bluuebunny/crossref_metadata_2025 huggingface dataset:
Formation of Pancreatic Duct Cells from Bone Marrow during Neonatal Development.
Authors: Wang Xiuli , Ge Shundi , McNamara George , Bhushan Anil , Crooks Gay M.
Published: October 2019

A debate has raged in recent years over whether cells within the bone marrow have the capacity to generate non-hematopoietic, specialized tissue in other organs. Previous reports have studied bone marrow transplantation in adult recipients; most models have found that tissue injury was necessary for organ engraftment of bone marrow and at best low levels of engraftment are seen. In the present study, we show complex ductal epithelial structures in the pancreas completely derived from bone marrow transplanted during the neonatal period in the absence of tissue injury. BRDU labeling showed that epithelial cells lining the ducts and islet cells were cycling at significantly higher levels in the neonatal than in the adult pancreas (P<0.0001). The contribution of transplanted bone marrow to neonatal pancreatic development was thus explored. To exclude the effect of injury on engraftment patterns, non-conditioned, immune deficient neonatal mice were used as bone marrow recipients. 5-10x106 bone marrow cells from male transgeneic GFP mice were injected into female neonatal Nod/Scid/b2Mnull mice 24 hours after birth through the superficial temporal vein. Two months after bone marrow transplantation, hematopoietic engraftment of donor cells was obtained in all transplanted mice, with 22.3 ±4.9% GFP+ cells observed in the peripheral blood (n=9 mice) and pancreas was analyzed with immunohistochemistry, FISH and confocal microscopy. Co-staining of GFP, with anti-cytokeratin (CK) Ab for epithelium, and FISH for the presence of donor Y chromosome indicated that up to 40% of ducts (mean 8.4%) contained epithelium derived from donor bone marrow. In approximately half of these donor derived ducts, there were clusters of large and small ducts, all expressing GFP as if whole branching structures were derived from donor bone marrow. In addition, 2.5% of islets contained cells that co-expressed GFP and Insulin. Donor derived duct and islet cells did not co-express hematopoietic markers excluding donor blood cell contamination. Unlike pancreatic damage models, no donor derived vascular endothelial cells were found in the study. In contrast, bone marrow transplanted into adult mice rarely generated ductal epithelium (0.7% ducts) or islet cells. These findings indicate that the neonatal period may provide a unique window during which mechanisms exist to recruit transplanted bone marrow to proliferating pancreatic tissue without tissue damage. The data also support that existence in bone marrow of pluripotent stem cells or epithelial precursors that can home to pancreas and differentiate into complex organ specific structures.
DDAVP-Induced Increase of Factor VIII Activity in Blood Is Likely Due To Release of Factor VIII That Is Synthesized by Endothelial Cells.
Authors: Xu Lingfei , Nichols Timothy C. , McCorquodale Stephanie , Dillow Aaron , Merricks Elizabeth , Ponder Katherine P.
Published: October 2019

Desmopressin (1-deamino-8-D-arginine vasopressin, DDAVP) is commonly used as a nonreplacement therapy for mild von Willebrand disease (VWD) and hemophilia A. In humans, IV injection of 0.3 μg/kg of DDAVP induces a rapid 2 to 5-fold increase in plasma levels of both von Willebrand factor (VWF) and Factor VIII (FVIII) within 30–60 minutes, which is due to release from Wiebel-Palade bodies (WPBs) in endothelial cells. The stored FVIII may be synthesized by endothelial cells, which express FVIII in vitro. However, hepatoma cells can also express FVIII in vitro, and liver transplantation can correct hemophilia A. Thus, the liver may be the major site of production of FVIII in vivo, thus, an alternative explanation is that endothelial cells take up FVIII from blood and store it in WPBs with VWF, which can be released after DDAVP. DDAVP is effective in humans and dogs, but not in mice. In this study, we tested the effect of DDAVP on hemophilia A dogs after neonatal hepatic gene therapy with a retroviral vector (RV) expressing canine FVIII (cFVIII). With this gene therapy approach, canine hepatocytes express high levels of a reporter gene from an RV, but no expression is observed in endothelial cells. Thus, the major site of FVIII synthesis is the hepatocyte in this model. Our hypothesis is that if DDAVP increases FVIII levels in this dog model, it would indicate that the FVIII increase is due to uptake from blood by endothelial cells. Alternatively, if no increase in FVIII occurs after DDAVP stimulation, it would suggest that the increase in normal dogs is due to synthesis of FVIII by endothelial cells. An RV that contains the liver-specific human α1-antitrypsin promoter and the canine B-domain deleted FVIII cDNA was generated. RV was given IV to two hemophilia A dogs at 8x109 transducing units (TU)/kg at 3 days after birth. The whole blood clotting time (WBCT) and APTT time in both dogs have been normalized, and the plasma cFVIII COATEST activity has been maintained at 100–200% of normal for 11 months to date. DDAVP was injected IV at 0.5 μg/kg into RV-treated hemophilia A dogs at 7 months of age. Two separate doses of DDAVP were given with an interval of one week. The same dose of DDAVP was given to normal dogs as controls (N=4). In normal dogs, both VWF and FVIII levels increased 40% and 50% between 15 to 60 minutes after DDAVP, respectively. However, FVIII levels were not changed in RV-treated dogs, although VWF levels increased 150% or 60%. Thus, our data suggest that the normal FVIII increase after DDAVP administration is due to release of FVIII that is synthesized by endothelial cells. These data also demonstrate that DDAVP will not be effective at increasing FVIII activity in patients that receive liver-directed gene therapy and only achieve partial correction. Such patients would need to be treated with factor replacement if bleeding episodes occur.
Proteomic Analysis of Waldenstrom Macroglobulinemia (WM) and IgM Monoclonal Gammopathy of Undetermined Significance (IgM-MGUS) Identifies Potential Targets for Drug Therapy.
Authors: Ghobrial Irene M. , Leontocich Alexey A. , Eckel Jeanette E. , Timm Michael , Ziesmer Steve , Hoering Antje , Cabanela Miguel , Nehme Alex , Greipp Philip R. , Fonseca Rafael , Gertz Morie A. , Witzig Thomas E.
Published: October 2019

The objective of this study was to analyze molecular changes that occur at the protein level in WM and IgM-MGUS in order to identify proteins dysregulated at disease progression and design new therapies. We employed antibody protein microarrays (BD Clonetech, CA) to measure changes in the patterns of protein expression between WM, IgM-MGUS and normal lymphoplasmacytic cells as control. The antibody array is a novel technique that assays protein differences directly by hybridizing fluorescently labeled protein mixtures from cell extracts onto a glass slide spotted with 512 different monoclonal antibodies specific for human proteins. CD 19+ and CD138+ purified lymphoplasmacytic cells were obtained from cryopreserved bone marrow samples of 5 newly diagnosed patients with WM and 5 patients with IgM-MGUS. Control plasma cells were obtained from 3 pooled CD19+ and CD138+ purified normal donor bone marrow cells. To assess differential expression, the mean of the ratios of Cy5/Cy3 for each sample were analyzed using the Clontech software to calculate an internally normalized ratio. The normalized data were analyzed by the Genespring software and changes of protein expression ≥2 fold in 60% of the samples as compared to normal control were identified in both WM and MGUS groups. There were 20 proteins upregulated in WM and 21 proteins in MGUS as compared to normal control cells These included proteins from the small GTPases such as the Ras, Rho and Ran families (Rab 4, MONA, RCC1, and CDC42GAP), Kinases/signaling proteins (ROK alpha, p62 dok, JAK, 14-3-3e), cyclin dependent kinase (CDK2), fatty acid synthase (HDAC3), NFkB pathway (IKKalpha, IKBe), and histone deacetylase. The WM and IgM MGUS differed in only 3 proteins using the 2-fold threshold. The WM samples showed overexpression of the heat shock protein Hsp90, the Ras family protein CDC25C, and the chemotaxis protein p43/EMAPII as compared to the MGUS samples. This is the first proteomic study of WM and MGUS patients. It identifies novel proteins dysregulated in WM that may serve in future clinical trials as targets for therapy such as Hsp90, NFkB, HDAC and fatty acid synthase. In addition, it identifies 3 proteins that identify disease progression from MGUS to WM, which could be useful for monitoring MGUS patients and designing targeted therapies to prevent progression to WM.
Phase 2 Study of Oblimersen Sodium (G3139; Bcl-2 Antisense; Genasense®) Plus Gemtuzumab Ozogamcin (Mylotarg®) in Elderly Patients with Relapsed Acute Myeloid Leukemia (AML).
Authors: Moore Joseph O. , Seiter Karen , Kolitz Jonathan E. , Stock Wendy , Yu Richard , Frankel Stanley R.
Published: October 2019

Patients with relapsed AML over the age of 60 have a poor prognosis. Gemtuzumab ozogamicin (GO) has been approved for older pts in first relapse, although many pts who attain complete remission (CR) do not fully recover normal platelet count (so-called CRp). In vitro studies have shown that oblimersen down-regulates Bcl-2 in AML cells and enhances apoptotic cell death induced by GO. We conducted a Phase 2 study to evaluate the safety and efficacy of GO combined with oblimersen for older pts with AML. Eligibility requirements included: age ≥ 60 yrs; AML in 1st relapse; ≥ 3 mos 1st CR duration; ≥ 25% CD33-positive AML cells. Pts received oblimersen at a dose of 7 mg/kg/d for 7 days by CIV beginning on days 1 and 15; GO was given at a dose of 9 mg/m2 IV over 2 hrs on days 4 and 18. A total of 48 pts were enrolled (ITT population), all of whom received at least 1 dose of oblimersen; 9 pts failed to receive the required 2 doses of GO (per-protocol population, n=39). The median age was 67 (range, 59 to 88 yrs). Duration of 1st CR: < 6 mos: 7 pts; (15%); 6 to 12 mos: 29 pts (60%); > 12 mos: 12 pts (25%). No. of prior regimens: 1 (17 pts, 35%); 2 or 3 (26 pts, 54%); ≥ 4 (5 pts, 10%). Among treated pts, 79% completed 21 days of protocol therapy. Overall, 12 pts achieved a major response, either CR (n=5) or CRp (n=7), for an ITT response rate of 25% and a per-protocol response rate of 31%. The median time to remission was 52 days. Ten of the 12 responders survived > 6 mos, whereas only 6 non-responders survived ≥ 6 mos. Serious adverse events for the oblimersen/GO combination were qualitatively similar to those reported for GO alone and included among other reactions: Grade 3-4 febrile neutropenia (42%) or thrombocytopenia 33%; nausea; fever; rigors, and dyspnea. Treatment-emergent adverse reactions led to discontinuation of protocol therapy in 10 pts (21%). The most common serious adverse event was febrile neutropenia (25%). One pt (2.1%) died during treatment (sepsis) and 16 pts (33%) died within 30 days of last study medication (infection, bleeding, respiratory failure, progressive AML, and other disease-related complications). No episodes of VOD were observed. Oblimersen can be safely combined with GO; however, pts enrolled in this study appear to have had more unfavorable characteristics at entry compared with prior studies using GO alone in pts with relapsed AML. Therefore, assessment of an incremental benefit from the addition of oblimersen will require a randomized trial.
Midazolam: Significant Pain Reduction in Patients Undergoing Bone Marrow Puncture - a Clinical Trial.
Authors: Naumann Ralph , Schneider Ellen , Einsle Franziska , Kiani Alexander , Jenke Andreas , Lerche Linda , Klut In-Maria , Ehninger Gerhard , Joraschky Peter , Koellner Volker
Published: October 2019

Purpose: Bone marrow puncture with biopsy and aspiration is an everyday routine technique in haematology. The investigation induces substantial pain and fear in many patients. To our knowledge, prospective studies in adults have not yet been published. The aim of our prospective four-arm single-center interventional study was to compare the effect of relaxation (music with facultative instruction on how to relax) with that of an analgetic drug (piritramide), an anxiolytic drug (midazolam) and placebo to reduce pain and anxiety on a double-blind basis.

Methods: 138 patients (60.9% male, 39.1% female, median age 59.5 years, range 21 – 81 years; 26.5% with benign disease, 73.5% with malignant disease) were assigned randomly to one of the four interventional arms taking into consideration the contraindications for piritramide and midazolam before iliac crest puncture. The dose of piritramide administered was 0.05 mg/kg body weight i.v. and that of midazolam 0.035 mg/kg body weight i.v. Local anaesthesia with 1% lidocaine at the puncture site envisaged was administered in each patient. The pain tolerance, pain intensity and fear intensity were measured using visual analogy scales (VAS) from 0 to 10 immediately after puncture (early evaluation) and after two weeks (late evaluation).

Results: 134 patients (97%) could be evaluated. 23% of the patients received relaxation exercise, 26% piritramide, 27% midazolam and 24% placebo. Midazolam was able to increase the pain tolerance and reduce the intensity of pain induced by puncture to a significant extent compared to the other three arms, but there was no difference in the intensity of fear. No differences were found between the early and late evaluation. Subgroup analysis did not show any differences in respect of gender distribution, indication for bone marrow puncture, incidence of complications (e.g. several attempted punctures or wish for a special alleviative treatment).

Conclusions: Midazolam was shown to reduce significantly the pain induced by bone marrow puncture. All subgroups benefited equally from midazolam administration. In accordance with the results of our study, we recommend that midazolam after appropriate information should be offered to every patient before carrying out the bone marrow puncture provided that midazolam is not contraindicated and that there is adequate monitoring of the patients after puncture.
Post-Transplant Positron Emission Tomography Using Fluorine 18-Fluoro-Deoxyglucose ([18F]FDG-PET) Correlates with Outcome in Patients with Aggressive Non-Hodgkin’s Lymphoma Undergoing High Dose Chemotherapy Followed by Autologous Stem-Cell Transplantation (HDC/ASCT).
Authors: Kassim Adetola A. , Mineishi Shin , Greer John P. , McVay Natalie , Koyama Tatsuki , Chinratanalab Wichai , Brandt Stephen J. , Jagasia Madan , Ruffner Katherine , Schuening Friedrich G. , Stein Richard S. , Vusirikala Madhuri , Goodman Stacey , Morgan David S.
Published: October 2019

INTRODUCTION: Compared with standard chemotherapy, treatment of patients with relapsed aggressive lymphoma with high-dose chemotherapy and stem cell transplantation (HDC/ASCT) increases progression-free survival (PFS) and overall survival (OS). [18F]FDG-PET (PET) has been demonstrated to be more precise than conventional radiologic imaging techniques for restaging after chemotherapy [Kostakogu et. al. Euro J Nucl Med. 2000]. However, the value of PET, particularly posttransplant PET to predict clinical outcome after HDC/ASCT has yet to be established.

METHODS: We analyzed 44 patients who had histologically proven non-Hodgkin’s lymphoma (NHL), in first and subsequent relapse, sensitive to conventional-dose salvage chemotherapy, and had HDC/ASCT between April 1999 to June 2004 and PET as part of there disease evaluation, pre (6–8 weeks before) and/or post [early-30 days (D30) and late-100 days (D100)] HDC/ASCT. Thirty (68%) had DLBCL (diffuse large B-cell lymphoma), four (9%) FCCL (follicular center cell lymphoma), four (9%) T-cell lymphoma, three (7%) MCL (mantle cell lymphoma) and three (7%) TCR-BCL (T-cell-rich-B-cell-lymphoma). Log-rank analyses were done to compare PFS of PET positive and negative cases in (a) patients with DLBCL and (b) all histology combined.

RESULTS: Thirty patients (68%) had pretransplant PET and thirty-nine (89%) had posttransplant PET. Fourteen (32%) patients had positive pretransplant scans, while 21 (48%) had positive post-transplant PET-scans. Three patients died and one patient had progressive disease before day 30 and were excluded. Mean observation time of remission cases were 691 days (range 129-1916 days). Among DLBCL patients pre-transplant PET scan did not correlate with PFS (p= 0.1332, figure 1), while posttransplant PET correlated with PFS (D30-PET p=0.0019; D100-PET p=0.007, combined p <0.0001, figure 2). Amongst all histology’s, pretransplant PET-scan did not correlate with PFS, posttransplant PET-scans also showed a positive correlation with PFS (D30 p=0.0001; D100 p=0.0212 and combined p= 0.0001).

CONCLUSION: PET has an important prognostic role in evaluating response of patients following HDC/ASCT. In our analysis, posttransplant PET-scan showed positive correlation with PFS, and may be useful in detecting patients who need additional treatments as early as 30 days post-transplant. This finding however will need to be confirmed in a prospective study.
Aberrant Expression of BAFF System Molecules on B-Cell Precursor Leukemia Modulates Tumor Cell Survival: Novel Targets for Therapeutic Intervention.
Authors: Maia Sara , Rao Sambasiva P. , Sallan Stephen E. , Nadler Lee M. , Cardoso Angelo A.
Published: October 2019

Identification of pathways that support the survival and proliferation of acute lymphoblastic leukemia (ALL) cells will provide novel targets for therapeutic intervention. BAFF and APRIL, with their receptors, BAFF-R, BCMA and TACI are members of the TNF family that play a critical role in the survival of mature B cells but not of normal B cell precursors. We sought to determine whether BAFF/APRIL and their receptors might be aberrantly expressed on B-cell precursor ALL. Surprisingly, tumor cells from 28 of 29 ALL patients expressed mRNA for at least one of these receptors. Using flow cytometric analysis, surface expression of BAFF-R, BCMA, and TACI protein was detected on 89%, 78% and 56% of B-lineage ALL, respectively. Binding assays using a BAFF fusion protein demonstrated that ALL cells expressed high-affinity receptors for BAFF. Stimulation of leukemia cells with BAFF fusion protein triggered the activation of the NFκB pathway with phosphorylation of NFκB2 p100 (Ser864), and its processing to p52. However, BAFF binding did not trigger phosphorylation of IKKα, suggesting that BAFF-mediated activation of NFκB on leukemia cells occurs in an IKK-independent manner. These observations demonstrate that B-cell precursor ALL express functional receptors for BAFF/APRIL ligands. To examine the biological significance of the BAFF system in precursor B-cell ALL, we performed survival and proliferation assays using a myc-tagged BAFF. Our results indicate that while BAFF promoted survival of B-ALL cells, it did not induce their proliferation. Moreover, BAFF binding to leukemia cells was accompanied by increased expression of Bcl-2. To determine the source of BAFF in the leukemic BM, expression of membrane-bound BAFF and APRIL was analyzed in both leukemia cells and cells from the BM microenvironment (BM endothelium, BM mesenchymal stem cells and BM stroma). Surprisingly, leukemia cells expressed BAFF in all patients tested, and APRIL was detected in 94% of the cases. BM microenvironmental cells were found to express membrane-bound BAFF but not APRIL. These findings demonstrate that leukemia B-cell precursors express functional receptors for BAFF system ligands. Importantly, we show that BAFF-mediated stimulation of leukemia cells triggers the activation of the NFκB pathway through an IKK-independent mechanism, and promotes the survival of leukemia cells. The differential expression of BAFF and APRIL on B-ALL cells and the BM microenviromental cells suggests that these ligands may act through both autocrine and juxtacrine mechanisms. Our studies indicate that the BAFF/APRIL ligand-receptor axis might provide new opportunities for therapeutic intervention in B-cell ALL.
Human Herpesvirus 8 K1 Suppresses Fas-Mediated Apoptosis and Binds to the Fas Complex.
Authors: Samaniego Felipe , Wang Shu , Young Daniel , Younes Anas , Wang Suizhao
Published: October 2019

HHV-8 is associated with the development of primary effusion lymphoma (PEL) and Kaposi’s sarcoma. We have previously reported that PEL-derived cell lines constitutively express the HHV-8 K1 gene, which encodes for a transmembrane protein with an immunoreceptor tyrosine-based activation motif (ITAM). ITAMs are present in receptors regulating cell activation and survival. K1 in transgenic mice, driven by a constitutive promoter, is associated with development of lymph node hyperplasia and lymphoma. Given its NF-kB activation inducing properties, we hypothesized that K1 contributes to transformation, in part, by suppression of apoptosis. To study the role of HHV8 K1 gene product, we generated K1 stably expressing cell line BJABK1 and vector alone cell line BJABXS, with retrovirus vector pLXSN, and packaging cell line PT67 NIH 3T3. We also expressed a truncated K1 lacking the ITAM (K1m). Transient transfections were used to expressed epitope tagged K1. Anti-Fas antibody (CH-11) was used to induce apoptosis and cells were monitored for membrane protein Annexin-V, DNA integrity, and morphological features of apoptosis. Caspase 8, cFLIP and Fas were monitored by immunoblot analysis and flow cytometry. K1 was expressed in mouse hepatocytes by intravenous inoculation of plasmids, expressing K1 and green fluorescent protein. K1-Fas complex interactions were analyzed in immunoprecipitation-immunobloting analysis. K1 is expressed in PEL cells and PEL primary cells. Anti-Fas antibody induced apoptosis was suppressed in BJABK1 cells compared with BJABK1m and BJABXS cells. K1 also suppressed apoptosis, which had been induced by Fas ligand but not apoptosis induced by TRAIL or radiation. K1 expressing cells showed consistent lower levels of procaspase 8 cleavage products. Immunoprecipitation-immunoblotting analysis indicated Fas and K1myc were bound to the same complex. Taken together, the data indicate that expression of HHV8 K1 can suppress Fas-mediated apoptosis. K1 and Fas bind to the same complex in several cell types and in vivo. We anticipate K1 regulates apoptosis through binding of the Fas complex and represents a novel form of apoptosis regulation mediated by cellular proteins designated to modulated Fas.
A Multi-Center, Open-Label Study To Evaluate the Safety and Efficacy of Pentostatin, Cytoxan, and Rituxan (PCR) in the Treatment of Previously Untreated or Treated Bulky Stage II or Stage III or IV, Low-Grade B-Cell NHL.
Authors: Mena Raul R. , Christiansen Neal P. , Nyman David W. , Markan Yudhishtra , Chowhan Naveed M.
Published: October 2019

Introduction: The decision to treat indolent B-cell NHL is often based on progressing bulky disease, worsening symptoms, and increasing hematologic derangement. When treatment is indicated, these lymphoproliferative disorders are very sensitive to combination chemotherapies. Combination therapy with pentostatin (P), purine analog, cyclophosphamide (C), alkylator, and rituximab (R), anti-CD20 monoclonal antibody, represents a promising new approach in the treatment of patients with low grade NHL. Most regimens have utilized fludarabine as the purine analog, but the myelosuppression and immunosuppression of these combinations frequently results in severe infections. We have previously reported our experience with pentostatin and rituximab in a cohort of 133 previously treated and untreated patients with Grade III/IV NHL or CLL (2005 Pan-Pacific Lymphoma Conference).

Methods: Fifty-five patients have been enrolled in this study with an expected accrual of 100. Patients are being enrolled through the Pharmatech Research Network. Eligibility criteria allows previously treated and treatment naïve patients diagnosed with bulky stage II or low-grade stage III/IV NHL (REAL classification). Treatment consisted of intravenous infusions of P (4 mg/m2), C (600 mg/m2), and R (375 mg/m2) on day 1 of a 21-day cycle for a total of 8 cycles. Clinical evaluation was performed after even-numbered cycles. Patients were stratified by disease and by prior treatment status.

Results: Forty-seven patients have been eligible for evaluation. NHL patients (median age 64, range 30–84) received a total of 236 cycles (median 6). The ECOG status was 0 (65%) and 1 (33%). The overall objective response rate was 70% (CR 21%, Cru 13%, PR 36%). There were 3 grade 4 neutropenias documented and there was a single grade 4 leukopenia. There were a total of 3 deaths all of which occurred within 30 days of the last dose. The first death was due to a second primary, NSCLC, diagnosed after treatment began. The second death occurred in a 81 year-old female (PR) due to CHF and the last death, due to CAD, occurred in an 84 year-old woman (SD).

Conclusions: This immunochemotherapeutic regimen is very active in indolent Grade III/IV NHL and the incidence of significant toxities was extremely low. Future trials evaluating the use of R as maintenance therapy following this PCR regimen may also be warranted with an eye toward increasing the overall survival of patients with NHL. The use of combination therapy in elderly patients should be used with caution due to associated co-morbidities.
Recent Decrease in Acute GVHD and Increased Relapse in Children with Leukemia Receiving Unrelated Donor Bone Marrow Transplants.
Authors: Davies Stella M. , Wang D. , Wang T. , Arora M. , Ringden O. , Anasetti C. , Pavletic S. , Casper J. , MacMillan M.L. , Sanders J. , Wall D. , Kernan N.A.
Published: October 2019

Unrelated donor (URD) BMT is an effective treatment for leukemia in children, but significant morbidity and mortality occur as a result of acute graft versus host disease (GVHD). In addition, relapse is a major cause of treatment failure in children with otherwise successful transplants. We used the database of the Center for International Blood & Marrow Transplant Research (CIBMTR) to determine whether improved HLA typing and prophylaxis have changed incidence and risk factors for acute GVHD, and the impact of GVHD on treatment-related mortality and relapse. We analyzed outcomes of 638 myeloablative URD BMT performed between 1990 and 2003 for the treatment of AML, ALL, CML or MDS. Peripheral blood stem cell and cord blood recipients were excluded as risk factors for GVHD might differ. All recipients were <18 years of age, and had available high resolution HLA typing for HLA A, B, C and DRB1. Overall, 45% of children developed aGVHD grades II–IV, and 27% aGVHD grades III–IV. The data showed that risk of aGVHD was significantly higher in recipients of T-replete compared with T-depleted grafts (OR 3.12, 95%CI 2.02–4.83; p< 0.0001). Survival was reduced in children with grades II–IV aGVHD compared to those without (OR 1.96, 95% CI 1.46–2.62; p<0.0001). However, the occurrence of aGVHD reduced risk of relapse in children with ALL (OR 0.36 95% CI 0.17–0.75, p=0.004) but not in children with AML (OR 1.23, 95% CI 0.50–2.98; p=0.65). Outcomes have changed significantly over time. The risk of aGVHD was higher in children transplanted in earlier years (1990–1998, n=365) compared to 1999–2003 (n= 220) (OR 1.93 95%CI 1.27–2.91; p=0.002), suggesting that current prophylactic regimens may be more effective. In contrast to this recent improvement in risk of GVHD, risk of relapse was lower in children transplanted 1990–1998 compared with 1999–2003 (OR 0.47, 95% CI 0.28–0.78; p=0.004), possibly reflecting changes in the patient population currently referred for transplant, or perhaps a reduced graft versus leukemia effect with better control of GVHD. As more intense risk adapted chemotherapy has improved outcomes of upfront treatment of leukemia, children who are referred for transplant may have more resistant disease, increasing the difficulty of controlling disease with standard irradiation and chemotherapy preparative regimens. Overall survival of children with URD BMT has not changed over time, although it is possible that current results reflect treatment of children with more resistant disease compared with 10 years ago.
Should Hematopoietic Stem Cell Transplantation in First Complete Remission Be Restricted to High-Risk Patients in Childhood Acute Myeloid Leukemia?.
Authors: Muramatsu Hideki , Nishio Nobuhiro , Hama Asahito , Yagasaki Hiroshi , Takahashi Yoshiyuki , Doisaki Sayoko , Watanabe Nobuhiro , Matsumoto Kimikazu , Kato Koji , Kojima Seiji
Published: October 2019

Hematopoietic stem cell transplantation (HSCT) has been used as an effective consolidation therapy for children with acute myeloid leukemia (AML) in first complete remission (CR). Although it is effective in relapse prevention, often it causes severe late sequelae, such as short stature and infertility. There is a recent trend to restrict the use of HSCT in first CR for high-risk patients. However, there is no study comparing which strategy is better, risk-adapted or general recommendation for HSCT in AML children in first CR. In our institutes, all such children in first CR were recommended either allogeneic or autologous HSCT until 1997. After 1998, patients were classified into three risk-groups. Low-risk patients (t(8;21) and inv(16)) were not recommended to undergo HSCT. High-risk patients (−7, 5q-, Ph1, t(16;21) and remission failure) were recommended to undergo HSCT. Intermediate-risk (other karyotypes) patients received HSCT in first CR if a suitable donor was available. In this study, we retrospectively compared the prognosis of 66 patients who were diagnosed with de novo AML between 1991 and 1997 (group A; n=37) and between 1998 and 2003 (group B; n=29). AML with Down syndrome and AML-M3 were excluded. The median (range) age was five (0–15) years. FAB classifications were M0 (n = 1), M1 (n = 10), M2 (n = 22), M4 (n = 6), M4E (n = 5), M5 (n = 14), M6 (n = 0), and M7 (n = 8). Chromosome analysis data were t(8;21) (n = 18), inv(16) (n = 4), 11q23 (n = 10), other abnormalities (n = 14), normal karyotype (n = 14), and unknown (n = 6). Induction chemotherapy comprised VP-16, cytarabine, and mitoxantrone. Sixty-three of 66 patients (95.5%) achieved CR. HSCT in first CR was done in 24 patients (64.9%) in group A and seven patients (24.1%) in group B (p = 0.0044). Age, sex, WBC count at diagnosis, FAB classification and chromosomal abnormalities did not differ between the two groups. Fourteen (five in group A and nine in group B) patients relapsed. Six (three in group A and three in group B) of them were salvaged by HSCT. Both 5-year event-free survival (EFS) and overall survival (OS) were statistically higher in group A than in group B (5-year EFS: 83.8 ± 6.1% versus 62.1 ± 9.0%, p = 0.0404; 5-year OS: 91.6 ± 4.7% versus 71.6 ± 8.5%, p = 0.0364). Although intensified chemotherapy without HSCT for low-risk AML patients is desirable to avoid the late complications of HSCT, our analysis showed that the introduction of risk-stratified treatment strategy significantly worsened the chances of EFS and OS. Our risk stratification based on chromosomal abnormalities only may be insufficient to identify low-risk AML children. Development of more sophisticated risk classification, including molecular markers, may be required to identify true low-risk patients who should avoid HSCT in first CR.
MHC-Matched Stem Cell Transplantation in Rhesus Macaques Using a Costimulation Blockade-Based Immunomodulation Platform.
Authors: Kean Leslie S. , Hamby Kelly , Deane Taylor , Pearson Thomas C. , Larsen Christian P.
Published: October 2019

A strategy for producing high-level hematopoietic chimerism after non-myeloablative conditioning has been established in the rhesus macaque. This strategy relies on hematopoietic stem cell transplantation after induction with a non-myeloablative dose of busulfan and blockade of the IL2-receptor in the setting of mTOR inhibition with sirolimus and combined CD28/CD154 costimulation blockade. Hematopoietic stem cells derived from bone marrow and leukopheresis products both were found to be successful in inducing high-level chimerism. When transplants were performed between animals that were totally unmatched at the MHC, mean peripheral blood peak donor chimerism was 81% with a median chimerism duration of 145 days. Additional immune modulation strategies, such as pre-transplant CD8 depletion, donor-specific transfusion, recipient thymectomy or peritransplant deoxyspergualin treatment did not improve the level or durability of chimerism in the setting of these MHC-unmatched transplants. Recipient immunologic assessment suggested that chimerism occurred amidst donor-specific down-regulation of alloreactive T cells, and the reappearance of vigorous T-mediated alloreactivity accompanied rejection of the transplants. Furthermore, viral reactivation constituted a significant transplant-related toxicity and may have negatively impacted the ability to achieve indefinite survival of transplanted stem cells. In order to address the dual complications of lack of indefinite chimerism and viral reactivation, we sought to increase the level of MHC matching between donor and recipient transplant pairs. This necessitated a large-scale analysis of an NIH-sponsored rhesus macaque colony in order to determine the familial relationships and MHC-disparity between potential transplant pairs. We have analyzed over 500 animals from this colony, which has resulted in the first primate transplant series between donors and recipients with known degrees of MHC disparity. Our results suggest that viral reactivation is vastly improved when transplant occurs with even one shared MHC haplotype, but that the current immunomodulation strategy is insufficient to achieve immune tolerance in these haplo-identical transplants. However, we have observed long-term mixed chimerism in fully MHC matched transplant pairs that is stable even years after withdrawl of costimulation blockade-based immunomodulation. These results suggest that costimulation blockade may be an important addition to current immunomodulation strategies for producing stable engraftment after nonmyeloablative pre-transplant preparation, with potential application to non-malignant hematologic diseases and genetic disorders.
Correlation of Peripheral Blood Involvement Measured by Four-Color Flow Cytometry and the Achievement of Molecular Response in Mantle Cell Lymphoma Treated within the Prospectively Randomized Intergroup Trials of the European MCL Network
Authors: Pott Christiane , Boettcher Sebastian , Gesk Stefan , Siebert Reiner , Klapper Wolfram , Hoster Eva , Ritgen Matthias , Unterhalt Michael , Rottenkolber Marietta , Hiddemann Wolfgang , Kneba Michael , Dreyling Martin
Published: October 2019

Two prospectively randomized intergroup trials of the European MCL Network investigating the impact of different combined immuno-chemotherapy protocols for patients with stage II–IV mantle cell lymphoma (MCL) >< 65 yrs followed by autologous stem cell transplantation (PBSCT) for patients <65 yrs and a rituximab or interferone maintenance treatment for patients >65 yrs are currently performed.

Patients and methods: 180 German patients with diagnostic peripheral blood (PB) involvement detectable by consensus IGH PCR were analysed for t(11;14) translocation by FISH and 4-colour Flow Cytometry (FC) at diagnosis as well as molecular response (MR) by quantitative IGH-RQ-PCR after induction. The results were compared to clinical parameters at diagnosis and PFS.

Results: Patients had a median age of 61 years, an elevated LDH in 37%, B-symptoms in 41% and extranodal involvement in 37%. According to the MIPI risk factor score 26% had an adverse, 34% an intermediate and 40% a good prognosis. PB involvement by MCL was demonstrated in 67% (74/111) by t(11;14) FISH and in 97% (152/156) by FC. Detection of MCL cells by FC in PB did not correlate with clinical parameters as stage, LDH, extranodal involvement or bone marrow infiltration. However, median % of MCL cells measured by FC differed significantly with clinical stage (p<0.0001), LDH (p=0.0096) bone marrow infiltration (<0.0001) and MIPI prognostic index (0.0002). MR after induction treatment was achieved in 60% (60/100) of patients and correlated with BM infiltration at diagnosis (p=0.0117). There was a trend for improved PFS in patients achieving MR (PFS 90% vs. 75% at 15 months after induction) however, the observation time is still too short for definitive evaluation.

Conclusion: FC is highly sensitive for detection of PB involvement in MCL and improves staging accuracy. Achievement of MR might be an early indicator for treatment outcome after immuno-chemotherapy in MCL with the potential of defining prognostic subgroups.
Low Dose Rituximab in Adult Patients with Idiopathic Thrombocytopenic Purpura.
Authors: Zaja Francesco , Battista Marta L. , Pirrota Maria T. , Palmieri Salvatore , Montagna Michela , Vianelli Nicola , Cavallin Margherita , Bocchia Monica , Defina Marzia , Ippoliti Micaela , Ferrara Felicetto , Patriarca Francesca , Avanzini Maria A. , Regazzi Mario , Isola Miriam , Soldano Franca , Baccarani Michele , Fanin Renato
Published: October 2019

Rituximab 375 mg/sqm weekly for 4 weeks has significant activity in patients with idiopathic thrombocytopenic purpura (ITP). In this setting, different biological and clinical evidence suggests the possible use of lower doses of Rituximab. Twenty-eight adult patients, median age 43 years (range 16–71 years), with previously treated, active and symptomatic ITP were treated prospectively with Rituximab at the fixed dose of 100 mg iv weekly for 4 weeks. Exclusion criteria were positive HIV, HBsAg and pregnancy test, any B-cell lymphoprolipherative disease or other malignancies. Response assessment was evaluated considering the rate of overall and complete responses (OR = platelet count ≥ 50 x 109/L; CR= platelet count ≥ 100 x 109/L and discontinuation of steroid therapy, if present), the time to response (TTR; time necessary to reach a platelet count ≥ 50 x 109/L), the time to complete response (TCR; time necessary to reach a platelet count ≥ 100 x 109/L) and the duration of response. B-cell count and pharmacokinetics (PK) were monitored and related with clinical outcome. Stepwise logistic regression was used to assess whether response was associated with age, gender, diagnosis-Rituximab interval and basal CD20+ve lymphocytes. Results were considered statistical significant when P ≤ 0.05. All patients completed the therapeutic program receiving the four infusions of Rituximab as scheduled, none experiencing short term toxicity. All patients achieved B-cell depletion. OR and CR were achieved in 21/28 (75%) and 12/28 (43%) patients, respectively. CR rate was associated with younger age (OR=0.92 CI95%[0.85;0.99]). The median TTR and TCR were 31 and 44 days, significantly longer then those observed with standard dose in patients with similar characteristics (Haematologica2003;88:538). After a median follow-up of 11 months (range 3–18), 7/21 (33%) patients relapsed, and 3 needed further treatments. PK data showed a concentration time-course profile of Rituximab that was super-imposable, once corrected for the difference in the dose, to that observed previously in patients treated with standard dose, diseases with a median value of 4.1 micrograms/milliliter (range 0–11.9), 12 weeks after the start of treatment. In patients with ITP, low dose Rituximab led to short and mid-term response rates similar to standard dose but with slower timing of response.
A Comparative Proteomic Analysis of Platelets from Healthy Human Patients and Chronic ITP Patients Receiving AMG 531, a Thrombopoiesis-Stimulating Protein.
Authors: Sloan John M. , Coburn John P. , Bertino Amy M. , Wood Valerie , Kuter David J.
Published: October 2019

AMG 531 is a novel thrombopoiesis-stimulating “peptibody” consisting of an Fc carrier domain linked to 4 identical peptides that bind to and activate Mpl, the human thrombopoietin receptor. It has proven efficacy in raising platelet counts of patients with chronic ITP, and is being evaluated in chemotherapy-induced thrombocytopenia and myelodysplastic syndrome. While platelets from healthy subjects receiving AMG 531 reportedly behave normally in platelet aggregation studies, the effects of this medication on protein expression profiles are not known. The platelets from 12 healthy patients without known platelet defects and 9 chronic ITP patients receiving AMG 531 were isolated and their proteins extracted. Proteins with isoelectric points between pH 3 and 7 were separated by two-dimensional gel electrophoresis and stained with Sypro protein detection reagent. These gels were scanned and compared using Progenesis SameSpots software. In this pH range, there were approximately 5000 unique spots detected, representing individual proteins, isoforms of proteins or cleavage products. On multivariate analysis, approximately 11% of these proteins were expressed at levels significantly different between healthy patients without a known platelet disorder and chronic ITP patients receiving AMG 531. Principal component analysis substantiates these differences and a protein dendrogram has been created. The most significant protein expression differences are identified by tandem mass spectrometry. It is not known if these differences in protein expression represent younger platelets, stimulation of protein synthesis by AMG 531, or altered platelet protein expression as a reflection of the ITP. There is no known clinical correlate of these protein changes.
Reversible Bony Changes after Treatment with Bevacizumab in a Child with Cutaneovisceral Angiomatosis with Thrombocytopenia Syndrome: A Case Report and Review of the Literature.
Authors: Smith Angela R. , Jane Hennessy , Margaret Heisel Kurth , Stephen Nelson C.
Published: October 2019

Cutaneovisceral Angiomatosis with Thrombocytopenia (CAT) syndrome is a rare vascular disorder involving the skin and gastrointestinal tract. Clinical features of CAT syndrome include multiple, discrete skin lesions, gastrointestinal bleeding and thrombocytopenia. CAT syndrome is typically treated with angiogenesis inhibitors like corticosteroids and vincristine. Bevacizumab is a relatively new antiangiogenic agent that is FDA approved as a first-line agent for metastatic carcinoma of the colon or rectum in combination with a 5-FU based regimen. Bevacizumab is a humanized monoclonal antibody that binds all five isoforms of human vascular endothelial growth factor (VEGF) and prevents its interaction with the VEGF receptor. There is currently very little clinical pediatric experience with antiangiogenic agents.

Case: Though subtly symptomatic from birth, our patient was diagnosed with CAT syndrome at four months of age after an episode of grossly bloody stools. After failed therapy with corticosteroids and vincristine, the patient was given bevacizumab 10mg/kg/dose every two weeks for four doses with excellent clinical response. After her fourth dose, a chest radiograph revealed incidental, asymptomatic metaphyseal lesions of the humeri. A complete bone survey revealed similar lesions in the metaphyses of nearly all of her long bones. (Figure 1) Given preclinical data suggesting these lesions could result from the use of VEGF inhibitors, bevacizumab was discontinued. All metaphyseal lesions subsequently resolved. (Figure 2) To our knowledge, this is the first report of bony toxicity in a human receiving bevacizumab. A thorough literature review on VEGF inhibitors revealed no reported cases of metaphyseal lesions or stunted growth in humans receiving VEGF inhibitors. Animal studies, however, suggest this may be possible. Growth plates consist of avascular cartilage that is eventually replaced by bone. This process requires invasion of the cartilage with metaphyseal blood vessels and has been shown to be directed by a VEGF gradient (Nature Medicine1999; 5(6): 623–628). Preclinical studies performed using VEGF inhibitors in monkeys demonstrated reversible physeal dysplasia of the distal femur in the monkeys with open growth plates (Toxicol Pathol, 1999; 27(1): 78–86). We hypothesize that use of the VEGF inhibitor, bevacizumab, in a small child with active epiphyseal growth plates caused the radiographic lesions we observed. It appears that this side effect is reversible, at least after short-term use. We propose that close monitoring for bony toxicity is required for all children receiving bevacizumab and that this drug should be used only with caution in children with active bone growth.

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Constitutively Activated STAT3 Promotes Cell Proliferation and Survival in the Activated B Cell Subtype of Diffuse Large B Cell Lymphomas.
Authors: Ye Bihui Hilda , Ding Beibei Belinda , Yu Jian Jessica , Yu Raymond Y.-L. , Mendez Lourdes M. , Shaknovich Rita , Zhang Yonghui , Cattoretti Giorgio
Published: October 2019

During B cell development, cell proliferation and survival are regulated by stage-specific transcription factors. Accordingly, distinct oncogenic pathways are employed by B cell lymphomas representing different stages of B cell development. Diffuse large B cell lymphoma (DLBCL) contains at least two main phenotypic subtypes, i.e. the germinal center B cell-like (GCB-DLBCL) and the activated B cell-like (ABC-DLBCL) groups. It has been shown that GCB-DLBCL responds favorably to chemotherapy and expresses high levels of BCL6, a transcription repressor known to play a causative role in lymphomagenesis. In comparison, ABC-DLBCL has lower levels of BCL6, constitutively activated NF-kappaB and tends to be refractory to chemotherapy. In this study, we investigated the relationship between BCL6 and STAT3 expression/activation in DLBCL and normal GC B cells. Our results demonstrate that BCL6 directly inhibits transcription of the STAT3 gene by binding to two BCL6 sites in its 5′ regulatory region. As a result, high level STAT3 expression and activation are preferentially detected in ABC-DLBCL and BCL6-negative normal germinal center B cells. Specifically, in tonsillar GCs, STAT3 expression and activation is restricted to a previously uncharacterized subset of BCL6−Blimp-1− B cells in the apical light zone. The location and phenotype of these cells suggest that they are in the process of exiting the BCL6-directed GC program and transitioning to a plasma cell differentiation process governed by Blimp-1. The reciprocal relationship between BCL6 and STAT3 is also conserved in DLBCL such that STAT3 expression and activation is preferentially associated with the BCL6-low, ABC subtype. Most importantly, inactivating STAT3 by either AG490 or small interference RNA in ABC-DLBCL cells inhibits cell proliferation and triggers apoptosis. These phenotypes are accompanied by decreased expression of several known STAT3 target genes, including c-Myc, JunB and Mcl-1, and increased expression of the cell cycle inhibitor p27. In addition to identifying STAT3 as a novel BCL6 target gene, our results define STAT3 activation as a second oncogenic pathway operating in ABC-DLBCL and suggest that blocking STAT3 may be potentially therapeutic in treatment of these aggressive lymphomas.