bluuebunny/crossref_metadata_2025 huggingface dataset:
Disparate Mechanisms of Antifolate Resistance Provoked by Methotrexate and Its Metabolite 7-Hydroxymethotrexate in Leukemia Cells: Implications for Efficacy of Methotrexate Therapy.
Authors: Albertioni Freidoun , Jansen Gerrit , Fotoohi Kambiz , Assaraf Yehuda G. , Rothem Lilah , Stark Michal , Kathmann Letje , Gregorczyk Jacek , Peters Godefridus J.
Published: October 2019

Methotrexate (MTX) is one of the leading drugs in the treatment of leukemia, but extensive metabolism to 7-hydroxymethotrexate (7-OHMTX) can limit its therapeutic efficacy. In this study we investigated whether 7-OHMTX itself can provoke antifolate resistance that may further disrupt MTX efficacy. For this purpose we developed resistance to 7-OHMTX as well as MTX in two human leukemia cell lines (CCRF-CEM and MOLT-4) by stepwise exposure to increasing concentrations of 7-OHMTX and MTX. Consequently, both leukemia cell lines displayed marked levels of resistance to 7-OHMTX (>10 fold) and MTX (>75 fold), respectively. The underlying mechanism of resistance in the MTX-exposed cells was a marked decrease (>10-fold) in reduced folate carrier (RFC)-mediated cellular uptake of MTX. This was associated with transcriptional silencing of the RFC gene in MTX-resistant CCRF-CEM cells. In contrast, the molecular basis for the resistance to 7-OHMTX was solely due to a marked decreased (> 95%) in folylpolyglutamate synthetase (FPGS) activity which conferred >100-fold MTX resistance upon a short term exposure to this drug. This is the first demonstration that 7-OHMTX can provoke distinct modalities of antifolate resistance as compared to the parent drug MTX.
Src Homology 2 Domain Binding as a Phosphoproteomic Approach to Signaling in Acute Lymphoblastic Leukemia.
Authors: Horstmann Martin A. , Nollau Peter
Published: October 2019

A general theme of orchestrated signal transduction is played by activated receptor phosphotyrosine kinases (PTK) and receptor PTK targets which propagate signals via recognition of sequence-specific phoshorylated tyrosines by so-called Src homology 2 (SH2) domains. SH2 domain interactions are used as a means of recruiting target proteins to activated PTKs and to translocate them to the plasma membrane where many effector proteins activated by receptor PTKs such as phospholipase C-γ or PI-3 kinase have their substrates. SH2 domains make up the most prevalent type of phosphotyrosine binding domains involved in signaling downstream of activated PTKs. SH2 domains are not only present in proteins with intrinsic enzymatic activity but also in adaptor proteins which shuttle effector enzymes to target signaling complexes. Increasing numbers of diseases are known to involve phosphotyrosine specific kinases and/or phosphatases going awry exemplified by the notorious ErbB2 receptor PTK in breast cancer or the Bcr-Abl PTK in CML. Currently, the tyrosine phosphorylation state in most acute lymphoblastic leukemias is undefined which is predicted to differ among the various subgroups and to be distinct from the signaling state of normal hematopoietic cells. To identify aberrant tyrosine kinase or phosphatase activity in the various types of acute lymphoblastic leukemia is of great interest since enzymes in general make good targets for drugs. A novel SH2 domain binding approach is presented which can detect distinctive profiles of tyrosine-phosphorylated proteins in complex mixtures of cellular proteins. A battery of SH2 domains is employed as probes in a competitive far-Western blot based assay to identify specific tyrosine-phosphorylated sites which reflect active signaling pathways in a cell. A further refinement of this technology is under way with DNA-tagged probes being developed which allow for multiplexing and high throughput quantitative assessment of SH2-domain binding by quantitative PCR or microarray technologies.
Murine Melanomas Expressing High Levels of SDF-1 Repel and Escape Control by Tumor Specific T-Cells.
Authors: Vianello Fabrizio , Papeta Natalia , Kircher Moritz F. , Toner Mehmet , Weissleder Ralph , Irimia Daniel , Poznansky Mark C.
Published: October 2019

Most antigenic tumors are able to evade the immune system despite T cell recognition of tumor specific antigens. The chemokine, stromal derived factor- 1 (SDF-1 or CXCL12) is constitutively expressed by both normal and neoplastic tissues and is involved in tumor growth, metastasis and modulation of the anti-tumor immune response. We have previously shown that human T cells are repelled by the chemokine SDF-1, in a concentration-dependent and CXCR4 receptor-mediated manner. We proposed that the repulsion of tumor-specific T cells from a tumor expressing SDF-1 may represent a novel mechanism of immune evasion. B16 melanoma cells stably expressing OVA were genetically modified to constitutively express SDF-1 using a bicistronic MSCV2.2 retroviral transfer vector encoding murine SDF-1α and EGFP. SDF-1 generated by transduced cells was 58 ± 28.5 ng/ml/1x106 cells/24 hours as measured by ELISA. Immunization of mice with irradiated B16/OVA cells lead to destruction of B16/OVA.MSCV control tumors but not tumors expressing high levels of SDF-1 (p=0.0001). Failure to reject B16/OVA.SDF-1 tumors was associated with a reduction of T cell infiltration compared to control B16/OVA.MSCV (p=0.001). Similarly, early recruitment of adoptively-transferred OVA-specific CD8+ T cells into B16/OVA.SDF-1 tumors was significantly reduced in comparison to B16/OVA.MSCV control tumors as demonstrated by immunohistochemistry and real-time magnetic resonance imaging of nanoparticle labeled CTLs (p=0.018). Transferred memory OVA-specific CD8+ T-cells demonstrated antitumor activity against B16/OVA.MSCV control tumors but not against B16/OVA-SDF-1 tumors (p=0.04). Isolation and FACS analysis of tumor-infiltrating lymphocytes showed a 4-fold reduction of T cells in B16/OVA.SDF-1 compared to B16/OVA.MSCV control tumors (p=0.005). The in-vitro killing activity of tumor specific CD8+ T cells against B16/OVA.SDF-1 tumors was significantly reduced compared to B16/OVA.MSCV control tumors when measured in 51Cr release assay performed in a flat-bottom well system where cytotoxicity was dependent on effector cell migration (p=0.0004). Preincubation of OT-I CD8+ T cells with the CXCR4 antagonist AMD3100 restored their cytolytic activity against B16/OVA.SDF-1 cells (p=0.045). These data support the thesis that the active movement of tumor-specific T cells away from SDF-1, termed fugetaxis, may provide a novel mechanism by which a tumor evades challenge by immune effector cells and raises the possibility of the development of a novel immunotherapeutic approach to cancer.
Flow Cytometric Expression of bcl-2 by CD10+ B-Cells Reliably Distinguishes Follicular Lymphoma from Follicular Hyperplasia.
Authors: Kaleem Zahid , Leonard Ronald L.
Published: October 2019

The diagnosis of follicular lymphoma (FL) is often straightforward in most biopsy specimens. In a minority of cases, however, a differentiation from follicular hyperplasia (FH) can be challenging, especially in small needle core biopsies. The difficulty can be compounded in cases of FL with non-diagnostic immunoglobulin light chain ratios and cases of lymphoma without any significant surface expression of either immunoglobulin kappa or lambda light chain. The expression of bcl-2 by follicle center B-cells is often used to distinguish FL from FH using immunohistochemistry. The interpretation of bcl-2 expression patterns by immunohistochemistry, however, can be difficult because of normal expression of bcl-2 by T-cells present in reactive lymphoid follicles and by normal expression of bcl-2 by mantle zone B-cells. We have utilized multiparameter flow cytometry to evaluate the expression of bcl-2 in cases of FL and FH with successful results. This study includes consecutive biopsy specimens from well-documented cases of FL (n = 12) and FH (n = 16). Other lymphoma types were not included. Cytoplasmic expression of bcl-2 was evaluated on CD10+CD19+ B-cells using permeabilization techniques on a four-color Beckman-Coulter Epics-XL flow cytometer. Appropriate controls were run in each case. All cases of CD10-expressing FL showed significant expression of bcl-2 by CD10+ B-cells in contrast to no significant expression of bcl-2 by CD10+ follicle center B-cells of reactive follicles (Positive predictive value, 100%; Negative predictive value, 100%). Although our patient population is small, the results indicate that the expression of cytoplasmic bcl-2 on CD10+ B-cells by flow cytometry reliably distingushes FL from FH using CD10 gating. This method is superior to immunohistochemical evaluation of bcl-2 in cases with CD10 expression. A minority of FL, however, do not show bcl-2 expression (not seen in our series) and such cases must be taken into consideration when evaluating a clonal B-cell population with CD10 but no bcl-2 expression.

DIAGNOSIS CD45+CD19+(%) CD45+CD19+CD10+(%) bcl2+(%) in CD10+ gate FL grade 1/3 65 65 38 FL grade 1/3 68 44 67 FL grade 1/3 72 72 99 FL grade 1/3 88 62 99 FL grade 2/3 72 68 99 FL grade 1/3 59 59 95 FL grade 1/3 96 84 94 FL grade 1/3 59 59 95 FL grade 1/3 45 38 99 FL grade 2/3 76 74 76 FL grade 1/3 54 47 90 FL grade 2/3 35 34 98
A Population-Based Nested Case-Control Study of Prediagnostic Serum Antibodies to JC Virus and BK Virus and Risk of Non-Hodgkin’S Lymphoma.
Authors: Rollison Dana E. , Halsey Neal A. , Shah Keerti V. , Vascidi Raphael P. , Helzlsouer Kathy J.
Published: October 2019

Viral infections such as HIV, EBV, and HCV have been associated with increased risk of non-Hodgkin’s lymphoma (NHL). We conducted a nested case-control study to investigate the association between prediagnostic serum antibodies to JC virus (JCV) and BK virus (BKV) and subsequent risk of NHL.

Methods: Two research serum banks were established in Washington County, MD, with more than 45,000 volunteers contributing blood samples collected in 1974 and 1989. Incident cases of NHL diagnosed through 2002 (n=170) were identified among participants by linkage to population-based cancer registries. Two controls were matched to each case (n=340) on age, sex, and date of blood draw. Pre-diagnostic circulating IgG antibodies to JCV and BKV were measured using virus-like particle (VLP) enzyme-linked immunosorbant assays (ELISA). Associations between JCV and BKV antibody seropositivity and NHL were estimated using conditional logistic regression.

Results: Overall, neither serum antibodies to JCV (odds ratio (OR) = 0.83, 95% confidence interval (CI) = 0.56–1.23) or BKV (OR = 0.98, 95% CI = 0.64–1.48) were associated with an increased risk of NHL. Results were similar across NHL subtypes. Examining time to diagnosis suggested a possible increase in NHL risk associated with JCV antibodies, but not BKV antibodies, among individuals diagnosed 12–20 years after the time of blood draw. Among a subset of individuals who donated blood in both 1974 and 1989, those who experienced an increase in JCV antibody levels over time were more than four times as likely to develop NHL as compared to those whose antibody levels steeply declined (OR = 4.59, 95% CI = 1.30–16.25).

Conclusion: The finding of increased risk with increasing JCV antibody titers suggests a possible association between reactivation of JCV infection and subsequent NHL.
High Efficacy and Good Tolerability with Rituximab In Vivo Purging Followed by High Dose Chemotheraphy and Autologous Peripheral Blood Stem Cell Transplantation (APBSCT) in Non-Hodgkin’s Lymphoma (NHL).
Authors: Shi Yuankai , Yang Sheng , Han Xiaohong , Liu Peng , He Xiaohui , Ma Jun , Ren Hanyun , Cen Xi’nan , Zhou Shuyuan , Wang Chun
Published: October 2019

Purpose: High-dose chemotherapy (HDC) supported by APBSCT has been shown to be superior to standard therapy in NHL. However, many patients relapse due to minimal residual disease (MRD) in vivo or in the graft. Rituximab has the potential to clear both blood and bone marrow of malignant CD20+ cells, prompting this multicenter trial of in vivo purging with rituximab and HDC with APBSCT in China.

Methods: Cyclophosphamide 4g/m2 was used as the mobilization regimen, CY/TBI, BEAM or CBV could be used as HDC at the discretion of the institution. Four infusions of rituximab (375 mg/m2) were given: one day before mobilization, one day before harvesting, one day before transplantation and on day 8 after transplantation. BCL-2/Ig-H translocation was measured as a marker of minimal residual disease in blood or bone marrow before mobilization and during transplantation using real-time quantitative PCR.

Results: Thirty-one patients from 12 centers with histologically proven CD20+ NHL (28 aggressive, 3 indolent NHL) were enrolled. Twenty-four patients were previously untreated, and 7 patients had relapsed disease. Median yields of CD34+ cells and mononuclear cells were 5.9×106/kg and 4.4×108 /kg respectively. Median time to recovery of WBC >1.5×109/L, ANC >0.5×109/L and platelets >20×109/L after APBSCT was 10 days in each case. Median time to platelet recovery >50×109/L was 13 days. Generally, this therapeutic strategy was well tolerated with few side effects attribute to rituximab. All patients achieved a complete remission after APBSCT. At a median-follow-up of 12 months, overall survival and progression-free survival (PFS) are 87% and 73% respectively for all patients. In patients with aggressive NHL, overall survival and PFS are 85% and 73% respectively and in indolent NHL are 100% and 67% respectively. PFS and overall survival were slightly higher in previously untreated compared with relapsed patients (88% vs. 83% for PFS, 73% vs. 69% for overall survival). One of five 5 patients who were initially found to be PCR-positive and achieved PCR-negative status subsequently experienced progression accompanied by a return to PCR positivity. The remaining four patients are still in complete remission and are PCR negative.

Conclusion: These results suggest that the regimen of rituximab combined with HDCT and APBSCT is effective and well tolerated for the treatment of patients with NHL.
A Combination of Rituximab with Chemotherapy in Low Grade Lymphoma.
Authors: Katakkar Suresh B.
Published: October 2019

Introduction: The combination of rituximab with conventional chemotherapy is superior to rituximab therapy alone. Maintanence of chemo-immunotherapy, done for two years at a fixed interval based on pharmacokinetics of Rituximab, may give long term remission improving disease free intervals.

Rationale: MoAb therapy works not only by recruitment of FcR but also by complement dependent cytotoxicity(CDC) and growth arrest and/or apoptosis.

Methodology: This was an open label study. Pts with follicular and diffuse small cleaved-cell NHL stage III and IV were eligible. Induction chemotherapy consists of Cytoxan 650 mg/m2 iv, Vincristine 1mg/m2, Prednisone 50mg/m2 po for 7 days, repeated Q3 weeks. Rituximab was given 375mg/m2 iv Q week for 6 weeks. After 10 weeks of completion of Rituximab, pts were maintained on Rituximab 375 mg/m2 iv Q week for 6 weeks with only one chemotherapy in week 3. CT/PET scan done Q 3 months.

Results: 42 patients; 20 females and 22 males, median age 58. 18 stage III and 24 stage IV. 91%(38/42) went into CR with induction and 1st maintanence therapy. Remaining 4 went into remission after all 6 sessions. Only 3 have relapsed of which 1 is in CR again with reinduction. Thus 39/42 are in CR for a median of 47 months. The toxicity was minimal, with 5% fever chills and hypotension. All responded well to premedication with tagamet and corticosteroid; given iv. 2% incidence of neutropenia. Two patients had sepsis.

Conclusions: 1. The induction and maintanence with Rituximab with addition of chemotherapy is synergistic. 2. It has prolonged the disease free survival. 3. Time to disease progression is prolonged and the final duration of remission is not reached. 4. The toxicity is acceptable, though the treatment is expensive. 5. This chemo-immunotherapy may eliminate microscopic disease and avoid stemcell transplant and associated mortality and morbidity.
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

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

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

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

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

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

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

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

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.
Natural History of Skeletal-Related Events in Patients with Multiple Myeloma: Exploratory Analysis Based on Prior History of Skeletal Complications.
Authors: Hussein Mohamad A. , Kaminski Mary , Rosen Lee , Gordon David , Zheng Ming
Published: October 2019

Background: We previously reported the long-term efficacy and safety of 4 mg zoledronic acid compared with 90 mg pamidronate in patients with bone lesions from breast cancer or multiple myeloma (Rosen et al. Cancer. 2003;98:1735–1744). Bone lesions result in an increased risk of developing skeletal-related events (SREs), including pathologic fracture, spinal cord compression, radiation therapy or surgery to bone, or hypercalcemia of malignancy. It has also been suggested anecdotally that patients who have experienced a prior SRE may be at increased risk for future SREs, but no data exist to support this hypothesis. We report here the results of a retrospective exploratory analysis to assess whether patients with a history of SREs before study entry had a higher incidence of on-study SREs and to assess the clinical benefit of zoledronic acid compared with pamidronate in advanced multiple myeloma patients based on SRE history.

Methods: The subset of multiple myeloma patients treated with zoledronic acid (4 mg) or pamidronate (90 mg) every 3 to 4 weeks was retrospectively stratified according to history of SREs before study entry. A total of 353 patients with multiple myeloma were randomized 1:1 to zoledronic acid or pamidronate for 12 months, and 138 patients elected to continue blinded treatment for an additional year.

Results: Before study entry, 284 (~80%) patients had experienced at least 1 SRE and these patients were more likely to develop an SRE on study than patients without a history of SREs (time to first SRE hazard ratio = 2.22; P <.0001). Across treatment groups, 56% of these patients had at least 1 SRE on study (after 25 months) versus only 35% patients with no SRE before study entry. Additionally, patients with an SRE before study entry had a shorter median time to first on-study SRE (median, 293 days for zoledronic acid and 218 days for pamidronate) compared with patients who had no prior SRE (median, 504 days for zoledronic acid and not reached for pamidronate). Among patients with an SRE before study entry, the zoledronic acid group had a decreased percentage of patients with an on-study SRE compared with the pamidronate group (53% for zoledronic acid versus 59% for pamidronate; P =.291). Zoledronic acid also delayed time to first SRE by 75 days compared with pamidronate (median 293 days for zoledronic acid versus 218 days for pamidronate; P =.679).

Conclusions: This exploratory analysis indicates that multiple myeloma patients with a history of SREs are at significantly higher risk of developing subsequent skeletal complications. Early intervention for prevention of SREs may provide considerable clinical benefit to these patients.
Anti-Thymocyte Globulin Increases Dramatically Natural Killer Cell Interferon Gamma Production While Respecting Other Functionnal Aspects of These Cells.
Authors: Dalle Jean-Hugues , Menezes José , Wagner Eric , Champagne Martin A. , Duval Michel
Published: October 2019

Rational: The beneficial effect of NK cells after clinical hematopoietic transplantation has been seen when anti-thymocyte globulin (ATG) was used within the conditioning regimen. We therefore investigated the effect of ATG on human NK cells. Materials and methods: Percentages of CD3+ and CD3−/CD56+ among PBMC from either normal adult volunteers or cord blood were assessed by flow cytometry. Negatively purified NK cells were cultured for 2–4 days either alone or in the presence of rabbit ATG (Thymoglobuline, Sangstat) and/or IL-15, and then assessed for cell viability and functional properties: apoptosis were investigated by Annexin V staining. Cell proliferation was assessed by CFSE dye dilution. IFN-gamma secretion was quantified by ELISA. Cytotoxicity against K562 cells was studied by a 51Cr release assay. Binding of monoclonal antibodies to NK cells was assessed alone or after competition with whole ATG or ATG-Fab’2 fragments.

Results: The percentage of CD3+ cells in PBMC was over 65% before or after culture without ATG. In contrast, no T cell was detected after culture with ATG, whereas the percentage of NK cells increased from less than 15% to more than 50%. Both whole ATG and Fab’2 fragments bound NK cells while rabbit Fc fragments did not. ATG did not modify apoptotic and necrotic NK cells percentages. Proliferation was not affected by ATG. IFN-gamma production was dramatically increased by ATG treatment (figure). ATG respected IL15 induced NK cell cytolytic activity. Competition assays showed that ATG did not alter CD2, CD11a, CD16, CD56, CD57, CD62L, CD94, CD158a, CD158b, NKG2a, NKG2d, HLA-DR expression. CD18 and NKp46 expression was slightly decreased.

Conclusion: Our data show that ATG may play a role in the results of KIR mismatch hematopoietic stem cell transplantation. It may be possible that ATG confers functional advantages to NK cells versus T cells.

Figure Figure
A Possible Association between the Presence of Interleukin-4-Secreting Cells and a Reduction in the Risk of Acute Graft-Versus-Host Disease.
Authors: Takabayashi Maki , Kanamori Heiwa , Takasaki Hirotaka , Yamaji Satoshi , Koharazawa Hideyuki , Taguchi Jun , Tomita Naoto , Fujimaki Katsumichi , Fujita Hiroyuki , Fujisawa Shin , Maruta Atsuo , Ishigatsubo Yoshiaki
Published: October 2019

We monitored interleukin-4 (IL-4), interferon-gamma (IFN-g), and tumor necrosis factor alpha (TNF-a)-secreting cells using an enzyme-linked immunospot (ELISPOT) assay in a prospective study to assess the cytokine network after transplantation. Peripheral blood mononuclear cells were collected from 23 patients who received allogeneic stem cell transplantation, from before the preconditioning regimen to 56 days after transplantation. The frequency of IL-4-secreting cells was significantly higher in 5 patients receiving peripheral blood stem cell transplantation (PBSCT) than that in 18 patients who received bone marrow transplantation (BMT). Based on IFN-g and TNF-a release, there was a trend toward a decrease in the number of cytokine-secreting cells in PBSCT. Furthermore, patients who did not develop acute graft-versus-host disease (GVHD, n = 5) showed a significantly higher number of IL-4-secreting cells compared with those who developed acute GVHD (n = 18). These results indicate that the high percentage of IL-4-secreting cells may be responsible for the inhibition of acute GVHD. In addition, the increased percentage of IL-4-secreting cells may be responsible for the unexpected low incidence of acute GVHD in PBSCT, despite the presence of large numbers of mature T cells in the donor infusion.
Effects of Amotosalen Hydrochloride and Ultraviolet a Light on CD4 and CD8 Cells.
Authors: Jordan Catherine T. , Zimring James C. , Roback John D.
Published: October 2019

Background: Graft versus host disease (GvHD) and infections by opportunistic pathogens, such as cytomegalovirus (CMV), are causes of significant morbidity and mortality in recipients of allogeneic bone marrow transplants (BMT). Thus, there is a need for methods of graft engineering that inhibit the alloreactive T cells responsible for lethal GvHD without compromising the activity of antiviral T cells. In a murine MHC-mismatched BMT model, we have previously demonstrated that adoptive transfer of polyclonal T cells from donors immunized to murine CMV (MCMV) can decrease viral load but cause lethal GvHD. However, pretreatment of these cells with the psoralen, amotosalen hydrochloride, and ultraviolet A (UVA) light prevents GvHD without compromising antiviral response. We have previously hypothesized that these effects were due to differential sensitivity of naïve and memory T cells to amotosalen/UVA. Recent investigations have demonstrated that CD4 T cells are most directly responsible for lethal GvHD in this model. This observation suggested an alternative hypothesis, equally consistent with previous data, that the observed in vivo effects of amotosalen/UVA treatment are due to differential effects on CD4 and CD8 T cells. The assessment of this new hypothesis is the focus of the current studies.

Methods: Two models of T cell activation/proliferation were utilized to test the effects of amotosalen/UVA treatment on CD4 and CD8 cells: stimulation of B6.PL (H-2b) cells with concavalinA, and primary mixed lymphocyte reaction (MLR) between MHC-mismatched B6.PL (H-2b) and BALB/c (H-2d). Responder cells were pretreated with 2nM amotosalen and varying doses of UVA light (0–5 minutes). Proliferation of CD4 and CD8 cells was measured by flow cytometric analysis of CFSE-labeled responder cells.

Results: In both systems, CD4 proliferation was effectively eliminated by immediately prior treatment with amotosalen and UVA doses of 1 minute or higher. CD8 proliferation was eliminated by amotosalen and UVA doses of 2 minutes and higher. Both the amount of division on a per cell basis and the overall number of cells that initiated division followed similar trends.

Conclusions: These data demonstrate that both CD4 and CD8 T cells are sensitive to treatment with amotosalen/UVA and suggest a subtle difference in sensitivity of CD4 and CD8 populations. Since division of both CD4 or CD8 cells is inhibited at doses of amotosalen/UVA that prevent GvHD but allow anti-viral activity in vivo, it is unlikely that the observed differential sensitivity of T-cell subsets is sufficient to explain the in vivo effects of amotosalen/UVA treatment in this model. Using similar methodologies, ongoing studies are assessing the hypothesis that amotosalen/UVA has differential effects on naïve and mature T cells.