bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Adaptor Protein SLP-76 Is Not a Direct Substrate for BCR-ABL in the Chronic Myeloid Leukemia Cell Line K562.
Authors: Joseph Witzard , Luckashenak Nancy , Ramsey Kimberly , Clements James , Padmanabhan Swami 
Published: October 2019

Abstract
SLP-76(SH2 domain-containing leukocyte protein of 76 kD) is a hematopoietic adapter protein that is expressed in myeloid and T cells. SLP-76 is a substrate for tyrosine kinase in the src and syk family activation pathway required for T-cell receptor-mediated signaling. Cross-linking of the human FcgammaRIIa1 (CD32) in myeloid cells which contains an immune receptor tyrosine-based activation motif (ITAM) causes phosphorylation of SLP-76. Mice deficient in SLP76 develop fetal hemorrhage along with failure of T cell development and perinatal mortality. We have found that K562 cells express SLP-76. We hypothesized that SLP-76 or associated proteins may be substrates of Bcr-Abl in the K562 cell line and thus promote survival signals.
Materials and Methods: The K-562 cell line was obtained from the American collection of Cell Cultures. SLP deficient (−/−) KO mice were obtained from Dr. James Clements (Roswell Park Cancer Institute). Antibodies used include Sheep polyclonal IgG Anti-Human SLP 76, Peroxidase-conjugated Affinipure Donkey Anti-Sheep IgG at 0.8mg/ml, Mouse monoclonal IgG Anti-Phosphotyrosine, and Polyclonal goat Anti-mouse.
Immunoprecipitation and Immunoblot: Cells were either untreated or stimulated with purvanidate (phosphatase inhibitors). Purvanidate treated cells were used as a positive control. Spleenocytes isolated from a SLP-76 deficient mouse (KO) were used as negative control. Lysates were then subject to standard immunoprecipitation for SLP-76 followed by immunoblotting with a SLP-76 specific antibody. The denatured samples were then resolved by SDS-PAGE.
Results: SLP-76 is expressed in untreated and treated K562 with purvanidate and not detectable from lysates derived from SLP-76 KO spleenocytes. To assess the phosphorylation status of SLP-76 and any co-associated proteins in K562 cells, the SLP-76 blot was stripped and then immunoblotted for total phosphotyrosine content. SLP-76 does not appear to be constitutively phosphorylated in the K562 cells. However, significant tyrosine phosphorylation of SLP-76 was readily detectable in the purvanidate treated K562 cells.
Conclusion: The current studies reveal that although SLP-76 is indeed found in the K562 cell line expressing the Bcr-Abl oncogene. Somewhat surprisingly, despite the constitutive activation of the Bcr-Abl tyrosine kinase in K562 cells, we detected no obvious phosphoproteins co-precipitating with SLP-76 in the absence of purvanidate stimulation. Together, the lack of SLP-76 tyrosine phosphorylation and the lack of co-associated proteins in K562 cells suggest that SLP-76 is not a major player in the signal transduction pathways emanating from Bcr-Abl. However, a more stringent confirmation of this conclusion would require inhibiting SLP-76 expression in K562 cells and than assessing the growth and survival characteristics.
Figure Figure
Association between Vasculitides and Myelodysplastic Syndrome (MDS): A Report on 21 Cases.
Authors: Fain O. , Hamidou M. , Cacoub P. , Godeau B. , Stirnemann J. , Guillevin L. , Fenaux P. 
Published: October 2019

Abstract
Vasculitis rarely complicates solid tumors but is more common in hematologic malignancies, particularly MDS,. We report characteristics and outcome of 21 patients with both MDS and vasculitis.
Patients: We retrospectively collected data of patients with at least 1 malignancy (including MDS) and vasculitis diagnosed between 1992 and 2002 in 6 French centers. Vasculitides occurring during cancer remission were excluded, as were giant-cell arteritis, relapsing polychondritis (a well known association with MDS), cryoglobulinemia, infection- or drug-induced vasculitis.
Results: 60 patients, with median follow-up of 45 months were analyzed. Vasculitides diagnosed were: cutaneous leukocytoclastic (LV,45%), polyarteritis nodosa (PAN,36.7%), Wegener’s granulomatosis (WG,6.7%), microscopic polyangiitis (MPA,5%), Henoch-Schonlein purpura( HSP,5%). Cancers were hematological (66.7%) (including 21 MDS (35%) and (23%) lymphoid malignancies) and solid tumors (41.7%). Five patients had 2 cancers.
21 patients had vasculitis associated with MDS (median age 66 yr; M/F ratio 4.25).
FAB classification was: RAEB (n=11), RA (n=3) and CMML (n=7). Vasculitides were LV (n=9), PAN (n=9), MPA(n=1), WG (n=1), cutaneous granulomatous vasculitis (n=1).CMML were only associated with necrotizing vasculitis ( 6PAN, 1WG). Vasculitis and MDS were diagnosed simultaneously in 12(57%) cases, vasculitis was diagnosed after MDS in 8 (38%) cases (median interval: 11 months, range 1–45) and 6 months before MDS in 1 case.9 MDS progressed to AML. Vasculitides developed before or at the time of leukemic progression in 6 of them (median interval 3 months, range 0–12). Main vasculitis symptoms were: fever (47%), skin lesions(81%), arthralgias (62%), peripheral neuropathy (14%), renal involvement (43%)[microaneurysms 29%, renal failure 9.5%, glomerulonephritis 9.5%], inflammatory syndrome (81%), ANCA (20%). Vasculitis therapy was steroids (90%) +/− immune suppressors (33%). MDS treatment was: AraC( n:2), VP16 (n:3), hydroxyurea (n:2),allogeneic BMT (n:2), symptomatic (n:12). Vasculitis was cured in 10 (47%) cases. 17 (81%) patients died; only 1 death was linked to vasculitis. Regardless of the type, vasculitides associated with MDS had significantly more frequent renal involvement (p=0.02),steroid dependence (p=0.04), and less recovery (p=0.04) than vasculitides without MDS
Conclusion: Vasculitides associated to MDS appear to have distinctive features and more severe evolution.In addition, occurrence of vasculitides during the course of MDS may be associated with a high risk of rapid leukemic progression.
Chronic Lymphocytic Leukemia Associated with Autoimmune Diseases Often Discloses Activated Phenotype Pattern.
Authors: Duek Adrian , Shvidel Lev , Berrebi Alain , Bairey Osnat , Braester Andre , Rahimi-Levene Naomi 
Published: October 2019

Abstract
In autoimmune diseases the production of auto reactive antibodies is related to activation of normal B-lymphocytes. The high incidence of autoimmune disorders (AID) in CLL is well known, but the origin of auto antibodies is controversial. In the present study we postulate that Chronic Lymphocytic Leukemia associated with autoimmune diseases discloses activated phenotype pattern.
Out of the 891 CLL patients registered in the Israeli Study Group, we found 72 patients (8%) who presented at least one clinically expressed autoimmune disease at diagnosis.
Median age was 67 years old (range 45–87). 44% were male, 45 patients were at Binet`s stage A, 17 patients at Stage B and 4 at Stage C, and six patients were diagnosed with de novo PLL. The autoimmune disorders were as follows: Hashimoto`s disease (25), AIHA (11), Sjogren`s syndrome (10), rheumatoid arthritis (9), vasculitis (8), Grave’s disease (5), Evan’s syndrome (4), ITP (4), multiple sclerosis (2) and pernicious anemia, pure red cells aplasia, Raynaud`s syndrome, systemic erythematous lupus, ulcerative colitis (each-one). We found also 19 cases of positive direct antiglobulin test, 7 cases of positive rheumatoid factor, 4 cases with positive antinuclear antibodies, and 7 cases of hypocomplementemia. In 16 patients (22%) the morphology cell was consistent with activated lymphocytes (more than 30% of prolymphocytes). Immunophenotype analysis revealed increased expression (>30%) of activation markers CD38 or FMC7 in 27%. IgG was higher than 1000 mg/dl in 57%, while 5 cases found with IgM paraproteinemia.
Increased Beta2- microglobulin level, described by us correlating the activation of B-CLL cells, was increased in 78% of tested cases. The new marker ZAP 70 analyzed in seven patients and found positive in 5. Interestingly, Beta2m and CD38 wich usually define agressive disease were found predominatly in stage A patients with AID.
Our results suggest that in B- CLL associated with autoimmune disorders, the B CLL cells present some degree of activation. It is well known that B-lymphocytes in CLL are in a preactivated state and can be induced to differentiate into plasma cells and to stimulate residual normal B cells to produce high-affinity polyclonal autoantibodies with restricted specificity and pathogenic properties. This possibility would be in keeping with our observation.
Conclusion: our data showing activated lymphocytes morphology and /or increased expression CD38 or FMC7, high level of IgG and of Beta2-m in most of the cases, suggests that a state of activation of B-cells may correlate with the occurrence of autoimmune complication in B-CLL.
Intraclonal Immunoglobulin VDJ Diversification in Chronic Lymphocytic Leukemia Cells.
Authors: Dimitriadou Evangelia , Karali Vasiliki , Aggelopoulou Maria , Pangalis G.A. , Panayiotidis Panayiotis 
Published: October 2019

Abstract
The monoclonal Ig expressed on CLL cells was considered to be identical in all cells of the leukemic clone in a CLL patient. However, intraclonal VDJ gene diversification was recently reported in CLL B cells using SSCP analysis and sequencing of Ig PCR products exhibiting altered mobility. We used a different approach, by cloning directly monoclonal VHDHJH PCR bands from CLL patients and sequencing a large number of clones. Peripheral blood from five patients with typical CLL was used to extract DNA.. In one patient DNA from bone marrow and a lymph node biopsy was also analysed. PCR was performed with VH family specific primers and a consensus JH primer for 30 cycles. Monoclonal bands were ligated to TOPO vector and 25 to 70 insert containing clones were sequenced from both orientations. Sequenses contigs were aligned with DNASTAR and were compared with the germ line counterpart using BLAST and IMGT data bases. Intraclonal base variation was observed in 8–33% of the clones in all five patients. One to five base point-mutations /clone were found and there were mostly transitions (in 79% of mutations). In one patient with two monoclonal Ig sequences, (one hypermutated V3-7 and one unmutated VH3-23) intraclonal variation was observed in 69% and 8% of the clones respectivelly. In two patients clones with partially shared mutations were observed indicating the further diversification of a parental subclone. In the patient that VHDHJH clones from blood/bone marrow/lymph node were obtained, a difference in the frequency of diversified clones was noted. All samples were obtained from the patient within one month to exclude the possibility of time related mutations in the monoclonal Ig of the patient. In the patient’s VH1-69 unmutated sequence, 19 of 64 (29,6%) of bone marrow derived clones had additional mutations but they were found in only 12 of 71 (16%) peripheral blood derived clones. Five of 53 (9,4%) lymph node derived clones had additional point mutations. Low R/S ratio and targeting of FR rather than CDR regions is against the hypothesis of antigen driven somatic mutations in a fraction of the CLL cells in a given patient. The difference in diversification rate between different tissues in the same patient raises the question of the role of the patient microenvironment/stroma in partcipating in these mutations observed in CLL subclones. With the analysis of large number of clones it is possible that all CLL patients may have at least some degree of intaraclonal VHDHJH gene diversification.
Mutations Required for Lymphoid Leukemogenesis Are Not Required for c-Myc to Rapidly Induce Acute Myeloid Leukemia.
Authors: Li Qing , Luo Hui , O’Neal Julie , Kreisel Friederike , Inoyama Daisuke , Le Beau Michelle M. , Tomasson Michael 
Published: October 2019

Abstract
Dysregulation of c-Myc is one of the critical oncogenic events required for tumorigenesis in many tissue types. Although c-Myc has been shown to block differentiation of leukemic and normal myeloid cells and is activated by oncogenes commonly found in leukemia, its exact role in myeloid leukmogenesis remains elusive. When c-Myc is over-expressed in the lymphoid compartment, Eμ-Myc transgenic mice develop B-cell lymphomas only after a prolonged latency and require additional mutations to inhibit apoptosis, most commonly by disrupting the Arf-Mdm2-p53 pathway. Although this model has been well used to dissect pathways of Myc-induced lymphomagenesis, there is currently no efficient model with which to study the role of c-Myc in AML. In this study, we utilized a retroviral gene transfer system (MSCV-Myc) to introduce c-Myc into unfractionated murine bone marrow that was subsequently transplanted into syngeneic mice. Surprisingly, 100% of transplanted mice (N=110) from three different strains, developed acute myeloblastic leukemia (AML) characterized by rapid onset, hemiparesis, splenomegaly and infiltration of the marrow and spleen with myeloblasts. c-Myc protein level was comparable to the Eμ-Myc tumors. Disease latency was short (median survival=38 days). MSCV-Myc-induced tumors were oligoclonal, did not display significant cytogenetic abnormalities by SKY analysis, and were exclusively of myeloid origin. These tumors contained germline configuration of the Ink4A locus and wild type coding region of the p53, Arf and Ink4a. Also, the p53 target gene p21 in Myc expressing cells responded normally to gamma irradiation. These data demonstrate that c-Myc induced AML development without disrupting the Arf-p53 pathway and suggest that cooperating mutations were not required for the development of AML. In contrast, using the same retroviral system, lymphoid leukemia developed only if c-Myc and Bcl-2 were co-expressed or if Ink4a was mutated. Tumors from Bcl-2/c-Myc transplanted mice were comprised of a mixture of myeloid and lymphoid leukemic cells. Similarly, mice transplanted with c-Myc transduced Ink4a−/− bone marrow developed leukemia that displayed a mixed phenotype of myeloid and lympoid lineages. Lastly, factor independent methylcellulose colonies induced by Myc were exclusively of myeloid lineage suggesting as intrinsic difference between myeloid and lymphoid cells in response to Myc. Our data imply that dysregulation of c-Myc is sufficient to induce AML and provide direct evidence that Myc is an important downstream target in myeloid leukemogenesis.
Creatinine Clearance Is More Important Prognostic Factor Than ß2 Microglobulin in Multiple Myeloma.
Authors: Yun Jae-Pil , Suh Cheolwon , Lee Eunkyoung , Chang Jai Won , Yang Won Seok , Park Jung Sik , Park Su-Kil 
Published: October 2019

Abstract
In the new staging system of multiple myeloma (MM) by South West Oncology Group (SWOG), the concentration of serum β2 microglobulin (β2m) and serum albumin were focused as the most important prognostic factors for survival.
However, serum concentration of β2m has been known as an indicator of glomerular filtration rate. The aim of this study was to compare the prognostic value of the level of β2m with that of creatinine clearance (Ccr) in patients with multiple myeloma. Retrospectively, from January 1, 1996 to November 30, 2003, we reviewed 176 MM patients (M: F 110:66 mean age: 58.5±11.0) whose 24-hour urinary creatinine clearance was available at the time of diagnosis. We collected clinical data such as hemoglobin, serum creatinine, calcium, albumin, β2m, creatinine clearance before chemotherapies, and patients’ survival time. Pretreatment β2m was inversely related to Ccr (Spearman’s correlation coefficient = −0.781, P <0.01). In univariate analysis, relative risk (RR) of death was 1.047 (p< 0.001) for β2m and 0.985 (p< 0.001) for Ccr. But multivariate analysis using Cox’s proportional hazard model showed β2m was not significant prognostic factor in patients’ survival after adjustment for Ccr (RR 1.025, p=0.054) but Ccr was an independent risk factor of death after adjustment for β2m (RR 0.990, p=0.013). And univariate analysis identified that RR for β2m is not significant in 88 MM patients (66 patients with β2m ≥ 2.5mg/l) with relatively normal renal function (Ccr ≥ 50 ml/min) (RR = 1.110, p=0.306)
We could propose another new staging system with β2m replaced by Ccr in SWOG staging system. The stages were defined as: stage 1, Ccr ≥ 80ml/min; stage 2, 50< Ccr <80; stage 3, Ccr < 50 and albumin ≥ 3.0g/dl; and stage 4 Ccr < 50 ml/min and albumin< 3.0 g/dl. According to this proposed staging system, median survival time of each stage from 1 to 4 is 1475, 887, 513, and 196 days, respectively. (Log Rank test < 0.0001)
In conclusion, Ccr could be more important prognostic factor than the level of β2m in patients of MM and we propose that Ccr should be reassessed as the component of staging system of MM.
Mutations within the Protein Z-Dependent Protease Inhibitor (ZPI) Gene Are Associated with Venous Thromboembolic Disease: A New Form of Thrombophilia.
Authors: Harper Paul L. , Van de Water Neil S. , Ashton Fern , Tan Tina , Grady Anne O , Ockelford Paul A. , Browett Peter 
Published: October 2019

Abstract
ZPI is a recently characterised inhibitory serpin present in plasma. In vitro studies have shown that ZPI inhibits both factor Xa (in the presence of protein Z) and factor XIa. Deficiency of ZPI is predicted to enhance coagulation and may be a risk factor for venous thrombosis. To test this hypothesis we carried out mutation screening within the coding region of the ZPI gene in a cohort of 150 patients with a history of DVT or PE (first event before the age of 60 years) and 150 matched controls. Five PCR products were produced for each subject. Heteroduplex analysis was performed using dHPLC and the PCR products were sequenced directly if a heteroduplex mismatch was identified.
Sixteen mutations/polymorphisms were identified within the coding region of the ZPI gene. Two mutations were regarded as functionally significant as they produced stop codons at arginine 67 and tryptophan 303 and would be expected to result in the loss of ZPI activity. These stop codon mutations were found in 8 patients and 2 controls. Based on these results, a power calculation was performed that showed that an additional 100 patients and controls would be necessary to confirm that the stop codon mutations were statistically significant risk factors for thrombosis. Bidirectional allele specific PCR was developed to rapidly identify these stop codon mutations and a further 100 patients and 100 controls were examined using this technique. The stop codon mutations were found in 11 patients (W303X n=8: R67X n=3) and only 2 controls (R67X n=2) (two sided Fishers exact p=0.02) with an odds ratio = 5.7 (95%CI, 1.25 to 26.0). In addition two further non-conservative mutations were identified in two other thrombosis patients. These resulted in a serine to tyrosine (codon 122) and a phenylalanine to leucine (codon 124) change in the region homologous to the D helix of other heparin activated serpins. These changes, involving bulky aromatic amino acid residues, have the potential to disrupt the structure and function of the D helix.
We identified a further 12 mutations/polymorphisms (C454G, C574T, A603G, A647G, G752A, A947T, C1276T, G1277A, G1438A, A1617C, G1789T and C1811T (italics: not previously described)). The significance of these mutations is uncertain. We have identified mutations causing stop codons, which will result in loss of ZPI function, in 4.4% of patients who present with venous thrombosis before the age of 60 years, compared with an incidence of only 0.8% in controls. Further studies are ongoing to measure the plasma concentration of ZPI in the affected individuals. Our results support an association between mutations in the ZPI gene and venous thrombosis. We propose that ZPI deficiency as a result of these mutations is potentially a new form of thrombophilia.
Bisphosphonate Therapy Associated with an Increased Incidence of Mandibular/Maxillary Osteomyelitis in Multiple Myeloma Patients.
Authors: Thakkar Snehal G. , Isada Carlos , Englund Kristin , Tomford Walter , Karam Mary Ann , Reed Janice L. , Smith James , Hussein Mohamad A. 
Published: October 2019

Abstract
Bisphosphonates provide a meaningful supportive skeletal benefit for patients with multiple myeloma by inhibiting the resorbtion of bone thereby effectively reducing the incidence of pathologic fractures and hypercalcemia. Recently, bisphosphonates have been linked to osteonecrosis of the jaw in breast cancer and multiple myeloma patients. We report 14 cases of multiple myeloma patients being treated with bisphosphonates who developed periodontitis/osteomyelitis of the jaw.
All 14 patients were being treated with bisphosphonates for an average of 5 months (range: 3–9 months) prior to the onset of jaw symptoms. Thirteen of the 14 patients are 51 years or older. None of the patients had been irradiated in the jaw nor had obvious osseous manifestation of multiple myeloma in the jaw. Six patients were receiving zoledronic acid and 8 pamidronate. Organisms were isolated in 6/14 patients with the most common organism being actinomycosis. All patients remain on suppressive antibiotic therapy in the form of clindamycin.
The mandible and maxilla are bisphosphonate seeking bones as evidenced by scintigraphy. The local perpetuation of an infection in the mandibular/maxillary area in association with bisphosphonates is likely multi-factorial. This environment is continually exposed to stress trauma from repetitive motions. Bisphosphonates disrupt the normal bone homeostasis and this is complicated by the fact that the oral cavity is saturated with natural flora making this area vulnerable to infection. Additionally, in an acidic environment, such as one resulting from an infection, bisphosphonates are released locally from hydroxyapatite of bone into the surrounding environment and are cytotoxic to the local stromal cells. In the presence of inflammation and infection, there is evidence to suggest that bisphosphonates, especially the nitrogen-containing compounds, may actually up regulate the host inflammatory response with stimulation of IL-1 and IL-6. This generates a continuous cycle of inflammation and release of bisphosphonates from bone comprimising the healing process as has been previously reported.
We recommend that patients have a full dental examination at the time of diagnosis of the plasma cell dyscrasia especially if bisphosphonates are to be considered as part of the therapy. In addition, we suggest to hold bisphosphonates for a period of 3 months prior to invasive dental procedures to allow for the osteoclastic activity to recover, allowing for debris removal and lessening the chance of creating a fertile bacterial medium. Patients with multiple myeloma on bisphosphonate therapy whom experience dental/jaw discomfort should have an aggressive evaluation to rule out an infectious etiology.