bluuebunny/crossref_metadata_2025 huggingface dataset:
Complement-Induced Cell Death by Rituximab Depends on CD20-Expression Level and Acts Complementary to Antibody-Dependent Cell-Mediated Cytotoxicity.
Authors: van Meerten Tom , van Rijn Rozemarijn S. , Hagenbeek Anton , Ebeling Saskia B.
Published: October 2019

The CD20-specific monoclonal antibody rituximab (RTX) has significantly enhanced the overall survival of B-cell non-Hodgkin’s lymphoma but not of chronic lymphocytic leukemia. The level of CD20 expression on tumors has been related to response, but results of several studies are contradictory and no clear relationship could be established. The main effector mechanisms of RTX are thought to be complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), but the exact mechanism is still unknown. Part of the lack of a clear relationship may be explained by the absence of data on the combined contribution of CDC and ADCC.

To study the impact of CD20-expression level on CDC, ADCC and their relative contribution, we developed a system to manipulate CD20-expression levels on cells by transducing the CD20-negative CEM T cell line and developed a new assay to investigate the combined contribution of RTX-mediated CDC and ADCC. By limiting dilution of the transduced and selected cells, clonally-related CEM-CD20 cells were obtained with the number of CD20 molecules as the only variable parameter (mean fluorescence intensity range: 256–693, N=90). These were then subjected to RTX and human serum as a complement source. The results show that the CDC activity clearly depends on CD20-expression level and there is a linear correlation of CD20-expression level to RTX-mediated killing by CDC (p<0.001, r2=0.9125). To further strengthen the importance of CD20-expression level to RTX-mediated CDC, we transduced the already CD20-postive Raji Burkitt’s lymphoma cell line with the CD20-encoding retrovirus. The extra CD20 molecules thus obtained made the cells significantly more sensitive to human serum and RTX (p<0.001).

For ADCC, the CEM-CD20 clones were subjected to effector cells and RTX, which, in contrast to CDC, showed no correlation between CD20-expression level and cell kill via ADCC (p=0.1402, r2=0.1869).

For the combination of CDC and ADCC, as with CDC alone, results showed a correlation between CD20 expression and cell kill (p<0.0201, r2=0.6214). Importantly, CDC-resistant cells were sensitive to ADCC and vice versa. These findings indicate that CDC and ADCC act complementary.

In summary, these data show the importance of CD20-expression level for RTX-mediated cell kill and give new insights into the development of novel strategies to increase RTX-efficacy and the possibility to predict tumor response.
Cocktail Therapy Increased the Survival Rate of APL.
Authors: Zhou Jin , Meng Ran , Li Limin , Yu Jie , Yang Baofeng
Published: October 2019

Background Arsenic trioxide provides significant benefits in newly diagnosed and relapsed acute promyelocytic leukemia (APL) respectively. However, the high relapsed rate is still threatened the life of APL patients. Which regimen should be used to overcome or reduce the relapse in consolidated treatment is a key problem at present. We performed a pilot study about that.

Objective To Compare the effectiveness and security of cocktail therapy with single arsenic trioxide therapy in APL consolidated treatment.

Methods Sixty-Five APL patients, who once received arsenic trioxide treatment and obtained complete remission, were enrolled in this study. Patients were divided into two groups according to the different consolidated regimens. After reinforced treated with DA (daunomycin and cytarabine) or HOAP (harringtonine, vincristin, cytarabine and prednisone) for two course, Group A involved twenty cases received single arsenic trioxide consolidated, Group B included forty-five cases treated with the cocktail therapy, alternatively treated with arsenic trioxide, all trans-retinoic acid and chemotherapy (DA or HOAP). The relapse rates, the survival rates and the central nervous system infiltration rates in 3 years followed up were compared.

Results The relapsed rate of Group A was 55%, which was higher than that of Group B(17.8%). The re-remission rate after the first relapse in Group A was 22%, which was lower than that of Group B(42.8%). The central nervous system infiltration rate of Group A was 28%, which was higher than that of Group B(6%). The average survival time of Group A was 10.5±4.2months, which was shorter than that of Group B (22.5±5.5 months). The three-year survival rate of Group A was 15%, which was less than that of Group B (65.8%).

Conclusions Cocktail therapy —alternatively treated with arsenic trioxide, all trans-retinoic acid and chemotherapy will be the reasonable regimen for APL consolidated treatment. Which provided benefited on inhibiting relapse and central nervous system infiltration of APL.
Suppressor of Cytokine Signaling 3 (SOCS3) Regulates CXCL12-Induced Adhesion to VCAM1 during B Cell Development.
Authors: Silberstein Leslie , Le Yi
Published: October 2019

CXCL12 (SDF-1) induces prolonged FAK phosphorylation in bone marrow (BM) progenitor B cells, but only induces transient FAK phosphorylation in peripheral B cells. In addition, FAK signaling is tightly correlated with CXCL12-induced adhesion to VCAM-1. Given that the CXCR4 receptor expression is similar at all B cell stages, we hypothesized that progenitor and mature B cells possess different mechanisms to control FAK phosphorylation. Here we demonstrate that SOCS3 regulates CXCL12-induced FAK signaling through the ubiquitin-proteosome pathway. First we show that CXCL12 triggers increased FAK ubiquitination in human peripheral B cells and mouse spleen B cells, but not in human and mouse BM B cells. In accordance with this finding, SOCS3 expression is relatively low in progenitor BM B cells and high in peripheral blood and splenic B cells. Through over-expression of SOCS3 in the progenitor B cell line, REH, we demonstrate that SOCS3 co-associates with FAK following CXCL12 stimulation. Moreover, over-expression of SOCS3 results in decreased FAK phosphorylation (intensity and duration), and impaired CXCL12- induced chemotaxis and adhesion to VCAM1. Next, we isolated splenic B cells from MMTV-Cre SOCS3 fl/fl mice and found that SOCS3 deficient mature B cells have increased and prolonged FAK phosphorylation and also show enhanced chemotaxis and sustained adhesion to VCAM-1, similar to progenitor B cells. Flow cytometric analysis revealed that MMTV-Cre SOCS3 fl/fl mice have an increased number of IgM+, Ig D-, mature B cells in the BM compared to wild type mice. Taken together, our results suggest that regulation of FAK signaling through SOCS3 may be important for B cell maturation in the BM and for the egress of mature B cells into the circulation.
The Use of Hoechst Staining To Identify Hematopoietic and Other Adult Stem Cells.
Authors: Ogawa Rei , Fujimura Juri , Sugihara Hidemitsu , Suzuki Hidenori , Hyakusoku Hiko , Shimada Takashi
Published: October 2019

BACKGROUND: Hoechst staining has been used to identify hematopoietic stem cells, but it may also be useful in identifying other adult stem cells. Here we report our efforts to purify and characterize stem cells in the stromal vascular fraction of adipose tissue with Hoechst staining, also comparing our results with those of our studies using bone marrow.

METHODS: Stromal vascular fractions (SVFs) of adipose tissue and whole bone marrow (BM) were harvested from C57BL/6N mice, as were stem cells. Then the cells were stained with Hoechst 33342 and analyzed by flow cytometry and the number of cells in the side population (SP) counted. Moreover, surface antigens of SP cells were analyzed by flow cytometry using antibodies against CD44, 45, 45R, Sca-1, and c-kit, respectively, for 30 min on ice. Finally, the morphologic characteristics of cells in the SP of both BM and SVF were observed using electron microscopy.

RESULTS: The percentage of SP cells in BM was about 0.05 to 0.1% and that in the SVF was about 1.0 to 3.0%. The cell-surface antigens of BM expressed were CD44 (−), CD 45 (+), CD 45R (−), Sca-1 (+) and c-kit (+), while those of SVF were CD44 (−), CD 45 (−), CD 45R (+/−), Sca-1 (+/−) and c-kit (−). Upon electron microscopic observation, both BM and SVF cells in the SP were considered to be remarkably immature (immature cell organelles and a high N/C ratio).

CONCLUSION: The rate and expression patterns of cell-surface antigens in SP cells derived from BM were consistent with the results of previous reports. However, the same characteristics in SP cells derived from SVFs were clearly different. At present it is not clear whether cells in the SP of SVFs are adipose-derived stem cells. Indications were that there are 10 to 60 times as many immature cells in adipose tissue as in bone marrow. Moreover, it is possible that the great majority of cells in the SP of SVFs are not hematopoietic stem cells but unique adipose-derived stem cells. Finally, our studies suggest that Hoechst staining may be useful for identifying not only hematopoietic stem cells but also other adult stem cells.
Studies on Analogues of L-Cysteine and L-Cystine
Authors: WEISBERGER AUSTIN S. , SUHRLAND LEIF G.
Published: October 2019

Selenium cystine decreases the incorporation of S35 L-cystine by rat Murphy lymphosarcoma tumor cells both in vitro and in vivo. Selenium cystine also decreases the growth of the tumor in the intact animal.

Benzyl selenium cysteine does not inhibit the incorporation of S35 L-cystine by Murphy lymphosarcoma tumor cells in vitro nor does it affect tumor growth in the intact animal.

Thus the ability of selenium cystine to decrease the influx of S35 L-cystine into tumor cells in vitro is associated with the ability of this compound to inhibit tumor growth in the intact animal. Furthermore, the inhibitory effect of selenium cystine is not due solely to the presence of selenium within the molecule but is related to its structural identity with cystine.

The data are further suggestive of the importance of SH compounds in the growth of malignant cells.
Studies on Analogues of L-Cysteine and L-Cystine
Authors: WEISBERGER AUSTIN S. , SUHRLAND LEIF G.
Published: October 2019

Selenium cystine was administered orally to 2 patients with acute leukemia and to 2 patients with chronic myeloid leukemia. In all patients there was a rapid decrease in the total leukocyte count as well as a decrease in spleen size. This effect was observed in patients refractory to other chemotherapeutic agents as well as in the usually resistant types of leukemia. In patients with chronic myeloid leukemia the immature granulocytes disappeared much more rapidly than the mature granulocytes. The most striking and consistent effects were observed in acute leukemia. One patient who had become resistant to 6-mercaptopurine appeared to reacquire sensitivity to this compound after receiving selenium cystine.

These effects of selenium cystine on leukocytes correlate with the ability of selenium cystine to decrease the influx of S35 L-cystine by leukemic leukocytes in vitro. Other potentially effective analogues of cystine (or cysteine) may therefore be selected by this technic.

No changes were detected in any of the organs attributable to selenium cystine toxicity. The nausea and vomiting associated with the oral administration of selenium cystine was so severe that it was not possible to administer selenium cystine for a sufficient period of time to determine whether an appreciable remission can be obtained in leukemia. Further study is necessary to determine whether selenium cystine has any practical applicability in the chemotherapy of leukemia.

Although the mechanism of action of selenium cystine is not known, these striking effects of an analogue of cystine on leukemia are further suggestive of the importance of cystine (cysteine) in the metabolism of leukocytes.
Reticulocytopenia in Autoimmune Hemolytic Anemia
Authors: CROSBY WILLIAM H. , RAPPAPORT HENRY
Published: October 2019

1. In a series of 57 cases of autoimmune hemolytic anemia it was found that 25 or 44 per cent had a relative reticulocytopenia at times of hemolytic crisis. The mortality rate in this group was significantly higher than in those who showed a reticulocyte response consistent with the severity of anemia. Special significance is given to the high mortality rate among the patients with idiopathic AHA.

2. Four illustrative cases are presented.

3. There is a discussion of mechanisms that may be responsible for reticulocytopenia in the presence of severe anemia and erythroid hyperplasia of the marrow.
A Method for the Separation of the Different Morphologic Forms of the Blood Leukocytes
Authors: PHILIPPU ATHINEOS J.
Published: October 2019

A method for the separation of the different morphologic form of leukocytes is described.

The manipulations of the blood by this method are simple and the reagents used are easily acquired.

The leukocytes obtained are morphologically and functionally intact.
The Hemostatic Defect of Uremia
Authors: LARRAIN CAMILO , ADELSON EDWARD
Published: October 2019

The abnormalities of the hemostatic mechanism in three consecutive patients with acute renal insufficiency have been studied. In every case a prolonged clotting time in silicone and a prolonged bleeding time were found. In two cases defective prothrombin consumption was also present. Coagulation studies revealed no significant deficiency of plasma factors, no serious deficiency of platelet numbers or function and no well-defined anticoagulant effect to explain the changes. All three cases developed significant clinical bleeding at some time during the course of the renal insufficiency.
A Summary of Atypical Antibodies, Rare Genotypes, and ABO Hemolytic Disease Encountered in a One Year Survey
Authors: LEVINE PHILIP , ROBINSON ELIZABETH , STROUP MARJORY , MCGEE ROBERT , BUSHNELL LOUELLA N.
Published: October 2019

1. Among 13,000 selected blood specimens studied in 1954, there were 1882 which contained atypical antibodies, mainly anti-D.

2. Of the 177 sera with atypical antibodies other than anti-D, 85 contained anti-c, anti-E or anti-K and 18 sera contained anti-Lea.

3. There were 52 cases of intragroup hemolytic disease, more than half of which were due to anti-c.

4. Intragroup isoimmunization with production of antibodies other than anti-D was detected in 47 cases as incompatibility in the crossmatching. In 31 additional cases failure to detect incompatibility resulted in transfusion reactions.

5. There were 30 examples of very rare Rh genotypes due to the presence of rare chromosomes dCe (r'), dcE (r''), DCE (Rz), and the rarest of all, dCE (Ry). All told, there were 7 bloods which contained chromosome dCE (ry) including one—the first of its kind—which was homozygous.

6. It is recommended that rare genotypes be registered on a world-wide basis under the auspices of a suitable agency and that these bloods be made available as test cells or as compatible donors.

7. Sixty-two cases of ABO hemolytic disease are reviewed briefly. In all but four the mothers were in group O. Of the 55 group O mothers tested, all but 5 showed enhancement of the specific antibody in the indirect Coombs reaction.
Studies on the Pathogenesis of Splenic Anemia
Authors: GIBLETT E. R. , MOTULSKY A. G. , CASSERD F. , HOUGHTON B. , FINCH C. A.
Published: October 2019

Splenomegaly was produced in rats by repeated intraperitoneal injections of methylcellulose. Anemia and marked reticulocytosis resulted. Coombs tests were negative.

Comparative measurements of red cell life with Cr51 in normal, methyl-cellulose treated, and splenectomized animals showed a marked decrease of red cell survival in the methylcellulose rats. Slight but statistically insignificant increased survival could be demonstrated in the splenectomized animals.

Excessive red cell destruction occurred in the spleen as evidenced by rapid localization of Cr51-tagged cells in this organ. Direct correlation between severity of hemolysis and splenic size could be demonstrated. The hemolytic process decreased significantly after splenectomy.

Affected spleens showed red cell congestion of the pulp microscopically. Hemochromogen determinations revealed approximately twice the normal amount of hemoglobin per unit splenic mass.

Quantitative measurements of splenic phagocytosis showed a marked increases of total phagocytic mass with no significant increase of phagocytes per unit of spleen mass.

The mechanism of splenic anemia in these animals is thought to be a combination of an enlarged pulp compartment with resultant stasis and cell destruction.

Methylcellulose-induced splenomegaly may be considered as an experimental model for the study of the hyperfunctional spleen in human disease.
Some Observations on the Morphology of Erythropoietic Cells in Human Lead Poisoning
Authors: BERITIĆ TIHOMIL , VANDEKAR MILUTIN
Published: October 2019

Morphologic changes of erythropoietic cells in the bone marrow of 19 lead intoxicated patients are reported. In addition to clinical manifestations the toxic action of lead was verified by the laboratory findings: high lead blood values, increased coproporphyrinuria and characteristic findings in peripheral blood.

Qualitative changes of erythroblasts were constantly present in all smears examined. Abnormalities most frequently observed were: (a) basophilic stippling, (b) polyploidy, (c) nuclear alterations ("karyorrhexis"), and (d) aberrant chromosomes. The incideces of some of these changes have been given.

The mechanism by which these changes might be brought about is discussed; the role of interruption of cytokinesis and karyokinesis is emphasized.
Thalassemia in Sikhs
Authors: SIDDOO J. K. , SIDDOO S. K. , CHASE W. H. , MORGAN-DEAN L. , PERRY W. H.
Published: October 2019

1. The incidence of Thalassemia minor in the Sikh community of Vancouver, Canada, was computed to be six per cent from a study of 80 individuals, of whom five showed this condition.

2. To determine the incidence in blood relatives, the families of the above five propositi were then studied in detail. In addition, a sixth family, that of the original propositus was included. This made a total of 79 individuals, of whom 38 had Thalassemia minor and none had Thalassemia major.

3. It is suggested that a large pool of the Thalassemia gene existed in the Mediterranean basin and perhaps spread to Italy, Greece, Syria, Kurdistan, India, Thailand and China, countries which lie along the routes of early migration of races.
Ameboid Motility of Human Leukocytes
Authors: LAPIN JOSEPH , HORONICK ANDREW
Published: October 2019

This study presents a relatively simple and consistently reproducible technic for the quantitative evaluation of the ameboid motility of human leukocytes. Factors such as anticoagulants, time of centrifugation, the temperature of storage of leukocytes, the medium in which the leukocytes are suspended, and the use of tube or slide methods of observation with dark field or phase contrast microscopy, have been critically evaluated. The results of this technic, when applied to six normal donors and 36 ill patients, show that age, sex, anemia, general nutritional condition, diabetes and terminal disease have no clear cut effect on the vitality of the human leukocyte as measured by ameboid motility. It seems reasonable that this technic may be valuable in future studies on the leukotoxic effects of new drugs, as well as in studies of immunologic mechanisms in blood dyscrasias.
The Action of Leukocytosis-Promoting Factor on the Blood and Bone Marrow of the Guinea Pig
Authors: HARRIS P. F. , MENKIN V. , YOFFEY J. M.
Published: October 2019

Ten normal guinea pigs each weighing approximately 400 Gm. were given intraperitoneal injections of sterile pyrogen-free saline, and another ten were given injections of saline + 25 mg. of Leukocytosis-Promoting Factor (Menkin). The animals were killed 4 hours after the injection and quantitative studies made on the blood and the bone marrow.

LPF appears to give rise to a considerable discharge of granulocytes from the bone marrow, much greater than can be accounted for by the leukocytosis which develops. The administration of LPF is also associated with a marked increase in the marrow lymphocytes.
Antihemophilic Globulin Consumption During Blood Coagulation
Authors: DOUGLAS A. S.
Published: October 2019

(1) Antihemophilic globulin (AHG) consumption has been studied in normal blood maintained at 37 C. for one hour after collection. By the end of one hour the AHG was completely utilized.

(2) AHG consumption is defective completely in Christmas disease and heparin therapy, and to a lesser degree in phenylindanedione therapy. In platelet deficiency consumption of AHG is normal.

(3) A possible interpretation of these results is discussed in relation to the order of the interactions involved in blood thromboplastin formation.
Unusual Bleeding During Pregnancy: Report of a Case Due to Hypoprothrombinemia and Hypoproconvertinemia
Authors: SETNA SOONA S. , ALTMAN S. J.
Published: October 2019

A case of transient hypoprothrombinemia and hypoproconvertinemia associated with bleeding and occurring during the eighteenth and nineteenth weeks of a twin pregnancy has been described. The coagulation defect responded to intravenous K1, therapy but not to water soluble K. Death of one fetus and complete infarction of its placenta occurred during the eighteenth week. It is suggested that these events, or their cause, may have adversely influenced liver cell function and precipitated the severe deficiencies of prothrombin and proconvertin.
Confirmation of a Structural Abnormity in the Stroma of Erythrocytes from Paroxysmal Nocturnal Hemoglobinuria (PNH) After Hemolysis in Distilled Water
Authors: BRAUNSTEINER H. , GISINGER E. , PAKESCH F.
Published: October 2019

The presence of a structural abnormity in the stroma of erythrocytes from PNH after hemolysis in distilled water is confirmed.
Normal Bone Marrow Total Cell and Differential Values by Quantitative Analysis of Particle Smears
Authors: SANDKÜHLER S. , GROSS E.
Published: October 2019

1. A method is described which permits absolute, area-related bone marrow counts for the total nucleated cells and differential count. The technic is easy, the accuracy satisfactory.

2. Normal values for the marrow of young adults (18-45 years) are given.

3. The method permits comparison of any marrow cells independent from changes of the total cellularity, and it makes serial comparative marrow studies in diseases useful, especially in cases where the cellularity is greatly altered (aplasia, leukemia) and percentage-differential counts are almost impossible to judge properly.

4. Experiments in animals may be followed by absolute marrow counts, without alteration of the cells such as occurs in pipet- and shaking-methods. Experiments with ionizing radiation5 showed clearly the practical value of this technic.

5. A survey is given on some total cell counts in disease, showing the range of pathologic changes to be determined with the technic described. The range is from about 80,000 (in marrow atrophy) to 1.5 million (in small-cell leukemia).
Immunochemotherapy (R-MCP) Is Not Superior to Chemotherapy (MCP) Alone in Advanced Mantle Cell Lymphoma - 42 Months Follow Up Results of the OSHO 39 Study.
Authors: Herold Michael , Haas Antje , Srock Stephanie , Neser Sabine , Al Ali Kathrin H. , Neubauer Andreas , Doelken Gottfried , Naumann Ralph , Fietz Thomas , Freund Mathias , Rohrberg Robert , Hoeffken Klaus , Mohren Martin , Ittel Thomas H. , Kettner Erika
Published: October 2019

Introduction: Rituximab plus chemotherapy has been proved to be the standard in treating advanced follicular lymphoma. However in mantle cell lymphoma (MCL) the results are still controversial.

Methods: In a prospective randomised trial (OSHO#39) we compared the efficacy and toxicity of MCP chemotherapy(mitoxantrone 8 mg/m2 d 1+2, chlormabucil 3x3 mg/m2 d 1–5 and prednisolone 25 mg/m2 d q 4 weeks)versus MCP plus rituximab (375 mg/m2 d -1) in advanced indolent lymphoma and mantle cell lymphoma. Here we present the results of the MCL subgroup (n=90) among the 358 randomised patients (FL, MCL, LPL). Study endpoints included overall and complete response rate (RR + CR), progression free survival (PFS), event free survival (EFS), overall survival (OS) and toxicities.

Results: with a median follow-up of 43 months for the MCL subgroup we can provide relatively mature data. Concerning toxicities there was no striking difference between the treatment groups. The treatment results for the MCL patients are as follows:

Conclusions: Concerning all end-points R-MCP is not superior to MCP chemotherapy alone in advanced mantle cell lymphoma. Immunochemotherapy is obviously not the solution for this poor prognosis lymphoma entity.

Results R-MCP n=44 MCP n=46 p-value Response Rate 70,5% 63% .5074 Complete Responses 31,8% 15,2% .0822 PFS median 20,5 months 19 months .2482 PFS 42 months 31% 14% EFS median 19 months 14 months .1369 EFS 42 months 27% 11,5% OS median 56 months 50 months .4862 OS 42 months 60% 61%
BiovaxID Vaccine Therapy of Follicular Lymphoma in First Remission: Phase III Blinded Safety Update.
Authors: Stergiou Angelos M. , Neelapu Sattva S. , Casciano Roman , Jaffee Margo A. , Kwak Larry W.
Published: October 2019

Background: A previous single-arm Phase 2 study (Nature Med5:1171–7,1999), evaluated the ability of tumor-specific idiotype (ID) conjugated to keyhole limpet hemocyanin (KLH) administered with granulocyte-monocyte colony-stimulating factor - GM-CSF (BiovaxID vaccine) to induce complete remission (CR) and molecular remission (MR) in follicular NHL patients in first CR after chemotherapy. We reported (ASH 2005, Abstr 2441) that at 9.2 years follow-up, disease free survival (DFS) and overall survival (OS) were 45% and 95%, respectively; median DFS was 8.0 years. To date, there have been no additional reported mortalities in this cohort. Although these data are promising, improvements in the treatments and increasing survival for patients with follicular lymphoma have necessitated additional rigor in the evaluation of overall safety and overall risk-benefit. The ongoing Phase 3 study that opened in 2000 by the NCI is designed to evaluate the impact of this vaccine on DFS in previously untreated subjects who have attained CR/Cru. As of August 2007, 231 subjects have been enrolled to receive chemotherapy and 176 have been randomized to receive vaccine (ID-KLH or KLH-KLH).

Methods: Since Phase 3 trial inception in 2000, SAEs have been reported through a pharmacoviligance group and identified according to time period on the study (i.e. during chemotherapy, during vaccine, after vaccine, or unknown), causality, and relatedness to study agent.

Results: As of August 2007, 32 SAEs have been reported in the BV301 trial, 7 of which occurred during the vaccination segment of the study. At a blinded interim analysis of the Phase 3 safety data, of the 7 SAEs reported during the vaccine segment of the study, 3 are considered unlikely to be related to the study agent (chest pain, compression fracture, herpes zoster), 2 possibly related (arrhythmia supraventricular, chest pain), 1 unrelated (vomiting), and 1 unknown (febrile neutropenia). The 1 SAE, cerebral ischaemia, that occurred post vaccine is considered unrelated to the study agent.

Conclusions: Additional follow-up on the Phase 2 patients is needed, as well as similar analyses in other cohorts. Blinded Phase 3 safety data shows a positive safety profile for BiovaxID. Unblinded safety data will be needed to determine the final efficacy and safety profile.
Circulating CD4+CD25+ Regulatory T Cell and Th3 Cell of the Patients with Myelodysplastic Syndromes.
Authors: Shao Zonghong , Wang Huaquan , Xing Limin , Fu Rong , Tu Fengmei
Published: October 2019

Objective To investigate the quantitive and qualitive changes of peripheral blood CD4+CD25+ regulatory T cells and Th3 cells of the patients with myelodysplastic syndromes.

Methods Whole peripheral blood samples of 40 MDS cases and 19 nromal control were assayed by flow cytometer for detecting the numbers of CD4+CD25+ regulatory T cell and CD3+CD4+TGFβ+ T cell(Th3). Expression of FOXP3 and TGF-β of peripheral blood mononuclear cell(PBMNC) were analysed by reverse transcriptase-polymerase chain reaction(RT-PCR). Commercial ELISA kits were used to measure plasma concentrations of TGF-β 1 according to the manufacturer’s instructions.

Results. The percentage and number of CD4+CD25+ regulatory T cells in the CD4+T cell population were significantly higher in the peripheral blood of MDS patients than healthy controls[(21.5±2.2% vs 8.3±1.9%; P<0.05) and (76.1±20.6)×106/L vs (54.6±11.4)×106/L; P<0.05)]. The percentage and number of Th3 cells in the mononuclear cell population were significantly higher in the peripheral blood of MDS patients than healthy controls[(7.2±1.3%vs2.6±0.7%; P<0.01 and (63.5±15.2)×106/L vs (42.4±10.6)×106/L;P<0.05)]. The rate of expression of FOXP3 isolated from PBMNC was significantly higher in MDS patients than that in healthy controls (91.3% vs 52.6%; P<0.05%). But the expression of TGF-β of MDS patients didn’t significantly differ from that of healthy controls. The plasma level of TGF-β1 of MDS patients was significantly lower than that of healthy controls[(12.9±3.1)ng/ml vs (23.2±3.9)ng/ml; P<0.05)].

Conclusion The CD4+CD25 + regulatory T cells, Th3 cells and their inhibitive function increased in MDS, which might contribute to MDS clone escaping from immunosurveillance and gainning immunol privilege.
DCC-2036: A Novel Switch Pocket Inhibitor of ABL Tyrosine Kinase with Therapeutic Efficacy Against BCR-ABL T315I In Vitro and in a CML Mouse Model.
Authors: Van Etten Richard A. , Chan Wayne W. , Zaleskas Virginia M. , Evangelista Peter , Lazarides Katherine , Peng Cong , Li Shaoguang , Wise Scott C. , Petillo Peter , Flynn Daniel L.
Published: October 2019

Therapy with ATP-competitive ABL kinase inhibitors, including imatinib (Gleevec), dasatinib (Sprycel), and nilotinib (Tasigna), has revolutionized the treatment of chronic myeloid leukemia (CML), but a substantial proportion of patients develop resistance to these agents. A major mechanism of acquired resistance is mutations in the ABL kinase domain that render BCR-ABL insensitive to the drug; in particular, mutation of the ABL kinase “gatekeeper” Thr315 residue to Ile (T315I) confers pan-resistance to all ATP-competitive ABL inhibitors. To address this need, we developed and characterized a novel chemical class of compounds that bind to five structural pockets involved in the endogenous “switch” mechanism used by the ABL kinase to conformationally control its activity state. Using a structure-based drug design approach, diversity in these “switch pockets” between kinases can be exploited to develop inhibitors with high potency and specificity. The resulting ABL inhibitors have several attractive features, as they:

act through a non-ATP-competitive mechanism; avoid steric clash with Ile315; inhibit purified ABL that is either unphosphorylated (switch-off) or phosphorylated (switch-on) at Tyr393 with IC50 of 0.8–4.0 nM; co-crystallize with phospho-Y393-ABL WT and T315I; and have very prolonged residency time on the kinase (off-rate ∼400 min vs. 3 min for imatinib).

A lead compound, DCC-2036, is highly selective for ABL, FLT3, and SRC family kinases, and has favorable pharmacokinetic and toxicity profiles in animals. In vitro, DCC-2036 inhibited proliferation and induced apoptosis of Ba/F3 cells expressing BCR-ABL WT or several common imatinib-resistant mutants (Y253F, T315I, M351T) with IC50 from 5 to 25 nM, without appreciable inhibition of parental Ba/F3 cells in IL-3 (IC50 >1000 nM). In Balb/c mice injected with Ba/F3-BCR-ABL T315I cells, pharmacodynamic studies indicated >8 hour inhibition of phospho-Stat5 and phospho-ABL in the leukemic cells following a single oral 100 mg/kg dose of DCC-2036, whereas daily dosing of DCC-2036 significantly prolonged survival. In the mouse retroviral bone marrow transduction/transplantation model of CML, DCC-2036 at 100 mg/kg/d also reduced peripheral blood leukocyte counts and significantly prolonged survival vs. vehicle-treated control mice. These results demonstrate that ABL switch pocket inhibitors are a promising new pharmacologic weapon for the treatment of de novo and drug-resistant CML, including BCR-ABL T315I.
Valproate, a Histone Deacetylase Inhibitor, Enhances Purine Nucleoside Analogues Induced Apoptosis of B-Chronic Lymphocytic Leukemia Cells.
Authors: Bouzar Amel B. , Boxus Mathieu , Defoiche Julien , Techn Hervé Balon , Berchem Guy , Macallan Derek , Pettengel Ruth , Willis Fenella , Burny Arsene , Lagneaux Laurence , Bron Dominique , Chatelain Bernard , Chatelain Christian , Willems Luc
Published: October 2019

Resistance to chemotherapy and drug toxicity are two major concerns of Chronic Lymphocytic Leukemia (B-CLL) treatment by Purine Nucleoside Analogs (PNA, i.e. fludarabine and cladribine). We hypothesized that targeting epigenetic changes might address these issues and evaluated the effect of the histone deacetylase (HDAC) inhibitor valproate (VPA) at a clinically relevant concentration. We show that VPA acts in a highly synergistic/additive manner with 9-ß-D-arabinosyl-2-fluoroadenine-5′-monophosphate (F-ara-AMP, fludarabine, 1μM) and 2-chloro-2′-deoxyadenosine (CdA, cladribine, 1μM) to induce apoptosis of CLL cells. VPA, but neither fludarabine nor cladribine, enhances the production of Reactive Oxygen Species (ROS) and inhibition of ROS with N-acetylcysteine decreases apoptosis of CLL cells. VPA-induced apoptosis is caspase-dependent and involves the extrinsic initiation pathway. VPA stimulates hyperphosphorylation of p42/p44 Erk as well as overexpression of Bax, cytochrome c and Fas. These observations support a potential use of VPA alone or in combination with nucleosidic analogs in CLL therapeutic protocols allowing a reduction of their effective doses or improving their efficacy in PNA-resistant patients.
Anti-Angiogenic Effects of Sorafenib in Relapsed Chronic Lymphocytic Leukemia: Correlative Studies of a Phase 2 Clinical Trial.
Authors: Reganti Sowjanya , Kabba Edith , Stock Wendy , Wright John J. , Godwin John E. , Kini Ameet R.
Published: October 2019

Chronic lymphocytic leukemia (CLL) is a malignant B-cell disorder that remains a largely incurable disease. We and others have previously shown that angiogenesis is important in the pathogenesis of CLL. Sorafenib is a small molecule signal transduction inhibitor that exhibits anti-tumor activity through a number of mechanisms including blocking of the Raf/MEK/ERK pathway and inhibition of receptor tyrosine kinases involved in angiogenic signaling such as the VEGF receptors. The data presented here are correlative studies performed on a Phase 2 clinical trial designed to measure the overall response rate to oral doses of sorafenib in CLL patients who have failed previous nucleoside analog therapy (alone or in combination with alkylating agents) or monoclonal antibody therapy. Each cycle consisted of 400 mg of sorafenib administered orally twice a day for 28 days. Treatment continued until any of the withdrawal criteria (unacceptable toxicity, disease progression, patient’s decision to withdraw) were reached. Peripheral blood samples for correlative studies were obtained before initiation of therapy, after 12 weeks (beginning of cycle 4) and after 24 weeks (beginning of cycle 7), or when the patient went off-study for any indication. In these studies, we specifically focused on examining VEGF and the VEGF receptors VEGFR-1 (flt-1), VEGFR-2 (KDR), VEGFR-3 (flt-4), neuropilin-1 and neuropilin-2. These molecules were analyzed at the transcript level by RT-PCR and at the protein level by western blotting or ELISA. In addition we examined plasma levels of IL-8 by ELISA, since IL-8 has been recently shown to be an important mediator of cell survival and angiogenesis in CLL. Specimens were analyzed from a total of five patients. Plasma IL-8 levels were markedly decreased in all three patients who underwent therapy beyond 12 weeks. The two patients went off study at or before 12 weeks did not show any decrease in IL-8 levels. No decrease was noted between the pre-treatment and post-treatment plasma concentrations of VEGF for any of the patients. No consistent change in cellular expression was observed between the pre-treatment and post-treatment samples for VEGF, VEGFR-1, VEGFR-2 or VEGFR-3. Neuropilin-1 expression was decreased or absent in the post therapy samples in patients who received therapy beyond 12 weeks, while no change in expression was seen in the two patients who went off study at or before 12 weeks. Neuropilin-2 expression was not detected in any of the samples. These results suggest that sorafenib suppresses angiogenic pathways by inhibiting secretion of IL-8 and expression of neuropilin-1. Additional studies and samples are needed to confirm these interesting findings, and to further delineate the anti-angiogenic effects of sorafenib in CLL.
What Is the Role for Donor NK Cells after Nonmyeloablative Conditioning?
Authors: Baron Frederic , Petersdorf Effie W. , Sandmaier Brenda M. , Gooley Ted , Malkki Mari , Maloney David G. , Storb Rainer F.
Published: October 2019

Background: The potential role of donor NK cells after nonmyeloablative conditioning for allogeneic hematopoietic cell transplantation (HCT) is not defined. We investigated the impact of the kinetics of donor NK cell engraftment as well as the impact of missing recipient KIR ligands and the number of donor inhibitory and activating KIR genes on HCT outcomes in 282 patients (153 with HLA-matched related donors and 129 with unrelated donors) conditioned with 2 Gy TBI +/− fludarabine. Postgrafting immunosuppression consisted of cyclosporine and mycophenolate mofetil. Diagnoses were hematological malignancies (n=274) or solid tumors (8).

Methods: NK cells were isolated from peripheral blood by flow cytometry on days 14, 28 and 42 after HCT. The proportions of cells of donor and host origin were assessed by FISH or by VNTR-PCR. High-resolution HLA-typing was performed using oligonucleotide probe and/or direct sequencing methods. Donor KIR genotyping was performed using a commercial PCR-SSP kit (Invitrogen) following manufacturers protocol.

Results: High numbers of T (P=0.01) and CD34+ (P=0.009) cells in the graft, as well as lower numbers of donor inhibitory KIR genes (P=0.01) were each associated with higher levels of donor NK cell chimerism. There was a suggestion of an association between lower numbers of activating KIR genes and higher CD56 chimerism, however this was not statistically significant. NK cell chimerism levels were comparable in patients who had all KIR ligands present vs. in those who were missing any ligand, and there was no association between the specific missing ligand and NK chimerism. A day-14 NK cell chimerism level of < 50% was associated with increased risks of graft rejection (P=.009). Modeling chimerism levels as a continuous linear variable, there was no association between NK cell chimerism levels on day 14 and occurrence of grade II-IV acute GVHD. In contrast, high levels of donor NK cell chimerism on days 14–42 were associated with a lower risk of relapse (P=0.006) and better progression-free survival (P=0.003) in time-dependent analyses. The qualitative associations between donor NK cell chimerism and graft rejection, GVHD, relapse or progression-free survival did not change after adjustment for the presence of recipient KIR ligands nor after adjustment for the number of donor inhibitory or activating KIR genes. Finally, the 3-year cumulative incidence of relapse was 42% in patients who have all ligand for donor NK cell KIR, versus 38% in patients who miss one or more ligand for donor NK cell KIR (adjusted hazard ratio = 1.05; 95% confidence interval 0.65–1.68; p=0.85).

Conclusions: Robust engraftment of donor NK cells correlated with low risk of graft rejection, low risk of relapse and high progression-free survival but not with acute GVHD. The clinical importance of donor KIR inhibitory and activating genes on post-transplant donor NK chimerism merits further study.
Phenotypic and Functional Effects of Perifosine on Dendritic Cells.
Authors: Song Weihua , Hideshima Teru , Tai Yu-Tzu , Anderson Kenneth C. , Munshi Nikhil C.
Published: October 2019

Perifosine is a synthetic novel alkylphospholipid, a new class of antitumor agent which targets cell membranes and inhibits Akt activation. Perifosine inhibits multiple myeloma (MM) cell growth in vitro and in vivo mouse model. Currently perifosine is under the evaluation of phase II clinical trail in MM. Although perifosine has shown significant direct antitumor effects, its effect on immune system has not yet been clarified. The objective of this study is to evaluate the effects of perifosine on the activity of antigen presenting cells (APCs). Monocyte-derived dendritic cells (DCs) from normal human donors were used as the APCs, and mature DCs were obtained by the treatment of TNF-α and IL-1β. Perifosine was used at the concentrations of 2.5 uM, 5 uM and 10 uM for the treatment with DCs. We first evaluated the effect of perifosine on the survival of DCs. We observed that the perifosine treatment up to 48 hours had no effect on viability (>90%) of DCs, assessed by annexin V and PI staining. Alteration of phenotype by perifosine on DCs was further examined by flow cytometry. Our results demonstrated that with dose-dependent manner, perifosine led to a significant down-regulation of surface antigens on immature DCs at 24 and 48 hours, which associated to costimulation (CD40, CD80 and CD86), antigen presentation (HLA-ABC, HLA-DPQR) and maturation (CD83). However, we did not observed significant effect of perifosine on above surface markers on mature DCs. Since DCs play a crucial role on the regulation of Th1/Th2 immune responses by the production of IL-12, we next evaluated IL-12 secretion by DCs with and without perifosine treatment. Importantly, treatment with perifosine significantly decreased LPS-induced-IL-12 production, compared to untreated DCs (untrt vs. trt = 192.29 vs. 166.23 pg/ml (2.5uM), 111.19 pg/ml (5uM) and 44.886 pg/ml (10uM)) at 24 hours. To assess the effect of perifosine on DCs function on the regulation of T cell responses, we stimulated allogenic T cells with mature DCs with or without the pre-treatment of perifosine. The proliferation assay by 3H-TdR incorporation and IFN-γ production by ELISA indicated perifosine-treated DCs had no significant effect on the regulation of T cells function. Taken together, these results showed that DCs function are influenced by the treatment of perifosine. Our pre-clinical data therefore indicates the need to monitor immune functions in patients under the Akt inhibitor treatment.
Tetra-Arsenic Tetra-Sulfide Containing Triple-Agent Regimen as the First Line Therapy for Acute Promyelocytic Leukemia: Expeditiously Consecutive Complete Remission and Improved Disease-Free Survival.
Authors: Wu Tong , Zhao Jie , Jiang Bin , Wu Shu-Lan , Yang Ping-Di , Shan Fu-Xiang , Tong Chun-Rong , Lu Dao-Pei
Published: October 2019

Our previous study has demonstrated that tetra-arsenic tetra-sulfide (As4S4) treatment alone is highly effective and safe in both induction and maintenance therapy in acute promyelocytic leukemia (APL) patients (Lu DP et al, Blood2002; 99: 3136). In current clinical study, we explored whether As4S4 combined sequentially or simultaneously with all-trans retinoid acid (ATRA) and cytotoxic agents according to specific clinical condition as the first line therapy could expedite complete remission (CR) but in a safer way and further improve disease-free survival (DFS). The treatment-related toxicities were also studied. From April 2001 to May 2007, 114 patients with APL in a median age of 32.5 years (10–73 years) were enrolled including consecutive 68 newly diagnosed cases and 46 patients in hematological remission. For induction therapy, both As4S4 (60 mg/kg) and, if leukocytosis or retinoid acid syndrome was not a risk, ATRA (25mg/m2) were taken orally per day till CR. Cytotoxic agents such as mitoxantrone and/or hydroxyurea were added when WBC counts were more than 4 × 109/L. After hematological remission, 4 to 6 cycles of combined chemotherapy were given, and followed by alternatively oral use of As4S4 (3 cycles) and ATRA (1 cycle) in outpatient base for maintenance therapy until 4 years after diagnosis. PML/RARa transcript was monitored quantitatively by real-time PCR. Arsenic levels in blood and urine were also monitored with an atomic absorption spectrophotometer. Consecutive 68 newly diagnosed patients all achieved complete hematological remission in a median time of 29.5 days (14 – 62 days) without early death. The median time to achieve molecular remission was 61.5 days (34 – 120 days). Four out of 114 patients relapsed. One of them had obtained CR again. One patient died from leukemia relapse. Treatment with As4S4containing regimen was well tolerated. Mild gastrointestinal discomfort, transient elevation in liver enzyme, rash, and edema were noted in a few cases. Estimated DFS rate for 4 years was 94%, with a median follow-up time of 36.5 months (3–76 months). Our encouraging results show that faster consecutive CR, higher DFS rate and better quality of life have been achieved with the above regimen as the first line therapy for APL patients.
Phase II Multicenter Trial To Evaluate the Safety and Efficacy of Low-Toxicity Treosulfan/Fludarabine Conditioning Prior to Allogeneic Haematopoietic Stem Cell Transplantation in Patients with Acute Myeloid Leukemia.
Authors: Holowiecki Jerzy , Giebel S. , Beelen D. , Trenschel R. , Wandt H. , Schaefer-Eckart K. , Ruutu T. , Volin L. , Einsele H. , Stuhler G. , Uharek L. , Blau I. , Bornhaeuser M. , Zander A. , Kroeger N. , Aschan J. , Wojnar J. , Markiewicz M. , Freund M. , Casper J.
Published: October 2019

BACKGROUND: The toxicity of commonly used myeloablative regimens is the main factor limiting applicability and restricting upper age limit for allogeneic hematopoietic stem cell transplantation (alloHSCT). However, reduced intensity conditioning (RIC) protocols are associated with high risk of relapse. Previous dose-escalation trials revealed high-dose treosulfan-based conditioning an alternative treatment with myeloablative as well as antileukemic potential, accompanied by low non-hematological toxicity. The goal of this multicenter phase II trial was to evaluate safety and efficacy of treosulfan/fludarabine conditioning in AML patients.

PATIENTS AND METHODS: Seventy-five patients (median age 45 years, range 19–59) with AML in 1st (80%), 2nd (17%), or 3rd (3%) complete remission were treated with alloHSCT from either matched related (MRD, 40%) or unrelated donors (MUD, 60%). Preparative regimen consisted of treosulfan 14 g/m2/day on days −6 to −4, fludarabine 30 mg/m2/day on days −6 to −2, and in case of MUD-HSCT additionally ATG (Fresenius) 10 mg/kg/day on days −4 to −2. Graft-vs.-host disease (GvHD) prophylaxis consisted of cyclosporine and short-course methotrexate. 25% of the patients had an increased risk of toxicity for standard myeloablative conditioning regimens because of higher age or co-morbidities.

RESULTS: All patients engrafted after a period of absolute neutropenia with the median time to ANC >0.5 G/L and PLT >50 G/L of 21 (13–39) days and 19 (11–48) days, respectively. The cumulative incidence of complete donor chimerism was 72% on day +28 and 92% on day +100. Most frequent CTC grade 3 or 4 complications until day 28 after transplantation were febrile neutropenia (28%), infection in neutropenia (16%), and mucositis (5%). None of the patients experienced severe hepatic toxicity, including VOD. After a median follow up of 457 days (98–988), the probabilities of overall (OS) and disease-free survival (DFS) at 2 years equaled 67% resp. 58% (MRD-HSCT) and 58% resp. 45% (MUD-HSCT, p=NS). The 2-year cumulative incidence of relapse was 34% for MRD-HSCT and 37% for MUD-HSCT, whereas non-relapse mortality was 8% and 17%, respectively. Outcome was not significantly influenced by disease status (CR1 vs. ≥CR2) or age.

CONCLUSIONS: Conditioning therapy based on treosulfan at a dose of 3x14 g/m2 in combination with fludarabine is very well tolerated, resulting in a low NRM. The regimen demonstrated reliable efficacy in AML patients with confirmed first or subsequent remission. Further randomized studies are warranted to verify advantageous survival results.