bluuebunny/crossref_metadata_2025 huggingface dataset:
Myeloma Treatment Plan: Up-Front Dose-Dense Chemotherapy, Two Semi-High-Dose Autologous Transplants and a Non-Myeloablative Reduced Intensity Allogeneic Transplant.
Authors: Van Hoef Marlies E.H.M.
Published: October 2019

Introduction: Our non-myeloablative transplants for early stage disease program started in 2000 for breast cancer and recommends treatment by combined modalities consisting of intensive or dose dense induction, an autologous transplant strategy and a non-myeloablative reduced intensity conditioning transplant (Blood2001; 98 (suppl 1): 5241, 5242). In myeloma aggressive treatment from diagnosis has rarely been practised. Steroids with other low-dosed agents have been the standard for years and resulted in substantial mortality. The reason has been that the majority of patients are over 60 years at diagnosis of the disease (80% of women, 75% of males); myeloma start to reveal themselves around 40 years.

Methods: Several databases were searched to provide insight in the matter.

Results: A medline search on dose dense and myeloma showed no articles. Standard treatment in myeloma (melphalan and prednison or VAD) induce 6-10% (complete) responses; the newer agents thalidomide or bortezomib in combination with other agents induce 12 to 16% (complete) response, but the agents are quiet toxic.

Standard CHOP in myeloma has been reported to induce 60 to 65% partial response with very low toxicity (Nippon Ronen Igakkai Zasshi1991;28(4):504–508). CHOP-14 is well tolerated in older lymphoma patients and should be evaluated in myeloma. Two intermediate high-dose autologous transplants without prior therapy induced 25% near complete response (Blood2004;104:3052–7). No treatment related mortality was reported.

Non-myeloablative reduced intensity conditioning transplants after high-dose autologous transplants converted 18% complete remission to 70% complete remission and induced long-term disease free survival (Blood2002;100:755); this therapy was though associated with 11% risk on treatment related mortality, mainly attributable to graft versus host diseases. Disease free survival was high at give follow-up. GVHD is well manageable with available agents.

In over 75% the disease was lethal prior to the non-myeloablative transplant era.

Conclusion: Conventional therapy in myeloma has been characterized by few low-dose regimens; myeloma have for years been undertreated. To improve disease outcome a systematic treatment approach from initial diagnosis for any of the stages I to III disease is required and herein discussed.
Reduced Intensity Stem Cell Transplant (RIST) as Salvage Treatment for Relapse Following Myeloablative Allogeneic Transplantation in Adult Acute Myeloid Leukemia.
Authors: Sica R. Alejandro , Tuscano Joseph M. , Law Lisa Y. , Richman Carol M.
Published: October 2019

Relapse of acute myeloid leukemia (AML) following myeloablative allogeneic stem cell transplantation portends a dismal prognosis. Therapy aimed at enhancing graft-versus-leukemia (GVL) effect, e.g., by donor leukocyte infusion, has limited success in AML, and a second ablative transplant is associated with prohibitive mortality in adults. From April 2001 to March 2006, nine patients, ranging in age from 21 to 57, with high risk myeloid malignancy (7 AML and 2 advanced myelodysplasia) and overt bone marrow relapse less than one year after ablative busulfan/cyclophosphamide conditioning have been treated with a cytoreductive regimen of fludarabine (30 mg/m2/day) and cytarabine (2g/m2/day) for 5 days (-7 through -3) and G-CSF administration (5 ug/kg daily starting day -8) with or without idarubicin (8 mg/m2 days -7, -5, and -3) (8 cases) or fludarabine 30 mg/m2 for 3 days and 200 cGy total body irradiation (1 case). G-CSF mobilized peripheral blood stem cells from their original HLA-matched donor (8 siblings, 1 unrelated) were infused. Graft versus host disease (GVHD) prophylaxis was mycophenolate mofetil for 30 days and cyclosporine with a rapid taper. The mean onset of relapse after the initial ablative transplant was day 172 (range 106–271). Fludarabine-based therapy was well tolerated with no treatment related mortality. Full donor chimerism was established by day 72 (range 26–113) in 6 patients (67%). Five patients died: 2 from relapse without GVHD at day +30 and +301 after RIST (one patient with complex and one with Ph+ cytogenetic abnormalities), 3 from relapse with evidence of GVHD. Four patients survive: one has relapsed at 91 days after RIST and is receiving alternate therapy, while three patients (30%) survive in complete remission at 100+, 635+ and 1795+ days after salvage RIST. In 5 cases (56%), the duration of complete remission after RIST was longer than after the initial ablative transplant. We conclude that fludarabine-based RIST is a safe and effective salvage therapy offering a chance for increased survival with low morbidity in patients relapsing after ablative transplant. In addition, RIST therapy has resulted in long-term disease free survival in over 20% of cases in this study.
The Relationship between CRP, Citrulline and Bacteremia among Patients Undergoing Autologous Peripheral Blood Stem Cell Transplantation for Multiple Myeloma.
Authors: Herbers Alexandra H.E. , Donnelly J. Peter , de Pauw Ben E. , Blijlevens Nicole N.A.
Published: October 2019

Background:

Infection remains a leading cause of morbidity and mortality in hematopoetic stem cell transplant (HSCT) recipients. The objective of the present study was to identify the relationship between C-reactive protein (CRP), serum citrulline as an indicator of gut mucosal barrier injury, and bacteremia in patients undergoing autologous peripheral blood (PB)HSCT for multiple myeloma.

Methods:

29 consecutive patients given high dose melphalan as conditioning therapy for a PBHSCT according to a national protocol from May 2004 to May 2005 were included in the analysis. Blood cultures were obtained at least twice weekly during three weeks of hospital admission; bacteremia was defined by any positive blood culture. SPSS for Windows version 12 was used to perform statistical calculations. CRP values were transformed to log10 whereas the actual citrulline values were used.

Results:

The mean age of the patients was 55 (range 33–65) years and the mean length of hospital stay was 23 (17–34) days. Blood cultures of 19 patients yielded bacterial growth. For the overall study population a specific pattern of citrulline levels that was not correlated with the occurrence of bacteremia was observed. At baseline the levels (mean ± SD) amounted to 25.4 ± 6.2 micromol/L, after 1 week 14.1 ± 4.7 micromol/L, after 2 weeks 7.1 ± 3.5 micromol/L and after 3 weeks 9.3 ± 5.2 micromol/L. The corresponding CRP levels showed a high SD pointing at a patient-specific level. The respective values (mean ± SD) were: 5 ± 3 mg/L at baseline, 8 ± 10 mg/L lst week, 95 ± 86 mg/L 2nd week and 47 ± 83 mg/L 3rd week. A strong correlation (p<0.01) was found between positive blood cultures and low citrulline (7.6 ± 3.8 micromol/L); for CRP such a relation was not established.

Conclusion: The occurrence of bacteremia coincides with low serum citrulline levels, but not with CRP levels. Extending our observation might be of help to establish a lower limit of citrulline levels that is indicative of an imminent bacteremia.
Endogenous Arteriolar Fibrinolysis Requires Urokinase Plasminogen Activator and Leukocyte Urokinase Plasminogen Activator Receptor-1.
Authors: Bai Xufang , Gross Peter L.
Published: October 2019

Background: In venous thrombi, urokinase associated with leukocytes activates plasminogen to initiate endogenous fibrinolysis. Leukocytes also interact with arteriolar thrombi.

Objectives: We hypothesized that urokinase plasminogen activator (uPA) activity would also be delivered to arteriolar thrombi by leukocytes.

Methods: Using established techniques of high-speed intravital fluorescence microscopy to observe the cremaster muscle of mice, we measured platelet and fibrin accumulation in thrombi generated by a laser injury to arterioles. The accumulation of platelets in a thrombus is quantitated by measuring the fluorescence from anti-CD41 antibody tagged with a fluorescent secondary antibody and the presence of fibrin is detected using a fluorescence-tagged anti-fibrin antibody (T2G1).

Results: In C57BL/6 wild type mice, fibrin increased to a maximum at about three minutes after thrombus formation and then decreased, such that by eight minutes after thrombus formation fibrin is present at 34% of its maximal value. When wild type mice are pretreated with epsilon-aminocaproic acid (16 mg/kg IV), the decrease in thrombus fibrin content was less (to 54% of maximal 8 minutes after thrombus formation), implying that the fibrin loss is at least partially the result of plasmin activity. Thrombus fibrin accumulation in tissue-plasminogen activator deficient mice (Plat) was greater, peaking at 229% the amount found in wild type mice, but the amount of fibrin present at eight minutes after thrombus formation was not dissimilar from wild type mice (45% ± 5% v. 34% ± 3%, P=0.09). In uPA-deficient mice (Plau) and uPA-receptor-1-deficient mice (Plaur), the amount of maximal thrombus fibrin was 189% and 273% that of fibrin content in wild type mice. Also, although thrombus fibrin did decrease over time in Plau and Plaur mice, it decreased to only 51% and 48% of maximal (each were P < 0.005 v. wild type) at eight minutes after thrombus formation. When wild type leukocytes, isolated from blood by differential centrifugation and sucrose density gradient, were transfused into Plaur mice, thrombus fibrin loss was restored to wild type levels (38% of maximal at 8 minutes after thrombus formation, P=0.36 vs. wild type, P=0.016 vs. Plaur), implying that transfused wild type leukocytes, which express uPA-receptor-1, can rescue fibrinolysis in uPA-receptor-1-deficient mice.

Conclusions: These results confirm that endogenous fibrinolytic activity in vivo occurs soon after arteriolar thrombus formation. The plasminogen activators that are required for this fibrinolysis to occur are uPA, and the uPA-receptor-1 on leukocytes. These findings are compatible with the hypothesis that leukocytes can deliver plasminogen activator activity to arteriolar thrombi to initiate endogenous fibrinolysis.
Impact of Regulatory T Cells on Antigen Specific T Cell Response Using Recombinant Chimeric T Cell Receptors In Vivo.
Authors: Kofler David M. , Chmielewski Markus , Koehler Heike , Riet Tobias , Schmidt Patrick , Rappl Gunter , Hombach Andreas , Hallek Michael , Wendtner Clemens M. , Abken Hinrich
Published: October 2019

Recombinant T cell receptors with defined specificity against tumor cells are a promising experimental approach in the elimination of residual leukemia and lymphoma cells. It is so far unresolved whether regulatory T cells with suppressor activities impair the efficiency of cytolytic T cells grafted with a recombinant immunoreceptor. The frequency of regulatory T cells is highly increased in tumor patients and their suppressive function seems to play a role in the fail of an autologous T cell response against the malignant cells.

In this study we analyzed the antigen-triggered, specific activation of receptor grafted T cells in the presence or absence of regulatory CD4+CD25high T cells. CD3+ T cells were grafted with CEA-specific immunoreceptors containing the CD3-zeta signaling domain for T cell activation. Co-cultivation of receptor grafted effector T cells together with regulatory T cells repressed proliferation of the effector cells and decreased IL-2 secretion. Secretion of IFN-gamma and IL-10 was not impaired. Interestingly, the cytotoxicity of grafted effector T cells towards CEA-expressing tumor cells was not impaired by regulatory T cells in vitro.

To evaluate the relevance in vivo, we used a Crl:CD1 Nu/Nu mouse model to assess growth of CEA+ tumor cells in the presence of receptor grafted effector T cells and of regulatory T cells. Mice inoculated with tumor cells together with CD3+ effector T cells without immunoreceptor and regulatory T cells developed earlier tumors with faster growth kinetics compared to mice that were inoculated with tumor cells, CD3+ T cells and CD4+CD25- control T cells. Using effector T cells that were equipped with a recombinant CEA-specific CD3-zeta immunoreceptor, 2 of 5 mice developed a tumor in the presence of regulatory T cells while none of the mice developed a tumor in the absence of regulatory T cells.

Taken together, regulatory T cells obviously impair an antigen-specific, anti-tumor T cell attack in vivo. This seems to be due to repression of proliferation of the effector T cells and not to diminished cytotoxicity. These findings have major impact on the design of clinical studies involving adoptively transferred effector T cells.
A Review of the Economic Burden of Pleural and Pericardial Effusions in Cancer Patients.
Authors: Liou Sarah Y. , Stephens Jennifer M. , Tran Kimbach T. , Botteman Marc F.
Published: October 2019

OBJECTIVES: Pleural and pericardial effusions can lead to severe outcomes. Cancer accounts for an estimated 40% of all pleural effusions. About half of the effusions diagnosed in cancer patients are malignant, while the rest are nonmalignant and may occur as complications of the cancer treatments themselves. Pleural and pericardial effusions are associated with increased morbidity and mortality as well as high healthcare costs. The objective of this study was to review the economic burden of pleural and pericardial effusions in cancer patients.

METHODS: A systematic search of the English-language medical literature published between 1990 and 2006 was conducted. Additional publications and conference proceedings were retrieved from the article bibliographies and included in the review. Articles selected include prospective or retrospective studies specifically designed to examine burden of illness, direct medical costs, indirect costs, or cost drivers associated with pleural or pericardial effusions in cancer patients. All original costs were reported, with adjusted figures (to 2006 US dollars) presented in parentheses using the medical care component of the consumer price index from the US Bureau of Labor Statistics.

RESULTS: Of 15 studies identified, 11 met selection criteria and were reviewed in detail. Seven references reported data on costs associated with pleural or pericardial effusions in cancer patients. The cost per episode of pleural effusion ranged from $3,391 (2006 US $4,387) for outpatient treatment with pleural catheter to $20,996 ($37,341) for talc pleurodesis. The most common treatment for malignant pleural effusion is chest tube insertion and drainage with instillation of a sclerosing agent. Key cost drivers for significant pleural effusions included operating room costs, surgeon fees, and drugs such as sclerosing agents. Resources used for management of low grade pleural effusions include chest x-rays, physician outpatient visits, diuretics, and corticosteroids. For the treatment of pericardial effusion, the costs of performing pericardiocentesis and a pericardial window procedure were estimated to be $4,446 and $14,641 (2006 US$), respectively. Cost components for pericardial effusions, depending on treatment modality selected, included echocardiogram (3–10%), intensive care unit (17–56%), sclerosant (1–4%), surgeon fees (28–29%), anesthesia fees (20%), and operating room costs (31%).

CONCLUSIONS: Pleural and pericardial effusions lead to significant direct medical costs, contributing to the total cost of care among patients treated for cancer. These costs should be included in the economic evaluation of therapies that increase the risk of pleural and pericardial effusions. Given the scarcity of published analyses in this area, additional research is warranted to better understand the burden of pleural and pericardial effusions.
Blood Management Strategies Effect on Hemoglobin Levels in Hospitalized Patients.
Authors: Ford Patricia A. , Keck Gina R. , Ricci Angela F.
Published: October 2019

Phlebotomy for diagnostic testing is a notable source of blood loss, causing hospitalized patients to lose significant volumes of blood in a short period of time. Previous studies have documented correlations in phlebotomy blood loss, decline in hemoglobin and possible contributions to transfusion volume. One study reported average blood drawn for phlebotomy to be 12.4 mL/day for patients in medical wards, 33.5 mL/day in ICU patients, and 73.9 mL/day in patients with an arterial line (Smoller et al. NEJM.1986; 314: 1233–1235). Another study concluded that an average hemoglobin decrease of .79 g/dL was due to a similar volume of phlebotomy blood loss (Thavendiranthan et al. J Gen Intern Med2005; 20:520–524). Patients treated at the Center for Bloodless Medicine and Surgery (CBMS) at Pennsylvania Hospital decline blood products. Thus, to minimize iatrogenic blood loss, strategies were implemented including the use of pediatric tubes, reduced phlebotomy and the avoidance of central line wastage. In this study we attempt to analyze the adherence to and effectiveness of these strategies in preventing hemoglobin decline. We conducted a retrospective chart review of 46 out of 341 hospitalizations in the CBMS between October 2005 and July 2006, excluding ICU patients and those with blood loss such as surgical patients, patients with gastrointestinal bleeding, and dialysis patients. Erthropoietin (EPO) therapy was not given before or during the days evaluated. The analyzed population had a mean age of 58.7, an initial hemoglobin of 12.3 g/dL and a hospitalization period of 5.1 days. The absolute change between initial and final hemoglobin was on average .71 g/dL. Taking into account fluctuations during their stay, the average change from highest documented hemoglobin to lowest was 1.2 g/dL. In 30.4 percent of this population EPO therapy was subsequently initiated during their hospitalization. Therapy began at an average hemoglobin of 10.8 g/dL and was presumably the result of phlebotomy induced anemia. An average of 2.8 laboratory tests were ordered per day; 57 percent were chemistry panels or complete blood counts. These two tests were ordered almost daily. Thus, even at a CBMS where pediatric tubes are utilized and strict staff protocols are prescribed, the average iatrogenic blood loss is not appreciably different from a non-bloodless center without these regulations. Pediatric tubes can be costly and labor intensive and may not be the most important determinant of iatrogenic blood loss. Therefore, hospitals should work more diligently to eliminate daily and unnecessary phlebotomies by retraining house staff to keep patient blood loss at a minimum.
Evaluation of Once Weekly Subcutaneous Idraparinux Versus Standard Therapy with Heparin and Vitamin K Antagonists in the Treatment of Deep-Vein Thrombosis or Pulmonary Embolism - The Van Gogh Investigators.
Authors: Buller Harry R. ,   on Behalf of the Van Gogh Investigators
Published: October 2019

Background

Venous thromboembolism is treated with heparins, followed by a vitamin K antagonist. It would be desirable to replace this complex approach with one simple anticoagulant regimen.

Methods

We conducted two randomized, open-label trials involving 2904 patients with deep-vein thrombosis and 2215 patients with pulmonary embolism to compare efficacy and safety of idraparinux alone versus standard therapy, and document non-inferiority for efficacy. Patients received either idraparinux (2.5 mg once-weekly, subcutaneously), or low-molecular-weight heparin/unfractionated heparin with adjusted-dose vitamin K antagonists, for three or six months. The primary efficacy outcome was the three-month incidence of symptomatic recurrent venous thromboembolism (nonfatal or fatal).

Results

In the deep-vein thrombosis study the incidence of recurrence at day 92 was 2.9 percent with idraparinux and 3.0 percent in controls (odds ratio 0.98, 95 percent confidence interval 0.63 to 1.50), satisfying the pre-specified non-inferiority requirement. The six-month hazard ratio was 1.01. The incidence of clinically relevant bleeding at day 92 was 4.5 percent and 7.0 percent , respectively (P<0.01). By six months bleeding rates were similar. In the pulmonary embolism study the incidence of recurrence at day 92 was 3.4 percent with idraparinux and 1.6 percent in controls (odds ratio 2.14, 95 percent confidence interval 1.21 to 3.78), excluding non-inferiority.

Conclusion

In patients with deep-vein thrombosis, once-weekly subcutaneous idraparinux for three or six months had similar efficacy and safety as heparins and vitamin K antagonists, while, in pulmonary embolism, this regimen was less efficacious than standard anticoagulant therapy which had an unexpectedly low recurrence rate.
Anti-Leukemic Potency of Stapled BH3 Helices Correlates with Their Capacity for Bifunctional Activation of Apoptotic Pathways.
Authors: Walensky Loren D. , Pitter Kenneth , Morash Joel G. , Verdine Gregory L. , Korsmeyer Stanley J.
Published: October 2019

Selective targeting of apoptosis in vivo is a promising pharmacologic strategy for subverting cancer. BCL-2 family protein interactions constitute a critical control point for the regulation of apoptosis. Whereas multidomain anti-apoptotic proteins such as BCL-2 guard against cell death, multidomain pro-apoptotic proteins such as BAX constitute a gateway to cell death through mitochondrial damage. The BH3-only proteins function as death sentinels situated throughout the cell, poised to transmit signals of cell injury to multidomain members. BH3-only proteins deliver death signals via their alpha-helical BH3 domains, which are either neutralized by anti-apoptotic proteins or delivered, directly or indirectly, to the mitochondrial executioners BAX and BAK. By inserting hydrocarbon “staples” into native BH3 peptide sequences, we have generated a chemical toolbox of stabilized alpha-helices of BCL-2 domains (SAHBs) to dissect apoptotic pathways and develop prototype therapeutics. We previously demonstrated that a stapled peptide corresponding to the BID BH3 domain is a helical, protease-resistant, and cell-permeable compound that binds to anti-apoptotic targets with high affinity and exhibits anti-leukemic activity in vitro and in vivo. Using our expanded panel of compounds, we find that a stapled BAD BH3 likewise displays high affinity binding to select anti-apoptotic targets; however, BID SAHB is uniformly more potent than BAD SAHB in inducing apoptosis of a panel of leukemia cell lines. To explore the molecular mechanism underlying the differential potencies of BID and BAD SAHBs, we evaluated a model in which select BH3-only proteins directly engage pro-apoptotic multidomain proteins to trigger mitochondrial apoptosis. We detect and measure, for the first time, direct binding between select SAHBs, such as BID, and BAX. The observed interaction between BID SAHB and BAX triggered functional activation of BAX in vitro, resulting in mitochondrial cytochrome c release and FITC-dextran release from liposomes. The specificity of the BID SAHB-BAX interaction and its biochemical consequences is highlighted by abrogation of BID SAHB activity by point mutation and by competitive binding to anti-apoptotic BCL-XL. We confirmed the physiologic relevance of our observed in vitro interaction between BID SAHB and BAX by their co-immunoprecipitation from BID SAHB-treated leukemia cells. In contrast, BAD SAHB does not bind or activate multidomain pro-apoptotic BAX. These data provide an initial mechanistic explanation for the relative potency of BID SAHB in activating leukemia cell apoptosis. Thus, bifunctional SAHBs that directly engage both pro- and anti-apoptotic multidomain proteins may be more robust pro-apoptotic therapeutics, compared to compounds that selectively target anti-apoptotic proteins.
Evaluating the Performance of a Previously Reported Risk Score To Predict Venous Thromboembolism: A VERITY Registry Study.
Authors: O’Shaughnessy Denise , Rose Peter , Pressley Fran , Scriven Nicholas , Farren Tim , Nokes Tim , Arya Roopen
Published: October 2019

Accurate prediction of venous thromboembolic (VTE) risk remains a clinical challenge, and no prospectively validated risk scoring systems have been described. Recently, a computer-based alert to highlight hospitalised patients at risk of VTE that employed a simple risk score based on eight principal VTE risk factors was shown to improve patient outcome (N Engl J Med2005;352:969–977). The objective of this study was to assess the performance of this risk score in identifying those at risk of VTE.

The study cohort consisted of all patients who were enrolled in ‘VERITY’ up to 30th November, 2004. VERITY is a UK, prospective VTE treatment registry that enrolls patients presenting to hospital with suspected VTE (www.verityonline.co.uk). The vast majority of patients enrolled have no history of hospitalization and therefore patients will not have been risk assessed or have been given thromboprophylaxis, so essentially reflecting an untreated population.

The risk score was applied to the patient database and a score calculated for each patient for whom all 8 risk factors were known. Risk was scored as previously described: score=3 for cancer, prior VTE, hypercoagulability; score=2 for major surgery; score=1 for age>70 years, BMI>29, bed rest not related to surgery, use of hormone-replacement therapy or oral contraceptives. An increased risk was defined as a cumulative risk score of at least 4.

Of 27,179 patients presenting with suspected VTE, 6,124 had a positive diagnosis of deep vein thrombosis, pulmonary embolism, or both; in 15,980 cases the diagnosis was not confirmed. All 8 risk factors were known for 5,692 cases (1880 with confirmed VTE [31%] and 3812 VTE-negative cases [24%]). An increasing risk score was associated with an approximately linear rise in the proportion of patients with confirmed VTE; more than 50% of patients with a risk score ≥4 were diagnosed with VTE, rising to more than 71% in those with a score >7 (see figure).

Although this patient cohort is not identical to the population the risk score was developed for, these data suggest the value of this weighted risk score in identifying those at risk of VTE. Further refinement of this score, perhaps using more sophisticated modeling to increase the accuracy of the score to predict thromboembolic events, would be highly valuable for routine integration of VTE assessment into clinical practice.

Final diagnosis of VTE and weighted risk score (n=5,692) Final diagnosis of VTE and weighted risk score (n=5,692)
Osteoclasts but Not Macrophages or Dendritic Cells Support Long-Term Plasma Cell Survival.
Authors: Geffroy-Luseau Alexandrine , Jego Gaetan , Bataille Regis , Pellat-Deceunynck Catherine
Published: October 2019

Long-lived plasma cells (PCs) located within the bone marrow are the source of protective antibody for extended periods of time. Recent studies pointed out that PC survival depends on the ability of PCs to find a suitable niche. Bone marrow environment of PCs is constituted by cells of either mesenchymal (stromal cells and osteoblasts) or hematopoietic origin (macrophages, dendritic cells, osteoclasts (OCs)). In this work, we evaluated the ability of these five types of cells to support the long-term survival of human PCs in vitro.

Stromal cells were derived from normal bone marrow mononuclear cells and osteoblasts were SAOS2 cells (malignant osteoblastic cells). Macrophages, dendritic cells and OCs were derived from adherent circulating mononuclear cells with M-CSF, IL4 and GM-CSF or RANKL and M-CSF, respectively. Morphology, phenotype and functional features were used to characterize each cell type. On the other hand, plasmablasts (PBs) were generated from tonsil CD27+ B cells activated by CD40L expressing fibroblasts and then induced to differentiate by removal of fibroblasts in the continuous presence of IL2 and IL10. PBs were purified (by CD20 depletion) and cocultured with the five types of cells previously described.

To assess PC differentiation and survival in cocultures, we monitored the frequency of PCs by flow cytometry using an anti-CD38 and anti-CD138 double staining, along with Ig accumulation. Among cells of either mesenchymal (stromal cells and osteoblasts) or hematopoietic origin (macrophages, dendritic cells, OCs), only OCs allowed PBs to survive and differentiate into PCs that remained alive more than 14 days. Indeed, after a 5-day coculture with OCs, 100 000 PCs were alive (400 000 PBs were seeded at day 0) as compared to less than 10 000 PCs in the other culture conditions. Moreover, the total frequency of PCs after 14 days in culture with OCs was approximately 5% of the original input at day 0. PCs fully maintained their antibody secretory capacity since the level of secreted antibodies increased. Cellular interactions were required since PBs cultured with either OC conditioned medium or with OCs in transwell did not survive nor differentiate. Identification of membrane and soluble molecules supporting PC survival is now under investigation.

Our data indicate that the interaction with OCs is not limited to malignant PCs i.e., myeloma cells, and pointed out that OC is an essential partner of normal PC.
Methylseleninic Acid (MSA) Decreases DNA Synthesis, Induces Cell Death, and Sensitizes Both Rituximab Sensitive and Rituximab-Resistant Non-Hodgkin’s Lymphoma Cell Lines to the Anti-Tumor Activity of Cisplatin and Gemcitabine.
Authors: Hernandez-Ilizaliturri Francisco J. , Ali Minhas , Knight Joy , Rustum Youcef M. , Czuczman Myron S.
Published: October 2019

Selenium (Se) is an essential trace element with antioxidant properties. Examination of the dietary intake of Se in more than 25 countries found an inverse correlation with total age-adjusted cancer mortality. In addition, other investigator had shown that low serum levels of Se correlate with an adverse outcome in patients with diffuse large B-cell lymphoma (DLBCL) undergoing anthracycline-base chemotherapy. Various Se compounds had been developed and are undergoing preclinical testing such as MSA or Sodium selenite (SS). SS has been shown to decrease PKCg activity and induce apoptosis in murine lymphomas. In an attempt to further evaluate the role of Se in human lymphomas we studied the effects of MSA in a panel of rituximab-sensitive (RSCL) and rituximab-resistant cell lines (RRCL). Resistant clones were generated by chronic exposure of Raji, RL or DHL-4 cells to escalating doses of rituximab with (4RH) or without (2R) human complement. Functional assays were performed to demonstrate decrease in rituximab sensitivity on RRCL. In addition, resistance to chemotherapy (CDDP, doxorubicin, vincristine, etc.) agents was demonstrated in RRCL. Lymphoma cells were exposed in vitro to MSA (0 to 5mM) with or without CDDP (0 to 10mM) or gemcitabine (0 to 10mM). Following a 72 hour-period of MSA-drug exposure, changes in mitochondrial potential were determined by alamar blue reduction measured at 30 minutes intervals over a 48 hr period. In addition, changes in DNA synthesis and cell proliferation following drug exposure were performed using standard [3H]-thymidine incorporation assays. MSA induced dose-dependent cell death and a decrease in DNA synthesis in all the cell lines tested (both RSCL and RRCL). Up to a 50% reduction in mitochondrial potential and cell viability was observed in RRCL or RSCL exposed to 5mM of MSA. Incubation of RSCL and RRCL to 5mM of MSA induced synergistic cytotoxic and anti-proliferative effects when combined with CDDP or gemcitabine. Our data demonstrates that MSA is active against various RSCL and RRCL and potentiates the anti-tumor effects of chemotherapy agents. Our findings strongly suggest that MSA added to systemic chemotherapy (i.e. combination of gemcitabine and cisplatin) may result in a novel and potent salvage regimen against relapsed/refractory B-cell lymphomas.
Blast Transformation in a Patient with Primary Myelofibrosis Initiated from JAK2 V617F Progenitor.
Authors: Swierczek Sabina I. , Yoon Donghoon , Prchal Josef T.
Published: October 2019

Myeloproliferative disorders (MPDs) are caused by clonal proliferation arising from a single multi-lineage stem cell. The JAK2 V617F mutation has been reported in greater than 90% of patients with polycythemia vera (PV), and ∼50% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). However, several studies have indicated that the JAK2 V617F mutation is not an MPD initiating mutation but rather represents clonal evolution of these MPDs. Jelinek and colleagues first reported that most PV transformed acute leukemias are JAK2 V617F negative (Jelinek, Blood, 2005 106:3370). More recently, the role of the JAK2 V617F mutation in leukemic transformation in 27 patients with MPDs revealed that most JAK2 V617F -positive MPD patients transformed to a JAK2 V617F -negative AML (Theocharides, Blood, 2007 110:375); however, in the 4 patients with an apparent JAK2 V617F -positive leukemia clonality of leukemic blasts and mature granulocytes was not determined. Two models proposed by Theocharides et al may explain these findings. First, MPD and AML represent 2 independent clones that arose de novo from different progenitors. Second, MPD and AML are 2 subclones derived from a common progenitor. Here, we describe a woman with PMF with transformation to AML. We determined her JAK2 V617F mutation status by sensitive and quantitative real-time PCR (Nussenzveig, Exp Hematol, 2007 3:32). At the time of her transformation to AML, her normal appearing peripheral blood granulocytes were purified and the frequency of mutant JAK2 allele T was 6%. However, all FACS-sorter isolated CD34+ cells (enriched to 95% purity) were heterozygous for the JAK2 V617F mutation. To determine if MPD and AML clones arose de novo or from the same progenitor, we performed clonality studies using a newly developed sensitive and quantitative real-time PCR based on the X-chromosome inactivation principle using transcriptional clonality assays in granulocytes and CD34+ purified cells from peripheral blood at both stages of disease (see Swierczek et al, abstract, this meeting). When this woman’s PMF was first discovered, hematopoiesis was clonal, based on heterozygosity for three X-chromosome genes, FHL1, G6PD and IDS (Liu, Blood, 2003 101:3294) and their single allelic expression in granulocytes and platelets. At the time of leukemic transformation, both her granulocytes and leukemic CD34+ cells expressed all three identical isoforms from the same parental X chromosome. Our findings indicate that leukemic transformation does not invariably arise from a JAK2 V617F negative progenitor. This has important implication for therapy of MPDs with JAK2 V617F inhibitors, as these would not prevent leukemic transformation. It remains to be determined if the JAK2 background of leukemic progenitors is variable, and if there are differences between PV and PMF.
High Frequency of Surface Immunoglobulin IgG in B-Cell Chronic Lymphocytic Leukaemia.
Authors: Wiseman Catherine , Giustiniani Jerome , Mikkelsen Lene , Macey Marion , Goff Lindsey , Gribben John , Agrawal Samir
Published: October 2019

Background: A common diagnostic feature of chronic lymphocytic leukaemia (CLL) is the weak surface immunoglobulin (sIg) expression of the IgM/D isotypes. The occurrence of sIgG is believed to be an atypical finding in CLL.

Aims: To demonstrate that sIgG expression is a real and not artefactual by molecular and functional assays.

Methods: 194 unselected, consecutive cases of CLL were analysed during routine diagnostic testing for sIgG and sIgM. Flow cytometry was performed using fresh whole blood and red cell lysis (Pharmalyse solution, BD). For confirmation of these results and demonstration of functionality, we used 4-colour flow cytometry, PCR for mRNA detection, proliferation assay (3H incorporation), and calcium flux assay.

Results: 23% (44/194) of all CLL cases displayed sIgG, with 39 being sIgG+ alone. PCR results showed a distinct band indicating the presence of mRNA for IgG. Four-colour flow cytometry identified single cells that co-expressed CD19/CD160/sIgG and ruled out sIgM expression. In vitro proliferation assays demonstrated that sIgG is functional when triggered by F(Ab’)2 fragments specific for sIgG isotype. The strength of the signal in one case after stimulation via sIgG led to a 3.5-fold increase in cellular proliferation. Calcium flux assays also demonstrated that the IgG isotype was functional, one case showing a 50% increase in fluorescence intensity after stimulation via sIgG.

Discussion: 23% of CLL cases have sIgG expression. In addition to a high frequency of sIgG expression, this study has shown sIgG to be capable of signalling. If antigen-drive is important in CLL, an IgG B-cell receptor should have higher affinity for a putative antigen, which potentially would be a poor prognostic feature. However, such cases would also be expected to show mutated VH genes, indicating a better prognosis. Further study is required to elucidate the pathological significance of sIgG expression and its role in CLL.
Dose-Reduced Conditioning Followed by Allogeneic Stem Cell Transplantation in Patients with Myelofibrosis. Results from a Multicenter Prospective Trial of the Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT).
Authors: Kroeger Nicolaus , Holler Ernst , Kobbe Guido , Bornhaeuser Martin , Schwerdtfeger Rainer , Nagler Arnon , Bethge Wolfgang , Stelljes Matthias , Uharek Lutz , Wandt Hannes , van Os Marleen , Burchert Andreas , Corradini Paolo , Schubert Joerg , Kaufmann Martin , Dreger Peter , Wulf Gerald , Einsele Hermann , Gramatzki Martin , Zabelina Tatjana , Bacher Ulrike , Zander Axel , Heinzelmann Marion , Kvasnicka Michael , Thiele Juergen , Niederwieser Dietger , de Witte Theo
Published: October 2019

The major limitation of allogeneic stem cell transplantatiopn (SCT) in patients (pts) with myelofibrosis is the high treatment related mortality. We performed a prospective multicenter trial of a dose-reduced conditioning regimen, consisting of busulfan (10 mg/kg orally or 8mg/kg i.v), fludarabine (180 mg/m2) and anti-thymocyte globulin (ATG Fresenius: 30–60 mg/kg) followed by allogeneic SCT in pts with myelofibrosis. From 2002 to 2006, 104 pts with a median age of 55 years (range: 32–68) andlow risk with constitutional symptoms (18%) or intermediate risk (n= 58%) and high risk (n=19%) were included. Cytogenetic abnormalities and JAK2 mutation were noted in 22% and 48%, respectively. Bone marrow histology showed advanced fibrosis (MF 2 and 3) in all pts. All but 3 pts received peripheral blood stem cells as graft source either from related (n=33) or unrelated donor (n=71). All but one (1%) pts showed leukocyte and platelet engraftment after a median of 18 and 21 days, respectively. The median duration of leukocyte aplasia was 9 days (range: 3–21). Acute GvHD grade II to IV occurred in 19% and severe aGvHD III/IV in 7%, while chronic GvHD was seen in 32% of the pts. Non-relapse mortality at 1 year was 19% (95% CI: 11–27%) and significantly lower for pts younger than 50 years of age (0% vs 27%, p=0.004) and for pts with low risk vs intermediate/high risk disease (0% vs 27%, p= 0.02). The cumulative incidence of relapse at 3 years was 29% (95%CI: 15–43%). The 3 year overall (OS) and event-free survival (EFS) was 70% (95% CI 60–80%) and 55% (95% CI 42–68%). Significant factors for improved 3 year OS and EFS were age less than 50 years (92% vs 62%, p=0.003 and 79% vs 46%, p= 0,004) and low vs intermediate/high risk (100% vs 62%, p=0.01 and 72% vs 48%, p= 0.02), while no impact on survival was seen for cytogenetic abnormalities, JAK2 mutation status and donor (related vs unrelated). These prospective multicenter study show excellent outcome of an busulafan/fludarabine based reduced conditioning regimen followed by allogeneic SCT in pts with myelofibrosis.
Ex Vivo Graft Purging and Expansion of Autologous Mobilized Peripheral Blood Progenitor Cell (PBPC) Products from Patients with Multiple Myeloma (MM).
Authors: Yang Hong , Robinson Simon N. , Nieto Yago , Giralt Sergio A. , Jones Roy B. , Decker William K. , Xing Dongxia , Steiner David , Champlin Richard E. , Ng Jingjing , Thomas Michael W. , de Lima Marcos , Shpall Elizabeth J.
Published: October 2019

Introduction: Autologous PBPC transplantation has become the treatment of choice for selected MM patients. Autografts are often contaminated with MM cells which could be a source of post-transplant relapse. We elected to develop a graft manipulation procedure to purge tumor cells from the autograft without compromising its normal reconstituting activity. Previous studies have shown that bortezomib (Velcade, Millenium Pharmaceuticals, Inc., Cambridge, MA) can kill CD138+ MM cells, with little effect on CD138− B cells. However, the anti-CD20 monoclonal antibody, rituximab (Rituxan, Genentech, Inc., San Francisco, CA) has been shown to kill CD138− B cells, the putative MM “stem cells” population. Therefore we investigated whether an optimized combination of bortezomib and rituximab might effectively eradicate tumor cells from PBPC products. Previous studies1 have also suggested that tumor cells can also be selectively purged by ex vivo culture while expanding the normal hematopoietic progenitors. Thus, we incorporated an ex vivo culture step to optimize MM cell depletion and expansion of the reconstituting normal progenitors.

Methods: CD138+ cells were depleted from the thawed PBPC of MM patients using the midiMACS device (Miltenyi Inc, Auburn, CA). The CD138− cells were treated for 24 hrs with 10 or 20 μg/ml rituximab followed by 20nM or 80nM bortezomib for 16hrs. Cells were then washed and ex vivo-expanded using an allogeneic normal marrow donor-derived mesenchymal stem cell (MSC) co-culture technique as previously reported.2 At the end of culture, cells were evaluated for total viable cells, expression of CD138, CD20, CD19, CD34, CD45 by flow cytometry and colony-forming cell (CFC) content in methylcellulose assays (StemCell Technologies, Vancouver, BC).

Results: CD138+, CD138−/CD20+ or CD138−/CD20+/CD19+ cells were depleted in the MM PBPC products from patients when treated with bortezomib (20nM) and rituximab (20 μg/ml) followed by ex vivo culture.(Fig 1) Compared with input control, TNC increased by 6–72 fold and absolute numbers of CD34+ cells increased by approximately 3–9 fold. (See representative data Fig 2)

Conclusion: Treating CD138-depleted MM PBPC products with bortezomib, rituximab and 2 weeks of ex vivo culture depleted CD138+ malignant plasma cells and CD138− B cells (MM “stem cells”). An estimated >4 log tumor depletion from a mobilized PBPC product was achieved, while the use of ex vivo expansion culture not only preserved but increased the number of normal hematopoietic progenitors. Further refinements of this procedure are in progress and will be tested clinically.

Figure Figure Figure Figure
Syk Kinase Inhibition by R406 Inhibits BCR Mediated B Cell Proliferation and Interferes with BCR Signal Transduction in Primary Tumor Samples.
Authors: Sharman Jeff P. , Irish Jonathan , Hatton Olivia , Martinez Olivia , Levy Ronald
Published: October 2019

Background: B cells require B cell receptor (BCR) mediated survival signals during differentiation and throughout maturity. However, the CD79a/b signaling subunit of the BCR lacks independent kinase function and requires Syk kinase activity to activate the majority of downstream signaling effectors in the BCR signal transduction cascade. We have previously shown that inactivating Syk kinase with R406, a novel small molecule tyrosine kinase inhibitor, selectively kills B cells from primary human tumor samples and B cell lines (Sharman et. al ASCO 2007, Abstract # 3600). In addition, we have shown that Syk signaling is abnormally rapid, intense, and long-lived in primary human follicular lymphoma B cells (Irish et. al Blood2006108(9) 3135–3142). We hypothesized that R406 would prevent BCR mediated proliferation of normal B cells and interfere with BCR signaling in primary B lymphoma tumor samples.

Methods: Normal peripheral blood B cells were evaluated with 3H-thymidine incorporation following BCR stimulation with anti-IgM and treatment with several concentrations of R406. Phosphospecific flow cytometry was used to evaluate signal transduction in single cell suspensions derived from primary lymphoma tumor samples and cell lines pretreated with R406.

Results: Peripheral blood B cells proliferate in response to BCR stimulation. This proliferation is inhibited by R406 with IC50 of approximately 100 nM and IC100 at 1250 nM. Several concentrations of R406 were used to inhibit BCR signaling in primary tumor samples. At the lowest dose 50nM, there was little impact on downstream signaling in most samples. At the highest dose tested, 2500 nM, phosphorylation of SFK, Syk, Btk, c-Cbl, p38, Stat-3, Stat-5, Erk, and Akt was completely blocked in all patients. At this same dose, R406 did not inhibit Erk phosphorylation in response to PMA in cell lines.

Conclusions: At pharmacologically relevant concentrations, R406 prevents peripheral blood B cell proliferation in response to BCR stimulation and completely blocks phosphorylation of all proteins evaluated following BCR stimulation in primary tumor samples. Signaling from PKC to Erk which classically involves Ras, Raf, and MEK is undisturbed at maximum drug concentrations. These findings are consistent with the hypothesis that Syk determines the phosphorylation status of most proteins within the BCR signaling cascade following BCR stimulation. Since B cells require BCR mediated survival signals, the anti-BCR signaling effect of R406 may be effective against B cell neoplasms. This idea is currently being tested in a phase I/II clinical trial using an orally bioavailable form of R406 in patients with relapsed and refractory B cell NHL.