptosis. Thus, mutations in BIM sequence could lead to imatinib resistance independently of BCR-ABL mutations. In the current study, we performed BIM coding sequence analysis, using complementary DNA of leukocytes from CML patients mostly secondary resistant to imatinib. A total of 58 patients were analysed: secondary resistant to imatinib (n=40), or on the contrary sensitive to it (n=18). They were in chronic phase (CP, n=43), in accelerated phase (AP, n= 12), or in blastic phase (BP, n=3). Four patients were mutated in the tyrosin kinase domain of BCR-ABL. We did not find any mutation with amino acid change on the coding sequence of BIM. However, we observed a single nucleotide polymorphism (SNP) located in the fifth exon which encode for the BH3 alpha helix part of the protein. The impact of this previously described silent polymorphism C465T (rs724710) has never been studied before. We found the 3 following genotypes : C/C homozygous, T/T homozygous and T/C heterozygous. T/T genotype was the less frequent (It represents about 5% in the European population described in the international Hapmap project). Although not significant (10%, p=0.1468), we observed an increased T/T genotype frequency in the resistant population. In this resistant group, the T/T genotype frequency was increased in advanced phases (AP+BP, 20%) as compared to CP (4%, p=0.0016) or to sensitive cohort (5.6%, p=0.0153) (Fig.1). T/T genotype could affect RNA stability, translation speed and protein folding as described by Kimchi-Sarfaty et al. for the MDR1 gene (Science2007;315:525–8). This genotype can possibly be in linkage disequilibrium with others SNPs or abnormalities (i.e. in the promoter, in splicing sites) in the non coding sequence. The analysis of this polymorphism could be useful to predict the evolution of the disease during imatinib treatment and discriminate resistant patients particularly in advanced phase of the disease. Fig 1 : Genotypes distribution of the C465T polymorphism.

Fig 1. Genotypes distribution of the C465T polymorphism. Fig 1. Genotypes distribution of the C465T polymorphism.
Preliminary Functional Studies of hnRNPK in Progression of CML
Authors: Xiaoli Liu , Zhu Hongqian , Zhang Song , Du Qingfeng , Gong Junmei , Liu Zhi , Ouyang Zhao , Ouyang Lingyun
Published: April 2020

The blast crisis(BC) is terminal phase of chronic myeloid leukemia (CML), which is accompanied by an increase in both BCR/ABL mRNA and protein level and imatinib(IM) resistance. The BCR/ABL oncoprotein is responsible for inducing and sustaining the leukemic phenotype through its deregulated tyrosine kinase activity which is essential for recruitment and induction of signaling pathways leading to cytokine-independent proliferation, resistance to apoptosis, and impaired differentiation of BCR/ABL-expressing myeloid and lymphoid progenitors. However, the molecular mechanisms responsible for blastic transformation remain poorly understood, although a reasonable assumption is that the unrestrained activity of BCR/ABL in hematopoietic stem/progenitor cells is the primary determinant of disease progression. We had analyzed the changes of protein expression between the bone marrow mononuclear cells in CML-CP and that in CML-BC by the technique of proteome. Compared to CML-CP, the protein expression of heterogeneous nuclear ribonucleoprotein K(hnRNPK) increases in CML-BC. In present study, the over-expression of hnRNPK in CML-BC was verified by western blotting. Furthermore, the expression of hnRNPK increases in a new imatinib-resistant BCR/ABL-positive cell line, K562-R, than in the wild-type K562. After K562 and K562-R were treated by different inhibitors such as PD98059, LY294002, AG490 and imatinib, the protein expression and mRNA level of hnRNPK were detected by western blotting and QRT-PCR. Inhibition of BCR-ABL by imatinib significantly decreased hnRNPK expression in K562 and inhibition of MEK by PD98059, downstream of the BCR-ABL signaling cascade, decreased hnRNPK expression in both K562 and K562-R cells. In addition, down-regulation of hnRNPK by small double-stranded RNA specifically complementary to hnRNPK (siRNA-hnRNPK) can inhibite cell growth and induce maximal G2/M block at 48 hours in both K562 and K562-R cells, whereas cell appototis was observed only at 72 hours by AnnexinV-PI assay. The combination of siRNA-hnRNPK and imatinib induce more cell death in K562-R cells than either treatment alone. Our data therefore suggest that hnRNPK is regulated by the BCR-ABL/MAPK cascade in Ph+ CML. The results that down-regulating hnRNPK expression induced cell-growth arrest and subsequent cell death suggest the potential therapeutic utility of this strategy in patients with CML.
Systems Understanding of Synergism Between As4S4 and Imatinib in Treating BCR/ABL Leukemia Model and in Attenuating BCR/ABL Oncoprotein as Well as Related Regulatory Networks
Authors: Zhang Qun-Ye , Mao Jian-Hua , Liu Ping , Huang Qiu-Hua , Lu Jing , Zhang Yan , Weng Lin , Chen Quan , Chen Sai-Juan , Chen Zhu
Published: April 2020

The combination of Imatinib and arsenic sulfide (As4S4) exerts more profound in vivo therapeutic effects on chronic myeloid leukemia (CML) at both organism and cellular levels, as evident for the first time in this paper by significantly prolonged lifespan in mouse leukemia model bearing BCR/ABL and induction of apoptosis of BCR/ABL expressing cells. To address mechanisms underlying this synergy, we performed systematic analysis of the dynamic change of proteome, phosphoproteome and transcriptome in K562 cells after As4S4 and/or Imatinib treatment with the support of principal component analysis (PCA) and self-organization maps (SOM). Moreover, protein biochemistry experiments were performed to confirm the important conclusions from multiomics study. The integrated information indicated that As4S4 promoted the unfolding protein reaction (UPR) and activity of ubiquitination pathway, which can be considered as a major biochemical basis of the pharmacological effects of this ancient medicine. In this context, As4S4 was shown to target BCR/ABL through ubiquitination of key lysine residues and led subsequently to its degradation by proteasome. Meanwhile, Imatinib inhibited the PI3K/AKT/mTOR pathway with synergism of As4S4 and arrested cell cycle. Combination of the two agents synergistically decreased activity and quantity of BCR/ABL and activated intrinsic and extrinsic apoptosis pathways. These complex multi-molecular target and multi-pathway modifications at protein level, together with those at transcriptional regulation level, ultimately resulted in the synergistic attenuation of BCR/ABL oncoprotein and the therapeutic effects on CML model.
FLT3, CD32, PU.1, ERG, uPAR, and TAP2 Are Strongly Associated with the Progression of Chronic Myeloid Leukemia and Combination of Small Interference RNA of FLT3 and STI571 Synergistically Induced Apoptosis of K562 Cells
Authors: Kim Kyung Im , Ahn Kwang-Sung , Park Juwon , Lee Chansu , Kim Byoung Kook , Lee Young-Yiul , Oh Jung Mi , Yoon Sung-Soo
Published: April 2020

To characterize molecular mechanisms by which transition from chronic phase to blast crisis in chronic myeloid leukemia (CML) for developing novel therapeutic targets, we analyzed gene-expression profiles of leukemic cells from 12 patients in chronic phase and 9 patients in blast crisis using a 8.7K cDNA chip. We identified 89 genes that were up-regulated as well as 54 genes that were down-regulated in blast crisis of CML. The expression profile included oncogenes, tumor suppression genes, and human genes encoding proteins involved in transcription, signal transduction, metabolism, cell growth, differentiation, apoptosis and immune functions. 18 genes were selected among the up-regulated group for analysis using real-time PCR. Real-time PCR data indicated that the expression of FLT3 (p < 0.001), CD32 (p < 0.001), ERG (p < 0.001), uPAR (p < 0.001), MAD (p < 0.001) and TAP2 (p < 0.001) showed statistically significant difference between chronic phase and blast crisis. For further analysis, we utilized small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human FLT3. These small interfering RNA constructs significantly inhibited FLT3 expression at mRNA and protein levels in K562 cells. After treating both the FLT3 knockdown cells and control cells (FLT3 wild type) with STI571, MTT assay and the expression patterns of apoptosis related genes (PARP, caspase-3, Bax) were examined. MTT assay and caspase-3 activity assay showed that silencing of the gene for FLT3 significantly reduced cell viability and ultimately facilitated the induction of apoptotic cell death by STI571. These findings uncovered evidence of a complex signaling network operating down-stream of FLT3 that actively contributes to tumor progression. Thus, RNA interference-directed targeting of FLT3 can be a potential candidate anticancer agent in association with STI571 against chronic myeloid leukemia.
HHGV678, a Novel Tyrosine Kinase Inhibitor, Inhibits the Cell Growth of Imatinib-Sensitive and Resistant BCR-ABL Positive Cells
Authors: Qiu Lin , Wang Xiao-dan , Ge Fang , Wang Xiu-li , Zhang Bo-long , Ma Jun
Published: April 2020

Imatinib (IM) is a higherly effective targeted drug for CML. However, some CML patients, especially accelerated and blast crisis phase, often relapse due to drug resistance resulting from the emergence of IM-resistant point mutations within the BCR-ABL tyrosine kinase domain. This stimulates the development of new kinase inhibitors that are able to override resistance to IM. HHGV678 is a novel tyrosine kinase inhibitor and we employed IM-sensitive (K562 and 32Dp210) and resistant (K562R and fifteen 32Dp210 mutants) BCR-ABL+ cell lines to compare HHGV678 with IM on growth inhibition. In addition, synergistic effect of HHGV678 with IM was observed in 32Dp210 and 5 BCR-ABL mutants frequently observed in CML patients. MTT assay results showed that the estimated IC50 value of HHGV678 for K562 and 32Dp210 were 15.5 and 28-fold, for K562R and 15 BCR-ABL mutants, were 1.4–124.0-fold lower than that of IM, indicating that HHGV678 was a more effective than IM against cell growth of IM-sensitive and resistant cells. Using combination index analysis, HHGV678 displayed synergistic growth inhibition when used with IM in BCR-ABL mutants (M244V, Q252H, Y253H, E255K and T315I). HHGV678, combined with IM at their IC50 concentration induced apoptosis 2–5 fold higher than that of HHGV678 alone in BCR-ABL mutants respectively, by annexin-V staining. At the same condition, HHGV6787 resulted in remarkable decrease in CrKL phosphorylation as determined by western blot. We conclude that HHGV678 have significant activity against IM-sensitive and resistant BCR-ABL+ cell, especially when it combined with IM that warrant further investigation in clinical trials.
Preliminary Studies on the Role of a Small GTPase, RhoA and Its Regulation in Imatinib Resistance
Authors: Zhang Song , Du Qingfeng , Zhu Hongqian , Li Rong , Liu Zhi , Yang Jun , Wang Yu , Ouyang Zhao , Xiaoli Liu
Published: April 2020

Imatinib has impressive response rates and good tolerability quickly led to its adoption as frontline therapy for all patients with chronic-phase CML (chronic myeloid leukemia), but the therapeutic effect of imatinib is poor in the blast crisis, and imatinib resistance has become a major problem in CML. The possible mechanisms of imatinib resistance include the amplification of BCR-ABL fusion gene and its expression increase, the point mutant of BCR-ABL kinase domain and the effects of other tyrosine kinases such as Src, Hck and Lyn and so on. However, the second-generation tyrosine kinase inhibitors (such as nilotinib and dasatinib), which were developed to overcome imatinib resistance resulting from the point mutant or the activation of other tyrosine kinases, even can not prevent all patients with CML progression to drug resistance. So there would be the other potential factor in imatinib resistance. Our previous studies generated a new imatinib-resistant BCR/ABL-positive cell line, K562-R. The 50% inhibitory concentration of imatinib was 15-fold higher in K562-R than in the wild-type K562. The expression of RhoA gene is up-regulated in K562-R by microarray analyses. RhoA, a small GTPase (24KD), has been found overexpression in breast, colon, head and neck squamous cell carcinoma, bladderand testicular cancer, lung and gastric cancer. It plays an important role in the initiation as well as the progression of human cancer, but the potential role of RhoA related to imatinib resistance has yet been unknown. In this study, we firstly detect the biologic characteristic of K562-R cells with RhoA down-expression by RNA interference. When K562-R cells were transfected with 150nM siRNA-RhoA for 48 hours, the percentage of apoptotic K562-R cells is respectively 12.82% by AnnexinV-PI assay and 9.0% by Hoechst 33258 staining and both have significant increase, cell cycle analysis found significant G0/G1 arrest, the expression of CD29 increase and that of CD71 and GPA have no difference. Secondly, The K562-R cells were treated with three selective inhibitors, including PD98059 (Ras/MAPK inhibitor), LY294002 (PI3K/AKT inhibitor) and AG490(JAK/STAT inhibitor) for 2,4 and 8 hours and the expression of RhoA were analyzed by Western-Blotting. The expression of RhoA is arrested in the K562-R cells treated with PD98059 and AG490 and no different with LY294002. These results indicate that RhoA would be an important target in the down-stream of multi-signal pathways related to imatinib resistance and the potential function of RhoA in imatinib resistance involve in increasing of cell proliferation, resistance to cell apoptosis and changes of cell adhesion.