bluuebunny/crossref_metadata_2025 huggingface dataset:
Simultaneous Targeting of Aurora Kinases and Bcr-Abl by the Small Molecule Inhibitor PHA-739358 Is Effective in Imatinib-Resistant Mutations Including T315I.
Authors: Gontarewicz Artur , Balabanov Stefan , Keller Gunhild , Colombo Riccardo , Graziano Alessio , Bokemeyer Carsten , Fiedler Walter , Moll Jurgen , Brummendorf Tim
Published: October 2019

The emergence of resistance to Imatinib (IM) mediated by mutations in the BCR-ABL domain became a major challenge in the treatment of chronic myeloid leukemia (CML). Second generation Bcr-Abl inhibitors overcome most mutations except T315I, a frequent cause of clinical resistance, emphasising the importance of developing alternative therapeutic strategies. Here, we report studies performed with a novel small molecule inhibitor, PHA-739358, which selectively targets Bcr-Abl and Aurora kinases. PHA-739358 exhibited strong antiproliferative and pro-apoptotic activity against a broad panel of human BCR-ABL positive and negative cell lines and against mouse BaF3 cells ectopically expressing wild type (wt) or IM-resistant BCR-ABL mutants, including T315I. Pharmacological synergism of IM and PHA-739358 was observed in leukemia cell lines with low-level resistance to IM, whereas no synergistic effects were observed in BCR-ABL negative or highly IM resistant T315I mutated cells. Treatment with PHA-739358 significantly decreased phosphorylation of histone H3-(Ser10), a marker of Aurora B activity and of the small adaptor protein CrkL, an accepted down-stream target of Bcr-Abl. Hence, PHA-739358 acts via combined inhibition of Bcr-Abl and Aurora kinases. Moreover, strong antiproliferative effects of PHA-739358 were observed in CD34+ cells derived from newly diagnosed CML patients and from IM-resistant individuals in chronic phase or blast crisis including those harbouring a T315I mutation. Thus, PHA-739358 represents a promising new strategy for treatment of IM resistant BCR-ABL positive leukemias, including those harbouring the T315I mutation. Clinical trials investigating this compound in IM-resistant CML have recently been initiated.
Elevated Serum Interleukin-7 Levels Precede the Development of Acute Graft-Versus-Host Disease.
Authors: Dean Robert M. , Fry Terry , Mackall Crystal , Steinberg Seth M. , Hakim Frances T. , Fowler Daniel H. , Odom Jeanne , Foley Jason , Gress Ronald E. , Bishop Michael R.
Published: October 2019

BACKGROUND: Despite advances in transplantation immunology, there is no clinically practical tool to predict acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Murine models indicate that aGVHD is promoted by interleukin-7 (IL-7), a homeostatic cytokine for naïve CD4+ and CD8+ T-cells. We hypothesized that serum IL-7 levels might be associated with the development of aGVHD in patients (pts) undergoing allogeneic HSCT, and evaluated this using serum samples obtained for this purpose in a prospective clinical trial.

METHODS: Thirty-one pts underwent allogeneic HSCT from HLA-identical siblings. Pts received 1 to 3 pre-transplant chemotherapy cycles to induce profound, uniform host lymphopenia (CD4<100), followed by fludarabine-based reduced-intensity conditioning. GVHD prophylaxis was cyclosporine & methotrexate. Serum IL-7 was measured by ELISA at baseline and multiple time points from the day of transplant through 1 year after allogeneic HSCT. IL-7 levels were evaluated for associations with blood lymphocyte counts, aGVHD, and survival. Other variables examined for association with aGVHD were pt and donor age; CD3+ and CD34+ cell doses; donor gender; donor-recipient gender mismatch; donor or recipient CMV status, prior rituximab therapy; donor lymphoid or myeloid chimerism >95% at day +14; serum IL-15 levels; and levels of soluble tumor necrosis factor-α receptors 1 and 2 (sTNFR1 and sTNFR2).

RESULTS: Grades I, II, and III aGVHD occurred in 3%, 23%, and 19%, respectively; none had grade IV. Median (range) IL-7 levels rose from baseline 12.1 (0–46.9) pg/ml to 37 (13.3–79.2) pg/ml on day 0 before allografting, then fell to 12.0 (1.3–74.7) pg/ml by day +14; these changes were inversely correlated with absolute lymphocyte counts, CD3, CD4, and CD8 counts at baseline and day +7. The development of aGVHD was associated with IL-7 levels at day +7 (p=0.01) and day +14 (p=0.000033) post-transplant (Figure), along with the allograft CD34+ cell dose (p=0.012). Higher IL-7 levels at day +14 corresponded to more severe grades of aGVHD (p<0.0001).

Figure Figure

In logistic regression models, these factors jointly classified pts according to development or avoidance of aGVHD with a maximum sensitivity of 86% and a specificity of 100%. IL-7 levels were more strongly associated with aGVHD than were sTNFR levels or other parameters.

CONCLUSIONS: Determination of serum IL-7 levels in the early post-transplant period accurately predicted the risk of developing aGVHD after allogeneic HSCT and holds promise as a simple, reproducible test to select pts for pre-emptive therapy. These data support preclinical observations that demonstrate a critical role for IL-7 in inducing aGVHD and provide a rational basis for novel approaches to GVHD prophylaxis through modulation of the IL-7 homeostatic pathway.
Stem Cell Transplantation (SCT) Improves Survival in Acute Biphenotypic (ABL) and Acute Undifferentiated Leukemia (AUL): A Single Centre Experience.
Authors: Narayanan Sujaatha , Barnett Michael J. , Mourad Yasser R. Abou , Forrest Donna L. , Hogge Donna E. , Lavoie Julye C. , Nantel Stephen H. , Shepherd John D. , Song Kevin W. , Sutherland Heather J. , Toze Cynthia L. , Smith Clayton A. , Power Maryse , Nevill Thomas J.
Published: October 2019

Background: ABL and AUL are associated with an unfavourable outcome for patients (pts) treated with standard-dose therapy alone. Although high-dose therapy has been used successfully in this population, the optimal management of pts with ABL/AUL remains unclear.

Methods: A retrospective review was performed involving 24 adult pts with ABL or AUL who were treated in Vancouver between 1984 and 2006. Kaplan-Meier estimates were utilized for event-free survival (EFS) and overall survival (OAS) and a multivariate analysis was performed to determine factors predictive of outcome.

Characteristics: Utilizing the WHO criteria, 18 pts had ABL and 6 pts had AUL. There were 17 males and 7 females with a median age of 37 (range 22–75) years. Median white cell count (WCC) at presentation was 10.1 × 109(range: 0.9–196 × 109)/L. Seven pts had poor-risk karyotypes (3 pts complex, 3 pts with t(9,22), 2 pts with11q23 rearrangement, and 1 pt with monosomy 7),12 pts had standard-risk karyotypes, and 5 pts had an unknown karyotype. Induction chemotherapy consisted of Cytosine arabinoside (3–6/m2/day), Daunorubicin, Vincristine and Prednisone with one pt with t(9,22) also having received Imatinib Mesylate. Thirteen pts went on to receive high-dose therapy and SCT. Stem cell source was autologous in 3 pts (all with AUL) or a related (6 pts) or an unrelated donor (4 pts). Eight of 10 pts were in complete remission at the time of SCT, one was in relapse and one had primary refractory ABL. Conditioning was TBI-based in 10 pts and Busulfan-based in 3 pts.

Results: EFS and OAS estimates for all 24 patients at 3 years were 25% (95% CI 13%–50%) and 32% (95% CI 17%–58%), respectively. The non-relapse mortality (NRM) for the whole group at 3 years was 43% (95%CI 15%–61%). On multivariate analysis, when compared to pts receiving only standard-dose chemotherapy, pts who underwent high-dose therapy had significantly improved EFS [39% (95% CI 19%–77%) vs. 9% (95% CI 1%–59%), p=.03] and OAS [46% (95% CI 26%–83%) vs.14% (95% CI 3–74%), p=0.01], respectively. Age, WCC at presentation and cytogenetic risk group were not found to significantly influence outcome.

Conclusion: Although patient numbers are limited, this experience would suggest that patients with ABL/AUL who achieve complete remission with standard chemotherapy should be considered for high-dose therapy and stem cell transplantation. This approach provides them with the greatest probability of long-term event- free and overall survival.
Imatinib Mesilate Versus Allogeneic Bone Marrow Transplantation for Patients with Chronic Myeloid Leukemia in First Chronic Phase - A Brazilian Point of View.
Authors: Bittencourt Henrique , Funke Vaneuza , Fogliatto Laura , Magalhaes Simone , Setubal Daniela , Paz Alessandra , Macedo Antonio V. , Ruiz Jefferson , Azambuja Ana Paula , Silla Lucia M.R. , Clementino Nelma , Pasquini Ricardo
Published: October 2019

Imatinib has profoundly change indication of allogeneic bone marrow transplantation (alloBMT) worldwide for CML and is now the first line of treatment in most countries. However, in Brazil, Imatinib for CML in first chronic phase is provided by Federal Health Agency only for patients refractory/intolerant to interferon (IFN). In order to study results of treatment with imatinib and alloBMT in this particular scenario we retrospectively analyzed 266 patients treated for CML in first chronic phase (less than 60 years-old) in three different institutions in Brazil. End points were event free survival (EFS = absence of hematological response, lost of hematological/cytogenetic response, relapse in accelerated phase/blast crisis or death) and overall survival (OS). From jan/2001 to dec/2006, 176 patients received imatinib 400 mg after failure or intolerance to IFN. Median time from diagnosis to imatinib treatment was 19 months (range: 2 to 205). At the same period of time, 90 patients received an allo-BMT from an HLA-matched sibling (n=83) or an unrelated donor (n=7). Patients receiving peripheral blood stem cell or umbilical cord blood were excluded. Eighty-two patients received busulfan+cyclophsphamide as conditioning regimen and all patients received cyclosporin+methotrexate±steroids as GVHD prophylaxis. Median time from diagnosis to allo-BMT was 16 (range: 5 to 104) months. Gender distribution was similar between groups. Imatinib group had a higher median age (40.7 vs. 32.8 years, P<0.001). With a median follow up of 3 years, 5-year estimate EFS was 67.6% for patients receiving imatinib and 51.8% for patients receiving an allo-BMT (P=0.0002). Estimate overall survival at 5 years was 92.7% for patients treated with imatinib and 58.8% for patients receiving an allo-BMT (P<0.0001). Allo-BMT should be no more recommended as a first line alternative treatment, even if imatinib is available only for patients refractory/intolerant to IFN. Impressively, OS in Imatinib group were quite similar to those seen at the imatinib arm of IRIS study (Druker BJ et al. N Engl J Med2006; 355: 2408).
Human Leukocyte Antigens of A33, B58 or DR7 Decrease the Treatment-Related Mortality in HLA-Matched Sibling Hematopoietic Stem Cell Transplantation in Association with Heat Shock Protein 70-Hom Polymorphisms.
Authors: Kim Jin Won , Oh So Yeon , Kim Hye Jin , Yi Hyeon Gyu , Lee Kyung-Hun , Rhee Ji Young , Shin Eun-Soon , Piao Cheng Zhe , Kim Inho , Yoon Sung-Soo , Park Myoung Hee , Park Seonyang , Kim Byoung Kook
Published: October 2019

Human leukocyte antigens(HLA) are expected to influence outcomes or adverse effects in allogeneic hematopoietic stem cell transplantation through its immunologic function. However, the types of HLA and its mechanism to affect clinical outcomes are not well defined. In the other hand, heat shock protein 70-hom (HSP70-hom) plays an important role in protein folding and immune responses and was reported to influence the incidence of graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. And it was also reported that HLA types were associated with polymorphisms of HSP70-hom in several diseases. So, we evaluated the association between HLA types and HSP70-hom polymorphisms and identified the specific HLA types to affect clinical outcomes in allogeneic hematopoietic stem cell transplantation. We analyzed the DNA of patients and donors who underwent allogeneic hematopoietic stem cell transplantation from HLA-matched sibling donors at single institute between 1998 and 2005 for malignancy or aplastic anemia. The HSP70-hom polymorphisms, rs2227956 and rs2075800, were genotyped and HLA typing was conducted in 141 patients and their donors. Individual haplotypes were estimated from genotype data of the two HSP70-hom polymorphisms using the expectation maximization algorithm. The HSP70-hom polymorphisms of patients were completely identical to those of their donors. Patients(101) with TG haplotype (TG/TA, TG/TG or TG/CG) did not only show less treatment-related mortality but also had longer overall survival compared with those(40) with non-TG haplotype (TA/TA or TA/CG). (P=0.011, P=0.013,respectively) TG haplotype was associated with HLA types of A33, B58 and DR7.(P<0.001, P=0.002, P=0.039, respectively) Patients with HLA types of A33, B58 or DR7 showed less treatment-related mortality compared with patients without the these HLA types in multivariate analyses with age, sex, transplant method, stem cell source and risk group.(P=0.034, HR=0.397, 95% CI: 0.169–0.931) In conclusion, HLA types of A33, B58 or DR7 in HLA-matched sibling hematopoietic stem cell transplantation were protective for treatment-related mortality in association with HSP70-hom polymorphisms.

Figure Figure
Number of Prior Chemotherapy Regimens: Important Prognositic Factor of All-Cause Mortality after Allogeneic Hematopoietic Cell Transplantation.
Authors: Kiyokawa Tetsuyuki , Nagakura Shoichi , Hidaka Michihiro , Uike Naokuni , Hamaguchi Motohiro , Horibe Keizo , Sato Kensaku , Kawano Humio
Published: October 2019

BACKGROUND: Despite recent advances, mortality rates after allogeneic hematopoietic cell transplantation remain high and cannot accurately predicted.

PURPOSE: To determine the value of pretransplant factors in predicting all-cause mortality during first 1 year after allogeneic HCT.

PATIENTS AND METHODS: A retrospective review of 303 consecutive allogeneic hematopoietic cell transplants was performed with attention to mortality and pretransplant factors in three hematopoietic cell transplantation centers between 2000 and 2005 in Japan. Patients underwent transplantation from sibling (n=200) or unrelated donors (n=104) with myeloablative (n=188) or nonmyeloablative conditioning (n=116). Pretransplant factors and the hematopoietic cell transplantation-specific comorbidity index (HCT-CI: Sorror et. al.) were assessed with general linear model (GML) analysis.

RESULTS: Mortality before 1 year occurred in 145 of 303(48.3%) transplant recipients. The number of prior chemotherapy regimens and the HCT-CI were found to be useful predictors of early mortality. Kaplan-Meier estimates of survival as stratified by the HCT-CI (Fig.1) and the number of prior chemotherapy (Fig.2). The HCT-CI were useful for predicting outcomes. However, the sample size of patients with scores of 1 or more, captured by the HCT-CI did not exceed 15%. Futher, the number of prior chemotherapy regimens showed better suvival prediction.

CONCLUSION: The number of prior chemotherapy regimens will be a promising tool to identify patient at risk for all-cause mortality during first 1 year after allogeneic HCT.

Figure Figure
A Cellular Proteome Map of Human Multiple Myeloma.
Authors: Drach Johannes , Slany Astrid , Sagaster Verena , Gundacker Nina , Haudek Verena , Odelga Viktoria , Wimmer Helge , Zielinski Christoph , Gerner Christopher
Published: October 2019

Background and Aims: Molecular profiling identifies proteins characteristically deregulated in malignant diseases. Characteristic biomarkers may be useful to support diagnosis and patient stratification, while the recognition of aberrant cell activities and cell survival strategies may lead to the development of specifically designed pharmacologic strategies. We therefore performed proteomic profiling of primary human multiple myeloma (MM) cells in order to define the impact of protein expression abnormalities in MM.

Methods: Plasma cells were isolated from bone marrow of patients with MM or MGUS and forwarded to proteome analysis based on 2D gel electrophoresis in addition to shotgun analysis by nano-LC-MS/MS. Erythrocytes, platelets and plasma as well as quiescent and activated lymphocytes, monocytes, endothelial and dendritic cells from healthy donors were processed in an identical manner for comparative analysis. The resulting data were interpreted with the aid of a homemade SQL database.

Results: Among about thousand proteins identified in MM cells, we found aberrant expression of proteins involved in fatty acid beta-oxidation (VLCAD), unfolded protein response/ER-stress (ARMET protein, cytosol aminopeptidase), oxidative stress (ste20/oxidant stress responsive kinase 1), interferon response (MX1), iron uptake (transferrin receptor/CD71), DNA modification (transforming protein ERG, methyltransferase-like protein 7A), apoptosis and survival (apoptosis-inducing factor 1, hsp75), protein synthesis (ribosome-binding protein, Unc-13 D), cell adhesion (CD9/tetraspanin-29, LYRIC/metastatic adhesion protein), signaling (sts-1) and cell-cell interaction (Cystatin F, basigin/collagenase stimulatory factor, stem cell growth factor, small inducible cytokine B7, PD-ECGF/Gliostatin).

Conclusion: The presently applied proteome analysis strategy, based on the systematic investigation of purified primary human cells, allowed us to find characteristic alterations in MM cells. Several proteins directly relate to known aberrant cell activities such as elevated protein synthesis and secretion resulting in ER-stress, which makes the cells sensitive to proteasome inhibitor treatment. Work is in progress to use quantitative assessment of such proteins for patient stratification, identification of response predictors, and biomarkers for the distinction of MM and MGUS.
Overcoming Graft Rejection in Adult Patients with High-Risk Severe Aplastic Anemia Using Supplemental CD34+-Enriched PBSC in Combination with BM.
Authors: Cho Byung-Sik , Kim Sung-Yong , Kim Yoo-Jin , Eom Ki-Seong , Kim Hee-Je , Lee Seok , Min Chang-Ki , Kim Dong-Wook , Lee Jong-Wook , Min Woo-Sung , Kim Chun-Choo
Published: October 2019

The transplantation of a large number of stem cells can overcome the graft rejection with the pitfall of an increased incidence of graft-versus host disease (GVHD). We analyzed the clinical outcome of adult patients with severe aplastic anemia (SAA) at high risk for graft rejection who received a transplant with both bone marrow (BM) and CD34+-enriched peripheral blood stem cells (PBSC) to decrease the graft rejection. Between December 1999 and April 2006, 31 consecutive adult patients with SAA who were multi-transfused (more than 40 units of red blood cells ± platelets) and/or had a 3 years or longer period prior to transplant were investigated. The median age of patients was 39 years (range, 21–52) and 19 (61.3%) were women. The median interval from diagnosis to transplant was 62 months (range, 2–347). The median number of transfusions before transplant was 144 units (range, 20–680). Twenty-three (74.2%) patients received a prior immunosuppressive treatment and 3 (9.7%) prior allogeneic transplants. All patients received both BM and CD34+-enriched PBSC from sibling donors. PBSC were collected on days 5 and 6 of G-CSF (10μg/kg/day), and T cells were depleted using magnetic cell sorting (CliniMACS system, Miltenyi Biotec, Germany). Marrow harvesting was performed 48 hours after the last leukapheresis. Conditioning regimens consisted of cyclophosphamide/antithymocyte globulin + procarbazine (n=19) or fludarbine (n=9). Other 3 patients with a history of prior transplant received irradiatin based conditining regimens. GVHD prophylaxis consisted of cyclosporin and short-term methotrexate. Patients were transplanted with a median mononuclear cell dose of 1.35 (range, 0.43–4.05)×108/kg of body weight. The median CD34+ cell counts in the CD34+-enriched PBSC and unpurged BM were 7.99 (range 2.26–23.49)×106/kg and 4.41 (range, 1.34–15)×107/kg, respectively. With a median follow-up of 66 months, the 5-year probability of survival was 87.1%. The median time to neutrophils ≥0.5×109/L and platelet ≥20×109/L without platelet transfusions were 13 days (range, 10–32) and 16 days (range, 11–40), respectively. No patient developed graft failure. The cumulative incidence of acute GVHD over grade II and chronic GVHD were 9.7% and 16.1%, respectively. In conclusion, high rates of durable engraftment and excellent long-term survival were achieved by our approach which seems to play a role in overcoming the sensitization to histocompatibility antigens. Remarkably, the T-cell depleted PBSC apparently did not increase the incidence of GVHD. The high dose stem cell component therapy may be feasible for high-risk SAA adult patients.
Phenotypic and Functional Features of Vγ9/Vδ2 T Cells in Chronic Lymphocytic Leukemia Are Strongly Correlated with the Mutational Status of the Immunoglobulin Heavy Chain Variable Region (IgVH) and Metabolic Activity of the Mevalonate Pathway in Tumor Cells.
Authors: Coscia Marta , Peola Silvia , Matta Giorgia , Pantaleoni Francesca , Foglietta Myriam , Angelini Daniela , Chiaretti Sabina , Riganti Chiara , Guarini Anna , Drandi Daniela , Ladetto Marco , Bosia Amalia , Foà Robin , Battistini Luca , Boccadoro Mario , Fournie Jean Jacques , Massaia Massimo
Published: October 2019

Vγ9/Vδ2 (γδ) T cells have an important effector and regulatory role in innate and adaptive immunity against microbes, stressed cells and tumor cells. They represent less than 5% of peripheral blood lymphocytes, but can be activated and expanded in vitro by aminobisphosphonates (ABP) like zoledronic acid (Zol), as surrogates of their natural ligands. In this study, we have investigated the proliferative response of γδ T cells to Zol in 59 patients with chronic lymphocytic leukemia (CLL) at diagnosis. Proliferation of γδ T cells was observed in 30 patients (51%)(responders, R), whereas 29 patients (49%) were non-responders (NR). γδ T-cell subset distribution [e.g., naive (TN), central memory (TCM), effector memory (TEM), and terminally differentiated effector memory (TEMRA)] was well balanced in R patients, whereas TEM and TEMRA were largely predominant in NR patients. TEMRA of NR patients had a unique profile of NK receptors expression, characterized by the expression of the ILT4 inhibitory receptor, whereas R patients tended to have an higher expression of the costimulatory molecule NKG2D. Notably, HLA-G, a negative prognosticator in CLL and a well known ILT4 ligand, was more expressed in tumor cells of NR patients. This correlation prompted us to determine whether other tumor cell features were different in R and NR patients. Indeed, the latter showed significantly higher tumor cell counts, higher frequency of poor-risk cytogenetic abnormalities, and a significantly shorter median time to first treatment. Lastly, a very significant association was observed with the mutational status of the tumor immunoglobulin heavy chain variable region (IgVH), which was mutated (M) in 96% of R patients, and unmutated (UM) in 74% of NR patients. To gain further insight into this association, we evaluated the activity of the mevalonate pathway in tumor cells of M and UM patients. This pathway, that can be targeted by Zol, generates the phosphoantigens naturally recognized by γδ T cells. Bioinformatic and biochemical approaches showed that this pathway is more active in tumor cells of UM than M patients. Based on these data, we propose that tumor cells of UM CLL patients can more easily engage γδ T cells and, in the long run, exhaust their immune surveillance potential by driving their differentiation into functionally impaired TEMRA. In conclusion, we have identified a novel mechanism of immune escape which can contribute to the disease progression in UM CLL patients.
Tumor Associated Macrophages (TAM) in Skeletal Plasmacytomas of Patients with Multiple Myeloma.
Authors: Russell Stephen J. , Dogan Ahmet , Ong Hooi Tin , Vrana Julie , Dispenzieri Angela , Dietz Allan B. , Peng Kah-Whye
Published: October 2019

In multiple myeloma, the neoplastic plasma cells are either dispersed throughout the red marrow spaces of the axial and proximal appendicular skeleton or concentrated in discrete, well vascularlized plasmacytomas that stimulate osteoclastic bone resorption and erode into cortical bone. Interactions between bone marrow stromal cells (BMSC) and myeloma cells are clearly important in the pathogenesis of myeloma that diffusely infiltrates the bone marrow and where plasmacytomas are in contact with the bone marrow space. However, these interactions cannot be of major relevance at the centers of expanding plasmacytomas since these sites are devoid of osteoclasts, osteoblasts and other BMSC. Histological sections of skeletal plasmacytomas from 22 patients with multiple myeloma were therefore examined after immunostaining with anti-macrophage antibodies. Hematoxylin and eosin stained sections showed sheets of plasma cells interspersed with numerous small blood vessels. Immunostained sections revealed abundant CD68+, CD163+ macrophage infiltrates in all tumors, and scattered collections of CD3+ T lymphocytes. Tumor-associated macrophages (TAM) accounted for 2 to 12 percent of nucleated cells in these plasmacytomas and were evenly distributed through the parenchyma or clustered around CD34+ blood vessels. In general the TAM had dendritic morphology, and each dendrite was in close contact with multiple plasma cells. In a small percentage of cases, there was a subpopulation of TAM strikingly clustered around CD34+ positive blood vessels. However, vascular mimicry was not seen. Abundant murine CD68+ TAM were also found in subcutaneous KAS6/1 and MM1 myeloma xenografts in SCID mice. To determine whether cells of the monocytic lineage might be exploitable as carriers for systemic delivery of therapeutic agents to plasmacytomas, monocyte-derived dendritic cells were infected with oncolytic measles viruses and administered intravenously to SCID mice bearing subcutaneous KAS6/1 xenografts. Immunostaining for human MHC II antigen revealed that the infused DCs could localize to the KAS6/1 tumors where they heterofused with and transferred MV infection to the myeloma cells. This is the first report of TAM in skeletal plasmacytomas of myeloma patients. Further research into the role of TAM in supporting myeloma growth is warranted since they may offer many potential opportunities for the development of novel approaches to myeloma therapy.
Regulation of Chronic Lymphocytic Leukemia Cell Proliferation by CTLA4.
Authors: Mittal Amit K. , Rohlfsen Rae A. , Joshi Avadhut D. , Hegde Ganapati V. , Bociek R. Gregory , Joshi Shantaram S.
Published: October 2019

B-cell chronic lymphocytic leukemia (CLL) is the most common adult leukemia with heterogenous clinical course. Prognostic markers such as CD38 expression has proven to be reliable in predicting clinical outcome where high CD38 expression correlates with poor prognosis and low CD38 expression is associated with good prognosis. Recently, our lab has shown that expression of CTLA4 in CLL cells is inversely correlated with CD38 expression using microarray profiling and real time PCR. Microarray analysis showed significant differential expression of CTLA4 between high and low CD38 subgroups (n=49) (Joshi et al. Clinical Cancer Research, 2007, in press). However, whether CTLA4 plays a direct role in CLL pathogenesis or not is unknown. Therefore to study the potential role of CTLA4 in CLL, CTLA4 expression was down regulated in primary CLL cells from six different patients using antisense oligonucleotide. The down regulation of CTLA4 expression was confirmed by RT-PCR analysis. The proliferative capability of the CTLA4-down regulated CLL cells was determined using MTT assay as well as radiolabeled thymidine uptake assay at 24, 48 and 72 hours in culture. There was a significant increase in the proliferation of CTLA4-down regulated CLL cells compared to untreated control CLL cells or CLL cells treated with an irrelevant oligonucoeotide. To further understand the CTLA4 mediated regulation of CLL cell proliferation, expression levels of downstream target molecules of CTLA4 such as NFATC2 and c-FOS was studied using RT-PCR. There was a significantly increased expression of target genes NFATC2 and c-FOS, molecules in the CTLA4-downregulated CLL cells compared to control CLL cells. In addition, further analyses of the microarray data revealed that CD38 high expressing CLL cells (n=22) has significant (p<0.05) higher NFATC2 and c-FOS expression compared to CD38 low expressing CLL cells (n=29). This was further confirmed by real time PCR analyses (figure1). Thus, these results clearly demonstrate that CTLA4 is involved in the regulation of proliferation of CLL cells via regulating the expression of NFATC2 and c-FOS, the downstream signaling molecules involved in the cell proliferation. (This work was supported by the CLL Foundation, Houston, TX).

Figure Figure
Osteopontin in Hemophilic Synovitis.
Authors: Hakobyan Narine , Kaushal Megha , Enockson Candace , Valentino Leonard A.
Published: October 2019

Introduction Hemophilia is a congenital bleeding disorder due to the deficiency of blood coagulation factor VIII or IX. The joint is the most common site of serious bleeding. Repeated episodes of hemarthrosis lead to hemophilic synovitis (HS) and destructive hemophilic arthropathy (HA). The molecular changes responsible for HS and HA are not known. Osteopontin (OPN) has been implicated in the process that leads to bone destruction in other forms of arthritis. We hypothesized that OPN plays a similar role in HA.

Methods Hemophilic synovitis was induced in factor (F) VIII and IX deficient mice by causing traumatic (blunt trauma) and non-traumatic (needle puncture) hemarthrosis in the right knee joint of mice. The concentration of OPN was measured in plasma of mice five days after inducing hemorrhage. Expression of OPN in synovial tissue from affected knee joints was compared with control uninjured joints by DNA microarray and semi-quantitative RT-PCR analysis on different days following joint injury.

Results OPN concentration in the plasma of both FVIII and FIX deficient mice sacrificed 5 days after a single capsular puncture was increased 92–98% compared to control mice. In other experiments, traumatic hemarthrosis was induced three times at weekly intervals in the right knee joints of FVIII deficient mice. On day 21, synovial tissue from the knee joints of 20 mice was isolated, pooled and RNA from affected and control joints probed using microarray. The expression of OPN in right knee tissue was 7.5-fold greater compared to the left. These data were confirmed by RT-PCR analysis which showed that OPN was highly expressed in synovial tissue from mice 14, 21 and 30 days after single puncture injury and 21 days following three traumatic injuries.

Conclusion The concentration of OPN was increased in the plasma of mice following hemarthrosis and OPN expression was increased 7-fold in synovial tissue of mice with hemophilic arthropathy. Our data implicate OPN in the pathobiology of hemophilic arthropathy.

Figure Figure Figure Figure
Experience with a Modified Nijmegen-Bethesda Method for Measurement of Inhibitors in Hemophilia Patients: The CDC Inhibitor Surveillance Pilot Project.
Authors: Miller Connie H. , Platt S. Jean , Abshire Thomas , Brettler Doreen , Brockenstedt Paula , DiPaola Jorge , Massey Gita , Neff Anne , Shapiro Amy , Tarantino Michael , Wicklund Brian , Creary Melissa , Soucie J. Michael
Published: October 2019

A pilot project of a prospective surveillance system for coagulation factors VIII and IX inhibitors has been initiated at 9 U.S. Hemophilia Treatment Centers (HTCs). More than 500 patients have been enrolled. Risk factor and product exposure data are recorded at each HTC. A blood specimen is collected upon entry, annually, at product switch, or for clinical indication and tested centrally at the CDC, using a modified Nijmegen-Bethesda method. Plasma from specimens collected in 4.5mL evacuated tubes containing 3.2% sodium citrate is shipped overnight on cold packs. Specimens are heated to 56oC for 30 minutes to remove endogenous and infused FVIII and centrifuged. Specimens are initially screened for inhibitor using a single dilution of 3 parts patient plasma to 1 part normal pool plasma buffered with imidazole to pH 7.4 (BNPP). Specimens showing inhibition and those from previously positive patients are tested in multiple dilutions at 1 part patient dilution to 1 part BNPP. Dilution is in naturally FVIII-deficient plasma containing normal von Willebrand factor. After a 2-hour incubation at 37oC, FVIII remaining in the patient mixture is divided by FVIII remaining in a 1:1 mixture of BNPP and FVIII-deficient plasma and expressed as % residual activity (RA). %RA is converted to Nijmegen-Bethesda units (NBU) using a curve with one NBU equal to 50% RA. An inhibitor plasma of known titer is run with each assay as positive control. Split specimens shipped frozen and on cold packs showed a correlation of 0.998. 3:1 and 1:1 mixtures showed a correlation of 0.97. Almost 50% of the first 200 specimens received had measurable FVIII activity. The heating step was introduced to remove FVIII without damaging the antibody. 65 specimens went from >100% RA to a titer of 0–0.2 NBU after heating. Among 538 specimens tested for FVIII inhibitors, 435 (81%) were from patients with no previous history of inhibitor (shown below). 429 (98.6%) were < 0.5 NBU. The 6 specimens (1.4%) with > 0.6 NBU are under investigation as possible seroconversions.

Nijmegen-Bethesda Units in Patients with No Previous Inhibitor NBU No. Pts. (%) NBU No. Pts. (%) NBU No. Pts. (%) 0 220 (50.6%) 0.4 3 (0.7%) 0.8 1 (0.2%) 0.1 127 (29.2%) 0.5 0 0.9 0 0.2 59 (13.6%) 0.6 0 1.0–4.0 2 (0.5%) 0.3 20 (4.6%) 0.7 2 (0.5%) 5.0–19.0 1 (0.2%)

Among 121 specimens tested for factor IX (FIX) inhibitors, 113 were from patients with no previous history of inhibitor and all had NBU <0.2, using a comparable FIX inhibitor assay. Testing in a central laboratory by uniform methods will facilitate detection of new inhibitors and investigation of risk factors.
Epitope Identification and Generation of an Antigen-Specific T-Cell Clone in a Mild Hemophilia A Inhibitor Subject with Missense Genotype R593C.
Authors: James Eddie A. , van Haren Simon D. , Ettinger Ruth A. , Thompson Arthur R. , Kwok William W. , Voorberg Jan , Pratt Kathleen P.
Published: October 2019

The development of inhibitory anti-FVIII antibodies is a major clinical problem in hemophilia A. While less common in mild/moderately severe patients, the relative risk of inhibitor formation is elevated in patients with missense mutations in the FVIII A2 domain, especially those with an R593C genotype. In this study, T-cell responses to 14 FVIII A2 domain peptides with predicted DRB1*1101 MHC binding motifs were investigated using MHC class II tetramer reagents. CD4+ T cells were isolated from a hemophilic subject (DRB1*1101, 1302) with FVIII missense genotype R593C and stimulated with pooled peptides. This subject had developed a longstanding low titer inhibitor after receiving multiple FVIII infusions. Staining with fluorescent, peptide-loaded tetramers revealed that the hemophilic subject, but not an HLA-matched healthy control, had a DRB1*1101-restricted response to peptide A2589–608, which contained the wild-type R593 sequence. MHC class II tetramers with bound A2589–608 were used to sort antigen-specific T cells and then generate a T-cell clone recognizing this high avidity DRB1*1101-restricted epitope. FVIII residues 594–602 (FLPNPAGVQ) comprise a predicted high-affinity binding motif. Peptide binding assays confirmed that A2589–608 bound to recombinant, monomeric DRB1*1101 protein with high affinity. A peptide with the corresponding hemophilic sequence (A2589–608, 593C) bound with affinity that was threefold lower, but in a range that should be sufficient for presentation to T cells. The other 12 A2 peptides had a wide spectrum of binding affinities for the DRB1*1101 protein. Interestingly, prediction algorithms suggest that the peptides containing residue 593 occupy the MHC Class II peptide-binding groove in a register that places this residue just outside the p1 anchor position. Thus we anticipate that residue 593 modulates T-cell recognition of peptide A2589–608. This provides a potential explanation for inhibitor development in mild hemophilia A patients with a FVIII R593C genotype who are HLA-DRB1*1101. Immunogenicity of this FVIII region with respect to other HLA types is under investigation.
U.S. Inhibitor Pilot Project: Participant Characteristics and Infusion Log Adherence.
Authors: Creary Melissa S. , Soucie John M. , Miller Connie H. , Hooper W.C. , Abshire Thomas C. , Brettler Doreen B. , Pockenstedt Paula L. , DiPaola Jorge , Massey Gita , Neff Anne T. , Shapiro Amy D. , Tarantino Michael D. , Wicklund Brian M. , DiMichele Donna ,   Hemophilia Treatment Centers
Published: October 2019

Inhibitors are now considered to be the most important complication of clotting factor replacement therapy in hemophilia. In January 2005, the Universal Data Collection (UDC) project, which includes data on over 14,000 patients with hemophilia A or B, was amended to include prospective data collection on inhibitors. We report interim results of a pilot project involving 9 selected Hemophilia Treatment Centers in the U.S. Dedicated data managers collected risk factor and product exposure data from enrolled patients on a monthly basis. Tracked infusion log submissions were used to estimate patient adherence to infusion log completion. A blood specimen is collected at baseline, annually, at product switch, or for clinical indication and is tested centrally for inhibitor using the Nijmegen modification method. Results are reported and any above a threshold value are followed up with repeat testing and clinical correlation. As of August 2007, 514 male patients with hemophilia, ages 2–84 years (mean 23 yrs SD 17) had been enrolled. Of these, 415 (81%) had FVIII deficiency (64% severe, 20% moderate, 16% mild) and 99 (19%) had FIX deficiency (35%, 43%, and 22%, respectively). 85% of the patients were caucasian, 8% black, and 4% Hispanic. A previous history of inhibitor was present at enrollment in 71 (14%). Prophylactic treatment was used by 37% and 80% received product by home infusion. Historical exposure to product collected at enrollment was as follows: 14% had 0–20 exposure days (ED), 12% had 21–100 ED, 7% had 101–150 ED, and 64% > 150 ED. During follow-up, 6 patients have had inhibitor titer measurements over 0.4 BU. One of these was known to have an active inhibitor at enrollment. Confirmatory testing of remaining cases is underway. Mean adherence rate for patient infusion log completion in the 9 HTC’s was 45% (range 7% to 74%). Overall adherence increased with time on study from about 40% at one month to nearly 60% for those enrolled at least 10 months. In summary, enrolled participants represent a wide range of characteristics including age, race, disease severity and number of factor exposures. Interim findings indicate that prospective collection of factor infusion data from hemophilia patients necessary to appropriately monitor inhibitor formation, is feasible but labor intensive and requires patient adherence to completion and submission of accurate infusion logs. Because inhibitor formation is rare and the potential risk factors are many, large scale studies with long-term follow-up will be needed to fully assess risk factors for inhibitors.
Improved Therapy of Multiple Myeloma by Combining Milatuzumab (Anti-CD74 Humanized mAb) and Bortezomib: In Vitro and In Vivo Results.
Authors: Stein Rhona , Goldenberg David M.
Published: October 2019

Background: The humanized anti-CD74 monoclonal antibody, milatuzumab (hLL1, or IMMU-115; Immunomedics, Inc, Morris Plains, NJ), is in clinical evaluation for therapy of multiple myeloma (MM) after preclinical evidence of activity in this tumor type (Stein et al, Blood2004;104:3705). Here we examine the ability of milatuzumab to increase the efficacy of drugs in MM cell lines.

Methods: MTT cytotoxicity assays were performed on a panel of MM cell lines, including CAG, KMS11, KMS12-PE, and MC/CAR, to examine the effects of bortezomib, doxorubicin (dox), and dexamethasone (dex) alone and combined with milatuzumab or milatuzumab + crosslinking 2nd Ab (goat anti-human IgG, GAH). In vivo studies used a CAG-SCID mouse model of disseminated disease.

Results: Without drugs, crosslinked milatuzumab, but not milatuzumab alone, yielded significant anti-proliferative effects on the four MM cell lines. In combination studies, crosslinked milatuzumab produced significant reductions in the IC50 values of the anti-MM drugs. For example, in CAG, milatuzumab+GAH decreased the IC50 values 58%, 78%, and 98% for bortezomib, dox, and dex, respectively (P=0.0034, 0.0073, and 0.078, respectively). In vivo, milatuzumab at 100 μg/injection, 2x weekly for 4 weeks, starting 1 day after injection of CAG cells, more than doubled the median survival time (MST) from 42 days in untreated CAG-bearing SCID mice to 103 days. Combination therapy with milatuzumab and bortezomib or dox was compared to milatuzumab alone, with treatments initiated 5 days after injection of CAG cells. Bortezomib alone (1.0 mg/kg) increased MST from 33 to 44 days (P=0.0021 vs. untreated). Treatment with milatuzumab alone (100 μg/mouse) increased the MST to 73 days (P<0.0001 vs. untreated). When bortezomib and milatuzumab treatments were combined, the MST increased to 93 days (P=0.0441 vs. milatuzumab and P=0.0065 vs. bortezomib). Thus, the combination of milatuzumab and bortezomib increased survival significantly compared to either single treatment. Given alone, dox yielded little or no effect on survival compared with untreated animals, and there was no significant difference between milatuzumab monotherapy and milatuzumab plus doxorubicin in this model. In contrast, a milatuzumabdox immunoconjugate was found to be a highly effective therapeutic agent, with all mice achieving long-term survival. The inhibition of the NF-κB survival pathway of B-leukemic cells by milatuzumab supports its complementary effects when combined with drugs having different mechanisms of action, such as bortezomib.

Conclusions: The therapeutic efficacies of bortezomib, dox, and dex are enhanced in vitro in MM cell lines when given in combination with milatuzumab. In vivo, milatuzumab alone or especially in combination with bortezomib is highly effective in MM. (Supported in part by USPHS grant P01CA103985 from the NCI, and grants from the Thomas and Agnes Carvel Foundation and the Walter and Louise Sutcliffe Foundation.)
Preliminary Results of CNTO 328, an Anti-Interleukin (IL)-6 Monoclonal Antibody (mAb), in Combination with Bortezomib in the Treatment of Relapsed or Refractory Multiple Myeloma.
Authors: Manges Robert F. , Sutherland Heather J. , Jagannath Sundar , Orlowski Robert Z. , La Police David , DeWitte Mark , Lang Zhihui , Corringham Robert E. , Rossi Jean Francois
Published: October 2019

Background: Bortezomib represents a significant advance in the treatment of relapsed/refractory myeloma, but its efficacy is limited by a number of resistance mechanisms including activation of the anti-apoptotic heat shock protein (HSP) and stress response pathways. CNTO 328 is an anti-IL-6 chimeric monoclonal antibody shown to have anti - myeloma activities in vitro. Because IL-6 signaling augments the HSP response, downregulation of IL-6 signaling may enhance bortezomib’s clinical anti-myeloma activity. Pre-clinical studies demonstrated that this combination had an additive to synergistic effect in inducing apoptosis in IL-6-dependent and independent multiple myeloma cell lines (Voorhees et al., 2007, in press).

Methods: In this safety lead-in cohort of a pivotal trial (clinicaltrials.gov), CNTO 328, 6 mg/kg, was administered by IV every 2 weeks in combination with bortezomib, 1.3 mg/m2, given by IV on days 1, 4, 8, and 11 every 3 weeks. Pateints received a maximum of four, 6-week treatment cycles, after which the bortezomib schedule was reduced to four, once weekly doses in a 5-week maintenance cycle. When disease progression was documented, oral dexamethasone was added at 20 mg and given on the day of, and day after each bortezomib infusion, for a maximum of two cycles. All later cycles included 20 mg dexamethasone given only on the day of bortezomib administration.

Results: Preliminary results from the first 6 patients are available. Pateint characteristics included median age of 68 years; B2M > 3.0 mg/L, (n = 4 pateints); CRP > 3.0 mg/L (3); a median of 2 prior regimens (range 1 – 3), and prior bone marrow or peripheral blood stem cell transplantation in three patients. Using the EBMT response criteria, three patients achieved confirmed partial responses (PRs). Additionally, two patients had unconfirmed PRs. One patient discontinued prior to confirmation, while a follow up assessment for the other patient is pending. The median number of cycles administered was 3. Two of the patients ended treatment prematurely, 1 due to disease progression and 1 due to adverse events. The remaining 4 patients continue on study. Grade 3/4 adverse events (AEs) considered possibly related to CNTO 328 and bortezomib include neutropenia (2 patients), leukopenia (1), lymphopenia (1), and bloody diarrhea (1).

Conclusion: Treatment with anti-IL-6 CNTO 328 in combination with bortezomib has been evaluated for safety and efficacy in the first 6 patients of an ongoing pivotal trial for the treatment of relapsed or refractory multiple myeloma. Initial activity observed with this combination is encouraging, and enrollment is continuing to explore its full potential.
c-Myb Regulates CD9 Gene Expression during Megakaryopoiesis.
Authors: Mukai Harumi Y. , Kono Tomoko , Motohashi Hozumi , Yamamoto Masayuki , Kojima Hiroshi
Published: October 2019

The nuclear proto-oncogene c-myb plays crucial roles in the growth, survival, and differentiation of hematopoietic cells. We previously generated through insertion mutagenesis a c-myb gene knockdown (KD) line of mice. In the mice transgene was inserted 77-kb upstream of the c-myb gene and c-Myb expression was markedly decreased in megakaryocyte-erythrocyte lineage-restricted progenitors (MEPs) of the homozygous knockdown mutant mice (c-myb KD mice). The c-myb KD mice exhibited anemia, thrombocythemia, and splenomegaly and these abnormalities were reproducible in a co-culture assay of MEPs with OP9 cells, but abrogated by the retroviral expression of c-Myb in MEPs. To understand the transcriptional program that accompanies the decline of c-myb gene expression, we performed DNA microarray analysis with MEPs and identified 74 genes that are upregulated and 36 genes that are downregulated in the c-myb KD mice. Of these genes, expression levels 15 genes are actually changed significantly in bone marrow cells of the c-myb KD mice. These genes harbor c-Myb recognition elements in their regulatory regions. Especially, we found that the CD9 expression was upregulated in the c-myb KD mice. Reverse correlation of c-Myb expression with the CD9 gene expression was verified using a luciferase reporter assay and chromatin immunoprecipitation assay. Agonistic antibody of CD9 stimulated megakaryocytic colony formation. On the contrary, upon the bone marrow suppression with 5-fluorouracil recovery of platelet number was delayed in the CD9-null mice. Furthermore, proplatelet formation was impaired when we used CD9-null mouse megakaryocytes, and the size of proplatelets was smaller than those generated by wild-type megakaryocytes. These results thus demonstrate that c-Myb suppresses the CD9 expression in a steady-state condition, while in the stress megakaryopoiesis CD9 is derepressed and acts to induce the megakaryopoiesis. Elucidation of c-myb-based transcription network seems to be of important to understand the megakaryocytic differentiation.