ALBERT

Description: PROs provide patients’ own appraisal of their health status and can be used to measure aspects of health that are generally not captured by ‘traditional’ clinical measures such as physician assessments and test results. This study analyzed PRO data from a multicenter, pivotal phase II trial of bortezomib in 202 patients (pts) with relapsed, refractory MM (NEJM2003;348:2609–17). PRO questionnaires (EORTC-QLQ C30 and MY24, FACT/GOG-Neurotoxicity (Ntx) and FACIT-Fatigue subscales) were administered at the screening visit (baseline) and day 1 of cycles 3, 5, 7 and at the end of the study. Results: In this population with relapsed, refractory MM, poorer baseline pre-treatment multi-dimensional quality of life (QoL) scores were significantly correlated (range of r= 0.22 to r= 0.77) with fatigue, pain, dyspnea, appetite loss, neurotoxicity, MM disease symptoms, and treatment side effects. Clinical response to bortezomib (Complete Response or Partial Response) was associated with statistically significant (p

Description: The purpose of this study was to determine the facility and reliability of the World Health Organization (WHO) classification of myelodysplastic syndromes (MDSs) with several observers reviewing the same diagnostic specimens. We also wanted to determine if the WHO classification provided additional information about predictability of clinical response outcome. To accomplish these goals we reviewed 103 previously diagnosed cases of low-risk MDS. We found 92% interobserver agreement (P 〈 .001). Sixty-four of these patients had been entered into clinical trials using growth factors by the Nordic MDS Study Group. The WHO classification reliably predicted therapeutic response to the combination of granulocyte colony-stimulating factor (G-CSF) and erythropoietin (Epo). The response rate differed significantly between refractory anemia with ringed sideroblasts (RARS) and refractory anemia with multilineage dysplasia and ringed sideroblasts (RCMD/RS) with regard to therapeutic response (75% versus 9%; P = .003). Also, in the group of patients with less than 5% marrow blasts, there was a difference in median survival between patients with unilineage dysplasia (51% surviving at 67 months) and those with multilineage dysplasia (median survival, 28.5 months; P = .03). (Blood. 2004;103:3265-3270)

Description: Rituximab proved to be effective in relapsed and refractory indolent NHL as a single agent and generated impressive results in phase II studies in combination with chemotherapy. In a prospective randomized trial we compared the efficacy and toxicity of rituximab (375 mg/m² d 1) plus MCP-chemotherapy ( mitoxantrone 8 mg/m² d 3 + 4, chlorambucile 3 x 3 mg/m² d 3 – 7, prednisolone 25 mg/m² d 3 – 7 ) given every 28 days for a total of 8 cycles versus MCP (d 1 – 5) x 8 cycles alone in advanced indolent NHL and MCL. Efficacy endpoints included overall and complete response rates, event free survival, progression free survival, overall survival and toxicity. For response assessment classical definitions have been used. Between 10/98 and 09/03 we randomized 358 patients (pts) with advanced stage follicular lymphoma (FL) (grade 1 + 2), lymphoplasmacytic lymphoma and MCL to either R-MCP or MCP. The study arms are well balanced for all demographic factors. 201/358 pts (56%) had FL. Both regimens were well tolerated with a low incidence of serious adverse events. The overall response rate (RR) and the complete response rate (CR) for all pts was 85,5% and 42% in the R-MCP arm versus 65,5% and 20% in the MCP arm (p

Description: During lymphoid development, Notch1 plays a critical role in the T-cell/B-cell lineage decision, while Notch2 is essential for marginal zone B-cell (MZB) development. Notch pathway activation induces translocation of intracellular Notch (ICN) to the nucleus, where it interacts with the transcription factor CSL (CBF1/RBP-Jk, Suppressor of Hairless, Lag-1). In vitro, ICN binds Mastermind-like proteins, which act as potent Notch coactivators. Three MAML family members (MAML1-3) have been identified in mammals, but their importance in vivo is unknown. To investigate the function of MAMLs in hematopoietic development, we introduced a dominant negative (DN) mutant of MAML1, capable of inhibiting Notch1-4, in murine hematopoietic stem cells. DNMAML1 resulted in early inhibition of T-cell development and the appearance of intrathymic B cells, phenotypes consistent with Notch1 inhibition. The T-cell differentiation block was as profound as that produced by enforced expression of the Notch modulator Deltex1. In DNMAML1-transduced spleen cells, a dramatic decrease in MZB cells was present, consistent with Notch2 inhibition. In contrast, Deltex1 did not decrease MZB cell numbers. These results suggest a critical role for MAMLs during Notch-mediated cell fate decisions in vivo and indicate that DNMAML1, but not Deltex1, can be used to interfere with the function of multiple Notch family members. (Blood. 2004;104:1696-1702)

Description: Stage of the disease at transplant is critical for outcome after unrelated donor umbilical cord blood transplantation (UD-UCBT). The results of UD-UCBT in adults transplanted early in the course of their disease are unclear. Thus, UD-UCBT remains as the last resort for most patients. The major aim of this report was to study the outcome of a series of adult patients with hematologic malignancies undergoing UD-UCBT early in the course of their disease in a single institution. From May 1997 to May 2004, 40 patients in early disease stages underwent UD-UCBT. All patients received thiotepa, busulfan (orally in 29, intravenously in 11), cyclophosphamide, and antithymocyte globulin (Lymphoglobulin in 24 and Thymoglobulin in 16) as conditioning, cyclosporine and prednisone for graft-versus-host disease (GVHD) prophylaxis, and filgrastim to fasten engraftment. Diagnosis were chronic myeloid leukemia in chronic phase in 14 cases, high-risk acute lymphoblastic leukemia in 14 (12 in CR1, 1 in CR2, and 1 in CR3), high-risk acute myeloblastic leukemia in 8 (7 in CR1 and 1 in CR2), and high-risk myelodysplastic syndrome in 4 (3 untreated and 1 in CR1). Median age was 27 years (range, 16–46). The degree of HLA match (HLA-A and -B by serology and -DRB1 by high-resolution DNA typing) was 6/6 in 2 (5%), 5/6 in 18 (45%), and 4/6 in 20 cases (50%). The median number of nucleated and CD34+ cells infused was 1.8 x 107/kg (range, 0.9–4) and 0.8 x 105/kg (range, 0.1–5.7) respectively. Median time to PMN above 0.5 x 109/L and to platelets above 20 x 109/L was 22 days (range, 13–44) and 69 days (range, 32–188), and the cumulative incidence of myeloid and platelet engraftment was 90% (95% CI, 81–99%) and 70% (95% CI, 57–86%), respectively. Time to myeloid engraftment showed a direct relationship with the number of CFU-GM and CD34 cells cryopreserved (P = .02 and .01 respectively) and infused (P = .0001 and .0004 respectively). Platelet engraftment was faster in patients receiving grafts with a higher number of CFU-GM (P = .005) and CD34+ cells (P = .04), in those receiving Thymoglobulin (P = .02) and in those not developing acute GVHD above grade II (P = .04). Eight patients (20%) developed acute GVHD above grade II, and 9 of 25 patients at risk had extensive chronic GVHD. Patients receiving Thymoglobulin had a lower risk of acute GVHD (P = .0003). With a median follow-up of 33 months (range, 3–87), the probability of disease-free survival (DFS) at 3 years was 48% (95% CI, 30–66%) and was related directly to age (P = .004) and inversely to the development of acute GVHD above grade II (P = .004). The probability of DFS at 3 years was 66 % for patients younger than 31 years and 54% for those not developing acute GVHD above grade II. Cell dose, degree of HLA mismatch, and diagnosis did not clearly influence DFS. These results compare to those obtained after matched unrelated donor bone marrow transplantation, and suggest that UD-UCBT is a reasonable first-line option for adults with hematologic malignancies requiring transplantation and lacking a HLA-matched sibling donor.

Description: Chronic graft-versus-host disease (cGVHD) is an increasingly common cause of morbidity and mortality in allogeneic stem cell transplantation (alloSCT). Relative to acute GVHD (aGVHD), much less is understood about cGVHD. Using the B10.D2 → BALB/c murine cGVHD model, which shares critical pathologic features with human cGVHD, we find that radiation-resistant host T cells regulate cGVHD. We initially observed that recipients lacking all lymphocytes developed accelerated and more severe cGVHD. Using genetically deficient recipients, we determined that αβ+CD4+ T cells were required to regulate cGVHD. Increased cGVHD severity was not due to the absence of T cells per se. Rather, the potency of regulation was proportional to host T-cell receptor (TCR) diversity. Only CD4+CD25+, and not CD4+CD25-, host T cells ameliorated cGVHD when added back, indicating that host T cells acted not via host-versus-graft activity or by reducing homeostatic proliferation but by an undefined regulatory mechanism. Thus, preparative regimens that spare host CD4+CD25+ T cells may reduce cGVHD. Donor CD4+CD25+ T cells also reduced cGVHD. Depletion of CD4+CD25+ cells from the inoculum exacerbated disease, whereas transplantation of additional CD4+CD25+ cells protected against severe cGVHD. Additional CD4+CD25+ cells also promoted healing of established lesions, suggesting that their effects persist during the evolution of cGVHD.

Description: Recombinant human activated protein C (rhAPC) is a natural anticoagulant with potentially important anti-inflammatory properties. In humans with severe sepsis, rhAPC treatment reduces mortality, but mechanisms responsible have not been well characterized. Accumulation of activated neutrophils in the lungs and other organs during severe infection contributes to sepsis-induced organ dysfunction, including acute inflammatory lung injury. Because neutrophils express an APC receptor, we hypothesized that immunomodulatory effects of rhAPC occur, in part, via modulation of neutrophil responses. To examine this issue, we performed a double-blinded, placebo-controlled study of rhAPC in a human model of endotoxin-induced pulmonary inflammation. Administration of rhAPC significantly reduced leukocyte accumulation to the airspaces, independent of pulmonary cytokine or chemokine release. Neutrophils recovered from bronchoalveolar lavage fluid of volunteers receiving rhAPC demonstrated decreased chemotaxis ex vivo. Decreased neutrophil chemotaxis following exposure to rhAPC was confirmed in vitro. No differences were detected in gene expression, kinase activation, cytokine release, cell survival, or apoptosis of neutrophils recovered in the presence or absence of rhAPC. These studies demonstrate that rhAPC reduces both endotoxin-induced accumulation of leukocytes in the airspaces and neutrophil chemotaxis. These rhAPC-induced effects on neutrophil function may represent a mechanism by which rhAPC improves survival in patients with sepsis. (Blood. 2004;104:3878-3885)

Description: Multiple myeloma (MM), a malignancy of plasma cells, develops in the bone marrow, and generates devastating bone destruction. Along with enhanced bone resorption, clinical evidence has also suggested suppression of bone formation as a contributing factor to the bone loss in MM. In contrast to recent understanding on mechanisms of osteolysis enahnced in MM, little is known about factors responsible for impaired bone formation. A canonical Wingless-type (Wnt) signaling pathway has recently been shown to play a critical role in osteoblast differentiation. Therefore, in the present study, we aimed to clarify mechanisms of suppression of osteoblast differentiation by MM cells with a particular focus on a canonical Wnt signaling pathway. Because several secreted Frizzled related protein (sFRP) and DKK family members are known as soluble Wnt antagonists, we first examined the expression of sFRP-1, 2 and 3 and DKK-1 in MM cell lines including U266, RPMI8226 and ARH77. All cell lines expressed sFRP-2 and sFRP-3 mRNA observed by RT-PCR. However, sFRP-1 was not expressed in any cell line, and Dkk-1 was expressed only in U266 cells at mRNA levels. We next conducted Western blot analyses for these factors and detected only sFRP-2 in immunoprecipitants of conditioned media as well as cell lysates of all these cell lines. However, no other factors were found at protein levels. Furthermore, sFRP-2 mRNA and protein expression was detected in most MM cells from patients with advanced or terminal stages of MM with bone destruction including plasma cell leukemia (3/4 and 8/10, respectively). In order to examine a biological role for sFRP-2, we added recombinant sFRP-2 to MC3T3-E1 cell culture together with BMP-2. Exogenous sFRP-2 partially suppressed alkaline phosphatase activity but almost completely mineralized nodule formation enhanced by BMP-2. Furthermore, sFRP-2 immunodepletion significantly restored mineralized nodule formation in MC3T3-E1 cells suppressed by RPMI8226 and ARH77 CM. These results suggest that sFRP-2 alone is able to suppress osteoblast differentiation induced by BMP-2 and that MM cell-derived sFRP-2 is among predominant factors responsible for defective bone formation in MM. Because MM cell-derived factors such as DKK-1, IGF-BP4 and IL-3 other than sFRP-2 have been implicated as an inhibitor of osteoblast differentiation, sFRP-2 may act alone or in combination with such other factors to potently suppress bone formation in MM. Taken together, MM cells may cause an imbalance of bone turnover with enhanced osteoclastic bone resorption and concomitantly suppressed bone formation, which leads to devastating destruction and a rapid loss of bone.

Description: We evaluated the toxicity-profile, engraftment potential, and efficacy of fludarabine-based nonmyeloablative allogeneic HCT in patients with a variety of nonmalignant hematological disorders. Twenty three patients (median age 29 years; range 11–52) with nonmalignant hematological disorders including ATG refractory SAA (n=13), severe paroxysmal nocturnal hemoglobinuria (PNH: n=9), and pure red cell aplasia (PRCA; n=1) were transplanted from 5/99 – 8/2004 at the NHLBI. The majority of patients had an extensive transfusion history including 11/23 who had HLA allo-antibodies and 4/23 with allo-antibodies to RBCs. Conditioning with fludarabine (25 mg/m2 x 5 days), ATG (40mg/kg x 4 days) and cyclophosphamide (60mg/kg x 2 days) was followed by infusion of an un-manipulated G-CSF mobilized allograft from an HLA matched sibling (n=18), parent (n=2), or single antigen mismatched sibling (n=3). GVHD prophylaxis consisted of cyclosporine (CSA) either alone (n=2) or combined with mycophenolate mofetil (n=10) or mini-dose methotrexate (n=11). Despite a high prevalence of pre-transplant allo-immunization, all patients achieved sustained donor engraftment in both myeloid and T-cell lineages. Myeloid recovery (neutrophils 〉500cells/uL) occurred at a median 14 days post transplant (range 8–18 days). Conversion from mixed to full donor myeloid and T-cell chimerism occurred in all patients by 110 days post-transplant. CMV reactivation occurred in 11/21 patients at risk (KM probability 52%) without any cases of CMV disease. Grade II–IV and III–IV acute GVHD was the major transplant complication occurring in 13/23 (KM probability 60%) and 8/23 (KM probability 38%) patients respectively. Fourteen of 21 evaluable patients developed chronic GVHD (limited in 11 cases), which resolved completely with low-dose alternate day steroids and/or CSA in all but 1 case. One patient who received an allograft from his HLA matched father died 16 months post-transplant from complications related to chronic GVHD. With a median follow up of 25 months (range 1–64 months), 20/21 patients evaluable more than 100 days post-transplant survive in complete remission with full donor chimerism in all lymphohematopoietic lineages (KM probability of long-term survival 92.8 %-see figure ). Conclusion: Fludarabine-based nonmyeloablative transplantation achieves excellent donor engraftment and long-term disease free survival in heavily transfused and allo-immunized patients with ATG refractory SAA and other nonmalignant hematological disorders associated with bone marrow failure.

Description: Thrombosis can be initiated when activated platelets adhere to injured blood vessels via the interaction of subendothelial collagen with its platelet receptor, glycoprotein (GP) VI. Here we observed that incubation of platelets with convulxin, collagen, or collagen-related peptide (CRP) resulted in GPVI signaling-dependent loss of surface GPVI and the appearance of an approximately 55-kDa soluble fragment of GPVI as revealed by immunoblotting. Ethylenediaminetetraacetic acid (EDTA) or GM6001 (a metalloproteinase inhibitor with broad specificity) prevented this loss. In other receptor systems, calmodulin binding to membrane-proximal cytoplasmic sequences regulates metalloproteinase-mediated ectodomain shedding. In this regard, we have previously shown that calmodulin binds to a positively charged, membrane-proximal sequence within the cytoplasmic tail of GPVI. Incubation of platelets with calmodulin inhibitor W7 (150 μM) resulted in a time-dependent loss of GPVI from the platelet surface. Both EDTA and GM6001 prevented this loss. Surface plasmon resonance demonstrated that W7 specifically blocked the association of calmodulin with an immobilized synthetic peptide corresponding to the calmodulin-binding sequence of GPVI. These findings suggest that disruption of calmodulin binding to receptor cytoplasmic tails by agonist binding to the receptor triggers metalloproteinase-mediated loss of GPVI from the platelet surface. This process may represent a potential mechanism to regulate GPVI-dependent platelet adhesion.

Description: Abnormal tissue factor (TF) expression has been demonstrated on blood monocytes and circulating endothelial cells in humans with sickle cell anemia. We have now studied sickle transgenic mice to help define the biology of endothelial TF expression in sickle disease. Using immunostaining of tissue sections, we find that this is confined almost exclusively to the pulmonary veins. About 15% and 13% of these exhibit TF-positive endothelium in the wild-type normal mouse and the normal human hemoglobin (HbA)–expressing control transgenic mouse, respectively. The mild sickle mouse is indistinguishable from normal (∼ 14% positive), but TF expression is significantly elevated in the moderate and severe mouse models of sickle disease (∼ 29% and ∼ 41% positive, respectively). Exposure of the mild sickle mouse to hypoxia for 3 hours, followed by reoxygenation, converted its TF expression phenotype to that of the severe sickle mouse (∼ 36% positive). Pretreatment with lovastatin eliminated excessive expression of TF in the posthypoxic mild sickle mouse (∼ 16% positive) and in the more severe mouse at ambient air (∼ 21% positive). In addition to identifying tissue expression of endothelial TF in the sickle lung, these studies implicate reperfusion injury physiology in its expression and suggest a rationale for use of statins in sickle disease.

Description: Transferrin receptor 2 (TfR2) is a type 2 transmembrane protein expressed in hepatocytes that binds iron-bound transferrin (Tf). Mutations in TfR2 cause one form of hereditary hemochromatosis, a disease in which excessive absorption of dietary iron can lead to liver cirrhosis, diabetes, arthritis, and heart failure. The function of TfR2 in iron homeostasis is unknown. We have studied the regulation of TfR2 in HepG2 cells. Western blot analysis shows that TfR2 increases in a time- and dose-dependent manner after diferric Tf is added to the culture medium. In cells exposed to diferric Tf, the amount of TfR2 returns to control levels within 8 hours after the removal of diferric Tf from the medium. However, TfR2 does not increase when non–Tf-bound iron (FeNTA) or apo Tf is added to the medium. The response to diferric Tf appears to be hepatocyte specific. Real-time quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis shows that TfR2 mRNA levels do not change in cells exposed to diferric Tf. Rather, the increase in TfR2 is attributed to an increase in the half-life of TfR2 protein in cells exposed to diferric Tf. Our results support a role for TfR2 in monitoring iron levels by sensing changes in the concentration of diferric Tf.

Description: CD infection is a common complication in immunocompromised patients, including those undergoing HSCT, with reported incidences ranging from 4% to 13%. However, there has been no previous detailed report on the clinical impact of positive CD toxin in patients after allogeneic HSCT with current supportive therapies. Therefore, we retrospectively reviewed the medical records of 422 patients who underwent allogeneic HSCT with a variety of preparative regimens at National Cancer Center Hospital from January 2000 to April 2004. CD toxin in stool samples was measured by the Latex particle agglutination method, and selected patients were examined further by endoscopy. Positive results with CD toxin were observed at least once in 51 patients, at a median time of 40 days (range, −1 to 212 days) following allogeneic HSCT. Most patients had severe watery diarrhea, along with other symptoms including fever, nausea, anorexia and abdominal cramping, which mimics pseudomembrane colitis. Twenty-seven of the 51 patients (53%) underwent endoscopic examination, and macroscopic findings were as follows; normal in 4 (15%), extensive edema in 11 (41%), and mucosal sloughing in 12 (44%), while none had pseudomembrane formation. Histological diagnosis revealed that 26 cases (96%) were compatible with GVHD, including 2 complicated with Cytomegalovirus colitis. Thirty-three patients (65%) were treated with oral vancomycin for a median of 14 days (2 to 46 days), while the remaining 18 were followed conservatively without any specific medication. Forty-four patients were evaluable for follow-up, and, regardless of management, all subsequently became negative for CD toxin. These findings suggest that positive CD toxin in the stool detected during the course of allogeneic HSCT may only reflect nonsignificant colonization or proliferation of CD in the gut, and hence may not necessarily imply clinically relevant pseudomembranous colitis which would require intense medical treatment. Additionally, in patients undergoing allogeneic HSCT, signs and symptoms of intestinal involvement may simply reflect concomitant GVHD per se or a pathology closely related to GVHD.

Description: Identification of growth factors in neoplasias may be a target for future therapies by blocking either growth factor receptor interaction or the induced pathway. Using gene expression profiling, we identified overexpression of 2 receptors for a proliferation-inducing ligand (APRIL) and B-cell activating factor (BAFF) in malignant plasma cells compared with normal plasma cells. APRIL and BAFF are involved in a variety of tumor and autoimmune diseases, including B-cell malignancies. We confirmed the expression of BAFF and APRIL receptors (B-cell maturation antigen [BCMA], transmembrane activator and calcium modulator and cyclophilin ligand interactor [TACI], and BAFF-R) in a majority of 13 myeloma cell lines and in the purified primary myeloma cells of 11 patients. APRIL and BAFF were potent survival factors for exogenous cytokine-dependent myeloma cell lines and were autocrine growth factors for the RPMI8226 and L363 autonomously growing cell lines. These factors activated nuclear factor (NF)–κB, phosphatidylinositol-3 (PI-3) kinase/AKT, and mitogen-activated protein kinase (MAPK) kinase pathways and induced a strong up-regulation of the Mcl-1 and Bcl-2 antiapoptotic proteins in myeloma cells. BAFF or APRIL was also involved in the survival of primary myeloma cells cultured with their bone-marrow environment, and protected them from dexamethasone (DEX)–induced apoptosis. Finally, the serum levels of BAFF and APRIL were increased about 5-fold in patients with multiple myeloma (MM) as compared with healthy donors. Altogether, these data suggest that APRIL/BAFF inhibitors may be of clinical value in MM. (Blood. 2004;103:3148-3157)

Description: We have carried out HLA-matched unrelated donor hematopoietic cell transplantation (HCT) after nonmyeloablative conditioning in patients with hematologic malignancies who were ineligible for conventional transplantations because of age, comorbidities, or both. The nonmyeloablative regimen consisted of 90 mg/m2 fludarabine and 2 Gy total body irradiation given before and mycophenolate mofetil and cyclosporine given after HCT. This report compares, retrospectively, morbidity and mortality among 60 consecutive patients given nonmyeloablative conditioning (nonablative patients) to those among 74 concurrent and consecutive patients given myeloablative conditioning (ablative patients) before unrelated HCT. The Charlson Comorbidity Index was used to assess pretransplantation comorbidities. Even though nonablative patients had significantly higher pretransplantation comorbidity scores, were older, and had more often failed preceding ablative transplantations and cytotoxic therapies, they experienced fewer grades III to IV toxicities than ablative patients. Further, the incidence of grades III to IV acute graft-versus-host disease (GVHD) was significantly lower in nonablative patients. Both patient groups had comparable 1-year probabilities of chronic GVHD. The 1-year nonrelapse mortality rate was 20% in nonablative patients compared to 32% in ablative patients (hazard ratio = 1.4). After adjustment for pretransplantation differences between the 2 patient groups, the hazard ratio was 3.0 (P = .04). Multivariate analyses showed higher pretransplantation comorbidity scores to result in increased toxicity and mortality.

Description: MDS is a disease of the elderly marked by dysplasia of hematopoetic cell lines resulting in cytopenias and AML progression. There is currently no treatment approved to alter the natural history of the disease. Aberrant methylation associated with cancer is a potential target for pharmacologic therapy, and DacogenTM (decitabine) for injection (SuperGen, Inc.), a cytosine analogue, indirectly depletes methylcytosine after incorporation into DNA with subsequent inactivation of DNA methyltransferases. We report the results of a Phase III trial of decitabine (DAC) vs. supportive care (Supp.Care) in adult MDS patients with IPSS Intermediate (Int)-1 (31%), Int-2 (44%) and high risk disease (26%). Secondary MDS (14%) and previously treated (27%) MDS patients were not excluded. Bone marrows were assessed by a blinded, independent pathologist. 170 patients (accrued from July 2001 through April 2003 at 23 centers) were randomized 1:1 to either Supp.Care or DAC (a 3 hr infusion of 15mg/m2/hr every 8 hrs on 3 consecutive days every 6 wks for up to 10 cycles. The groups were comparable for numerous risk factors, including time from diagnosis (median 29 weeks for DAC and 35 weeks for Supp.Care). Kaplan Meier (KM) curves for time to AML progression or death showed early and clinically meaningful separation (consistent with clinical benefit) in favor of DAC in all patients (intent to treat; ITT), Int-2/High Risk, and High Risk patients. Using the Cox proportional hazards model for the ITT population, the probability of progression to AML or death was 1.68-fold greater for Sup.Care than for DAC (p=0.023). Median Time to AML or Death (Days) MDS group (n) DAC Supp.Care p-value 1Protocol specified test. 2Preferred test for analysis of early separation of KM curves. n=89 n=81 Wilcoxon1,2 Log rank1 All Patients (170) 338 263 0.046 0.204 Int-2/High Risk (118) 334 189 0.005 0.040 High Risk (44) 260 79 0.001 0.006 Treatment naïve (124) 354 189 0.005 0.034 Figure Figure Investigator reported response rate by International Working Group criteria was 25% (10% CR, 15% PR) for DAC vs. 0% for Supp.Care (p〈 0.001), with responses equally distributed across baseline subgroups. Time to response was 100 days and median duration was estimated at 〉9 months. Responders (CR and PR) vs. non-responders had a median survival of 678 days vs. 406 days (p= 0.038 Wilcoxon). There were no treatment related deaths. As expected, grade 3–4 toxicity (including hematologic toxicity, febrile neutropenia) occurred in more DAC patients than in Supp.Care patients. Most patients tolerated treatment well. DAC appears a promising therapy for MDS with manageable toxicity. Final results of independently reviewed response rates and clinical benefit (transfusion independence; Quality of Life) will be presented.

Description: Guiding antileukemic treatment in patients with acute myeloid leukemia (AML) is increasingly based on levels of minimal residual disease (MRD) which can be quantified with high sensitivity by multiparameter flow cytometry (MFC). The optimum checkpoint for determination of MRD during the course of therapy, however, has not yet been determined. We applied MFC using a comprehensive panel of antibodies to identify leukemia-associated aberrant immunophenotypes (LAIPs) at diagnosis and to quantify MRD by individually selected antibody combinations. The prognostic impact of MRD levels was assessed in comparison to cytogenetics and age. Patients received double induction, consolidation, and maintenance therapies and underwent allogeneic stem cell transplantation if they were younger than 60 years and had a matched related donor. In 286 patients with newly diagnosed and untreated AML MFC-based assessment for the presence of LAIP has been performed. The median percentage of LAIP-positive bone marrow cells at diagnosis was 16.04% (range, 2.54%–76.14%). All individual LAIPs were applied to 26 normal bone marrow samples to estimate sensitivity based on the median percentages of LAIP-positive normal bone marrow cells which ranged from 0.00% to 1.01% (median, 0.02%). A total of 550 follow-up samples has been analyzed in these patients at different checkpoints (CP1, up to day 21 after start of therapy, n=85; CP2, day 22–60, n=122; CP3, day 61–120, n=158; CP4, day 121–365, n=137; CP5, after day 365, n=48). In order to adjust for differences in the percentages of LAIP-positive bone marrow cells at diagnosis the logarithmic difference (LD) between diagnosis and follow-up was calculated for each follow-up sample. The median LDs at the respective checkpoints were: CP1, 2.02; CP2, 2.29; CP3, 2.39; CP4, 2.53; and CP5, 2.81. Separation of patients according to the respective median LDs resulted in differences in event-free survival (EFS; CP1: 21.1 vs. 9.1 months, p=0.0711; CP2: 14.2 vs. 9.3 months, p=0.0095; CP3: 30.9 vs. 13.5 months, p=0.0055; CP4: median not reached vs. 14.1 months, p

Description: DAC is a potent hypomethylating agent with clinical activity in patients with myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). VPA is a histone deacetylase inhibitor used as an antiepileptic agent. In vitro, the combination of DAC with VPA results in synergistic antileukemia activity at doses of VPA above 1mM. Based on this data, we have developed a phase I/II study of this combination for pts with leukemia. The phase I of the study followed a classic 3+3 design. The dose of DAC was fixed: 15 mg/m2 iv daily for 10 days. This was based on a previous phase I study (Blood2004;103:1635) that indicated that this schedule had an optimal toxicity-response profile in this population. Three dose levels of VPA were selected: 20, 35 and 50 mg/kg. VPA was given orally for 10 days concomitantly with DAC. 22 pts have completed the phase I portion of the study (median age 56 years, range 4–78, 20 pts AML, 2 MDS). At dose level 1 (20 mg/kg of VPA) no grade III-IV toxicity was observed. At dose level 2 (VPA 35 mg/kg), 2 out 6 pts developed grade III neurotoxicity. Both pts were receiving high doses of other neurotropic agents. After IRB approval, 3 mores pts were treated at this dose level with no significant toxicity. Subsequently, 3 pts were treated at the highest planned dose level (50 mg/kg) with no toxicity observed. This cohort was then expanded to a total of 10 pts. One pt developed grade III neurotoxicity. No other severe drug-related toxicities were observed, but 5 patients at all dose levels developed grade II sedation/somnolence. Pancytopenia was induced in all pts. At dose level 1, one pt with refractory AML achieved complete remission (CR) after the second course of therapy. This is now maintained for 5 courses. At dose level 2, a patient with HIV disease and relapsed AML achieved CR after the third course of therapy, and 2 pts with relapsed AML achieved complete marrow responses (marrow blasts less then 5%, no recovery of peripheral counts). Of 3 pts evaluable for response at dose level 3, 1 pt with MDS has achieved CR after 1 course, and 1 with relapsed AML a complete marrow response. Median free VPA levels pretreatment were 0, and 25 mg/L on both days 5 and 10 and returned to 0 prior to next course. Histone acetylation measured by Western blot was observed in 3 pts (25%), all at doses above 20 mg/kg of VPA. Reactivation of p21 expression was induced in 4 out 11 pts analyzed. Global hypomethylation measured using a bisulfite PCR LINE assay was induced in 1 out 3 pts so far studied. Based on the toxicity observed, the phase II portion of the study was initiated. This is restricted to pts with AML/MDS. Seven pts have been accrued to this phase, and 8 out the 10 pts at dose level 3 of the phase I are also evaluable. The response data of this pts will be updated at the meeting. In summary, the combination of low dose DAC and VPA up to doses of 50 mg/kg can be safely administered to pts with leukemia although it may be complicated by neurotoxicity. Clinical and biological activity was observed at all dose levels.

Description: The enucleated definitive erythrocytes of mammals are unique in the animal kingdom. The observation that yolk sac–derived primitive erythroid cells in mammals circulate as nucleated cells has led to the conjecture that they are related to the red cells of fish, amphibians, and birds that remain nucleated throughout their life span. In mice, primitive red cells express both embryonic and adult hemoglobins, whereas definitive erythroblasts accumulate only adult hemoglobins. We investigated the terminal differentiation of murine primitive red cells with use of antibodies raised to embryonic βH1-globin. Primitive erythroblasts progressively enucleate between embryonic days 12.5 and 16.5, generating mature primitive erythrocytes that are similar in size to their nucleated counterparts. These enucleated primitive erythrocytes circulate as late as 5 days after birth. The enucleation of primitive red cells in the mouse embryo has not previously been well recognized because it coincides with the emergence of exponentially expanding numbers of definitive erythrocytes from the fetal liver. Our studies establish a new paradigm in the understanding of primitive erythropoiesis and support the concept that primitive erythropoiesis in mice shares many similarities with definitive erythropoiesis of mammals.

Description: Twenty five patients have been enrolled on this trial to date. There were 14 males and 11 females aged 0.6–54 years. Patients’ diagnoses and stage included: NHL in CR2 or refractory (n=2), AML (n=7), including 5 pts with secondary AML, ALL 〉 CR3 (n=4), CML in CP2 (N=1) and high risk MDS (n=11) including 5 pts with secondary MDS. Eight pts had a matched related donor, 14 pts an unrelated donor and 3 pts a mismatched related donor. Cytoreduction consisted of busulfan (Bu) (0.8–1 mg/Kg/dose x 10 doses), melphalan (Mel) (70 mg/Kg/day x 2) and fludarabine (Flu) (25 mg/m2/day x 5). Graft rejection prophylaxis included rabbit ATG (Thymoglobulin) (2.5 mg/Kg/day x 2). Four pts tolerated only one of two doses of the ATG, 2 pts received equine ATG and one pt Alemtuzumab. Twenty one pts received G-CSF mobilized peripheral blood stem cell transplants that were T-cell depleted by CD34 selection and E-rosetting while the other four pts received Soybean agglutinin E-rosette depleted marrow grafts. Cell doses were 1.3–20.5 x 106 CD34 cells/Kg. and 0 -100 x 103 CD3 cells/Kg. Engraftment occurred in 24 pts. One pt suffered a graft failure; This pt had initial low busulfan levels, and received bone marrow derived stem cells with a low cell dose from a 5/6 HLA-matched unrelated donor. Acute graft-versus-host disease occurred in four pts: grade 1 (n=2) and grade 2 (n=2) and no pts developed any grade 3-4 severe GvHD. Two patients were diagnosed with chronic GvHD: localized (n=1) and extensive (n=1). Two patients developed sepsis early post BMT, with secondary multi organ failure and early mortality, while for the rest of the patients, regimen-related toxicity was acceptable. Relapse occurred in 9 pts. Mortality included 7 pts from relapse, two pts from sepsis and multi-organ failure, 3 pts from infections, and one pt from unknown causes. The overall survival (OS) and disease-free survival (DFS) at 2 yrs for the entire patient cohort were respectively 44% and 42 %; The DFS was 50% for patients with secondary MDS or AML. In summary, the cytoreduction with Bu Mel and Flu allowed consistent engraftment of T-cell depleted grafts and was associated with acceptable outcome for patients with secondary MDS or AML.

Description: Warfarin is effective in decreasing recurrent thrombosis in patients with antiphospholipid antibody syndrome (APS). Antiphospholipid antibodies (APAs) can influence the results of clotting tests in a subset of these patients, which can be a major obstacle in monitoring warfarin. In this study, 59 patients receiving warfarin for a diagnosis of APS were compared to 49 patients receiving warfarin for atrial fibrillation (AF) with regard to consistency between International Normalized Ratio (INR) results obtained from two Point of Care (POC) monitors and a standard plasma-based method used in the coagulation laboratory. INR results were obtained using a fingerstick for whole blood on the ProTime® monitor (International Technidyne Corp, ITC, Edison, NJ) and by venipuncture using both citrated and non-citrated whole-blood on a HEMOCHRON®Signature (Hr. Sig.) monitor (ITC). Parallel INR measurements were obtained on an MDA-180 analyzer (bioMeriéux, Durham, NC), using Simplastin-HTF. Additional tests included chromogenic factor Xa (CFXa) and tests for APAs (antiphospholipid, anti-β2-glycoprotein I [β2GPI], and antiprothrombin ELISA’s). Insufficient blood was obtained from 5 patients for testing with the ProTime® (3 AF, 2 APS). For an additional 5 patients, all with APS, sufficient blood was obtained, but an INR could not be determined by the instrument (8%). Single INR results on the ProTime® were obtained from 2 of these patients on repeat testing. The data were analyzed with the 100 patients who had at least one INR result with the ProTime®. Analyses included determination of the means of absolute differences between each method of obtaining INR results, correlations between INR results and CFXa results, and correlations with APAs results. Systematic differences were found for each INR method comparison, ranging from 0.24±0.23 to 0.41±0.29, with correlation coefficients ranging from 0.54 to 0.80. The differences were similar for AF and APS patients for all INR comparisons with the exception of the results comparing the standard plasma-based method with the ProTime®, which showed a mean absolute difference of 0.24 for AF patients and 0.39 for APS patients (p=0.01). Preliminary data analysis did not show perfect calibration between the CFXa and the different methods for obtaining INR results for either patient group. Correlation coefficients ranged from 0.3 to 0.7 when comparing CFXa to each INR method, and the best correlation was between the standard plasma INR and the CFXa for patients with AF (0.65). Review of testing for APAs at the time of INR testing revealed that 10 patients with AF had elevated antibody levels (20%; all by antiphospholipid ELISA only and most only minimally elevated), compared to 27 patients with APAs (45%, with 21 having elevated anti-β2 GPI antibody levels). The five patients with non-measurable ProTime® INRs had elevated anti-β2GPI levels and generally higher INRs with the Hr. Sig., but CFXa results were not supratherapeutic. In conclusion, these results suggest that in a subset of patients with APAs, the ProTime® will not yield any results and can not be used due to the internal QC set of the monitor. APA testing did not specifically identify which APS patients would most likely have problems with INR testing.

Description: Gemtuzumab Ozogamicin (GO) is an anti-CD33 antibody linked to the cytotoxic antibiotic calicheamycin. Its use in AML and acute promyelocytic leukaemia has been previously described and its hepatotoxic side effects highlighted. Haemopoietic stem cell transplantation is another modality used in the treatment of relapsed AML with graft versus leukaemic effect being implemented by T cells that are effective against the tumor specific antigens. However relapse can occur despite the presence of 100% donor derived T cells. We report prolonged disease free survival (DFS) following the use of GO/DLI. Patients with relapsed or refractory leukaemia AML (n=28) and myelodysplasia (n=6, RAEB1 n=5, RAEB2 n=1), median age 52 yrs (19–66) received treatment with gemtuzumab ozogamicin (GO) between January 2001 and February 2004 at King’s College Hospital. Six had previously untreated AML or MDS, 17 had AML/MDS in first relapse, 4 in second relapse, 6 were refractory and 1 was in second remission. Seventeen patients received GO with FLAG ± Idarubicin and 17 had GO alone. Complete remission (CR) was similar in both groups( 47% vs. 41%respectively). Twenty seven patients received single dose, 5 patients received 2 and 2 patients received 3 doses of GO. Twenty six patients received 9mg/m2, 4 received 6mg/m2, 3 at 3mg/m2 and 1 at 4.5mg/m2. Following GO 5 patients received conventional conditioning allogeneic HSCT (AlloHSCT), 8 received reduced intensity conditioning (RIC-HSCT) and 1 had autologous rescue. HSCT was done at a median time of 37 days (17–444) post-GO. Six out of 11 patients who underwent HSCT post GO with no DLI are alive in CR, median 810d (120–1080). Hepatotoxicity with bilirubin elevation grades 2–4 (NCI toxicity criteria)occurred in 14 patients associated with elevated AST grades 2–4 in 8 patients and hepatic veno-occlusive disease in 4 patients. Pulmonary haemorrhage occurred in 3 patients, contributing to death in one. Direct anti-globulin test positivity was observed in 6 patients causing significant haemolysis in 4. Eight patients who relapsed following HSCT received GO followed by donor leucocyte infusion. Blasts from these patients were mean 75.5% (85% median)CD33+. Three of the eight patients achieved complete remission, 2 are alive 240 and 810d post GO. Two other patients underwent repeat RIC-HSCT post-GO followed by DLI, both achieved CR, and 1 is alive at 990d. Chimerism data available for 8 patients showed 0–56% donor derived haemopoiesis pre treatment. Donor derived CD3+ cells were 26%and 47% in two patients and 100% in 2 patients pre GO/DLI. In the former two patients the first did not respond to this treatment, the second became 100% donor in the unfractionated bone marrow and in CD3+ fraction. In the latter two, unfractionated bone marrow was 0% donor derived and 56% donor derived pre treatment with these increasing to 77% and a 100% donor derived respectively with the CD3+ fraction remaining 100% donor. Five of these 8 patients acheived CR.Overall 13 patients (31.4%) achieved CR, 9 (25.7%) are alive 120–1080 days post-GO, median 810d. The overall median survival post-GO was 76 days (2–1080). Our results indicate that gemtuzumab ozogamicin in combination with DLI can benefit the subgroup of patients with AML /high risk MDS who have relapsed post haemopoietic stem cell transplant.

Description: Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin therapy that can result in significant morbidity and mortality. Immediate discontinuation of heparin followed by the administration of a direct thrombin inhibitor (DTI) is the standard of care. There are two DTIs approved by the FDA for this indication, argatroban and lepirudin. Argatroban is metabolized in the liver by hydroxylation and aromatization and requires dosage adjustment in patients with moderate hepatic impairment defined as a Child-Pugh score 〉6. The normal recommended starting dose of 2 mcg/kg/min is reduced to 0.5 mcg/kg/min in these patients. Objective : Our clinical observation was that the recommended starting doses of argatroban in the ICU resulted in elevated activated partial thromboplastin time (aPTT) values that persisted for a prolonged time. This may result in a higher bleeding risk and delay life-saving invasive procedures especially in the absence of an antidote. Therefore, our objective is to show that critically ill patients without significant liver disease require a lower dosage of argatroban than recommended in the manufacturer’s prescribing information and to identify factors that may affect this recommendation. Materials and Method : Retrospective chart review of patients admitted to a medical or surgical intensive care unit (ICU), diagnosed with HIT who received argatroban for more than 24 hours. SPSS version 11.5 was used for data analysis. Results : 65 patients (37 men) were analyzed. The mean age was 65.8 years. 43% had abnormal liver function tests (LFT’s) defined as ALT〉50 or bilirubin 〉1.5 (100% of the patients had a normal baseline INR), 24.6% had acute renal failure (ARF) and 36.9% were septic. 40% had one organ system failure (OSF), 40% had two and 20% three. The diagnosis of ARF, sepsis and the number of OSF was based on the ICU physician’s documentation and the patient’s active problem list. Excluding 3 patients with a history of liver disease (1) or acute liver decompensation (2), the mean argatroban dose for our ICU patients was 0.91 mcg/kg/min. Patients with ARF (n=16/65) required a significantly lower dose (0.65 mcg/kg/min, p=0.044), as did patients with sepsis (0.70 mcg/kg/min, p=0.03, n=24/65). Even in patients with normal LFT’s, dosage requirements were lower ranging from 1.2 mcg/kg/min with one OSF to 0.52 mcg/kg/min with three OSF (p=0.009). Discussion : Factors other than pre-existing liver disease appear to affect the dosage of argatroban in critically ill patients with HIT. Our mean dose to achieve a therapeutic aPTT was 0.91 mcg/kg/min with even lower doses in patients with ARF, sepsis or more than one OSF. This may be explained by the influence of ARF, sepsis, or OSF on argatroban metabolism by the liver that is not necessarily reflected in ALT, bilirubin, or even INR measurements. This metabolic effect may be due to passive hepatic congestion, accumulation of certain metabolites or interaction with the multiple medications used in these patients. Consideration should be given to initiate argatroban at a lower dosage in the critically ill patient.

Description: Background We examined data from acutely ill medical patients enrolled in The International Medical Prevention Registry on Venous Thromboembolism (IMPROVE) to determine factors that are independently associated with an increased risk of bleeding during hospitalization. Methods Patients ≥18 years old, hospitalized for ≥3 days with an acute medical illness have been enrolled consecutively since July 2002. Exclusion criteria are: therapeutic antithrombotics/thrombolytics at admission; major surgery or trauma during 3 months prior to admission; and venous thromboembolism (VTE) treatment within 24 hours of admission. Patients with bleeding immediately prior to, or at admission were excluded from this analysis. Factors considered were: age, ICU stay, reduced creatinine clearance, severe infection, cerebrovascular stroke, cancer, diabetes, severe renal failure, hypertension, lower limb paralysis, bleeding disorders, hemorrhagic stroke, thrombocytopenia, gastro-duodenal ulcer, hepatic failure, central venous catheter at admission, hormonal therapy for cancer, platelet count, BMI, immobility, and length of hospital stay. Factors increasing the risk of bleeding were identified by univariate analysis (P≤0.20) and included in a multiple logistic regression model. Factors with significance of P≤0.05 were retained in the model. Bleeding events were defined as major or clinically significant non-major according to published criteria (Büller HR et al. N Engl J Med.2003;349:1695–702). Results . Data were from 2816 patients enrolled up to 30 June 2004 in 34 hospitals in 10 countries. Patients were: 48% female, mean age 64 years, mean weight 72 kg, mean length of hospital stay 12 days, and 37% were immobile for ≥3 days (median duration of immobility 6 days, including immobility immediately prior to admission). Only 89 (3.2%) patients had in-hospital bleeding: 1.2% major, 1.9% clinically significant non-major, 0.1% unspecified. Factors that were independently associated with an increased risk of bleeding (major or non-major) in acutely ill medical patients are shown in Table 1. Conclusion Only 3.2% of acutely ill medical patients in IMPROVE had in-hospital bleeding. Major bleeding (1.2%) was similar to that observed in a major clinical trial on VTE prophylaxis, MEDENOX (1.0%; Samama et al. N Engl J Med.1999;341:793–800). Advanced age, contrary to the belief of many physicians, was not independently associated with an increased risk of bleeding. Final results will be presented with adjustment for the influence of VTE prophylaxis and antiplatelet drugs. Table 1. Factors independently associated with an increased risk of bleeding in acutely ill medical patients Factor OR 95% CI *compared with creatinine clearance 〉60 mL/min (normal renal function) Bleeding disorder 8.38 3.53–19.90 Active gastro-duodenal ulcer 4.26 1.86–9.76 Hepatic failure 2.97 1.26–6.96 Creatinine clearance

Description: The anti-epileptic drug valproic acid (VPA) acts as an inhibitor of histone deacetylases. In combination with retinoic acid (RA), VPA triggers myeloid differentiation of primary acute myeloid leukemia (AML) blasts in vitro. In vivo, VPA posses an antineoplastic activity as indicated by pre-clinical studies in murine models of leukemia, renal and lung metastatic tumors. Therefore, we have designed a phase II clinical study in which VPA was combined with RA (VPA-RA) in the AML treatment. Eigth chemotherapy-resistant or high risk AML patients not eligible for additional intensive therapy (median age 61.5 yrs), were treated at the Hematology Units of the Universities “La Sapienza” and “Tor Vergata” Rome-Italy. VPA (Depakin[Sanofi-Wintrop]) was administrated from day 1 to day 28, at the initial dosage of 10 mg/kg/die p.o. with dose escalation until optimal VPA plasma levels (80–110ug/ml). RA (Vesanoid [Roche]) at the dosage of 45 mg/m2 p.o./d, divided in two administrations, was added once the optimal VPA plasma levels were reached or at day 14 and continued until day 28. Four patients had a history of MDS, three patients had a FAB M0, M1 and M2 de novo AMLs, while the remaining case was a myeloid blast crisis (FAB M0) of a Ph+ve CML. Cytogenetic characterization in the other patients revealed normal karyotype in one case, a pseudodiploid [der(12)] in one, hyperdiploid (+8) in one, complex K with a 7q- alteration in one, while in the three remaining cases the karyotype was not evaluable. Pre-treatment leukemic infiltration ranged from 22% to 95%. VPA plasma level 〉60mg/ml was reached between 8 to 28 days (median 14.5 days). In three patients, VPA-RA treatment induced hyperleukocytosis (〉50x 109/l) at day 16, 21 and 24, respectively, that was treated with chemotherapy (HU in two cases and low dose Ara-C in 1 case). Hematological improvement (≥50% decrease in packed red blood cell or platelet transfusion requirement) was observed in one case, a stable disease in five cases and disease progression in two cases. Peripheral blood and/or bone marrow samples were collected at day 0,3,7,14,21,28 for morphologic, immunophenotypic, cytogenetic and molecular studies. All patients showed features of myeloid-monocytic and/or erythroid differentiation of the leukemic clone, as revealed by morphologic, cytochemical, immunophenotypic analyses and by Q-RT-PCR of myeloid gene expression (GATA 1, MPO, CSF2Rb, etc.). Of note that high degree of myeloid differentiation correlated with early achievement of therapeutic VPA plasma levels and histone hyper-acetylation, as measured by immunocytochemistry and immunoblotting using antiacetylated histone H3 and H4 antibodies. Finally, differentiation of the leukemic clone was proven by FISH analysis showing the presence of the +8 and 7q- in maturing elements in patients whose leukemia blasts carried these cytogenetic lesions. The VPA-RA combination is a well tolerated treatment that induces phenotypic changes of the leukemic clone through chromatine remodelling. Further studies are needed to optimise this regimen with the aim of improving clinical response in leukemia patients.

Description: Several clinical studies of adoptive immunotherapy with genetically modified (GM) donor T cells infused after allogeneic hematopoietic cell transplantation (HCT) showed limited in vivo function of the transduced T cells. Factors that have hindered successful translation to clinical trials include insufficient preclinical data in large animal models and the need for prolonged cell culture - up to 2 to 3 weeks for optimal oncoretroviral (OR) vector transduction and selection of T cells. In preparation for in vivo studies of GM T cells to facilitate engraftment in the preclinical dog model of allogeneic HCT, we compared transduction protocols with OR and lentiviral (LV) vectors that aimed to decrease the duration of ex vivo T cell culture necessary for stable transduction while maintaining T cell alloreactivity. Vectors expressed enhanced yellow fluorescent protein (YFP) under a constitutive promoter. We compared vectors pseudotyped with viral glycoproteins (GP) including vesicular stomatitis virus (VSV)-G (LV only), feline endogenous virus (RD114), and chimeric RD114 envelope GP fused with murine leukemia virus-A cytoplasmic tail (RD114/TR). Although T cells transduced with LV vectors without prior mitogenic or allogeneic stimulation had 14% – 30% transduction efficiency of predominantly CD4+ cells, transgene expression was not sustained in CD8+ cells after allogeneic stimulation (n=3). In order to transduce T cells that could generate GM alloreactive cytotoxic T lymphocytes (CTL), freshly isolated T cells were stimulated with allogeneic dendritic cells (DC) for 4 days prior to transduction. VSV-G, RD114 or RD114/TR pseudotyped LV had primary transduction efficiency of 1.2 to 9% (n=5). Only cells that were transduced with RD114 or RD114/TR pseudotyped vectors maintained stable YFP expression after 2° allogeneic stimulation. Next, OR YFP vector pseudotyped with RD114 transduced 15 to 36% of DC allo-stimulated T cells (n=3). Both CD4+ and CD8+ cell populations were transduced (CD4+: CD8+ ratio 1.4:1) and the mean YFP fluorescence intensity was increased 0.6-log compared to LV vectors (p=0.01). We then evaluated T cells transduced with OR RD114 pseudotyped vector in vivo. To determine if short-term culture and transduction of T cells facilitated engraftment of CD3-depleted marrow in the DLA-haploidentical HCT model, donor T cells were collected on day −7 prior to HCT, cultured with recipient CD34+ derived DC, and on day −4 cells were transduced with RD114 pseudotyped YFP OR vector. To date, one dog was transplanted after 920cGy total body irradiation with 2x108 CD3+ donor cells/kg (1:1 CD4:CD8 ratio) 25% YFP expression and 2-log CD3-depleted marrow (4x108 TNC/kg). No post-grafting immunosuppression was given. Donor YFP transduced T cells were detected in the peripheral blood daily after HCT, and peaked on day +7. After engraftment on day +8, GVHD developed and the dog was euthanized on day +21 with all-donor chimerism. YFP+ T cells were detected in GVHD affected target organs. Previously, transduced donor CTL cultured for 4 weeks and transplanted with CD3-depleted marrow in this HCT model failed to engraft in all 5 dogs studied. These preliminary results support the hypothesis that short-term culture and transduction of donor T cells with RD114 pseudotyped OR vector maintain in vivo alloreactivity and facilitate engraftment of CD3-depleted marrow in MHC-mismatched recipients.

Description: Background: Chronic lymphocytic leukemia (CLL) cells are weakly immunogenic, a property that may contribute to disease progression and inhibit the effectiveness of immunotherapies such as vaccines. Low surface expression of co-stimulatory molecules contributes to this poor immunogenicity. CLL cells express Toll-Like-Receptor-7 (TLR7), a powerful modulator of innate immunity. TLR7 agonists may be capable of enhancing the immunogenicity of CLL cells and thereby increasing T cell mediated killing of CLL. Methods: Circulating CLL cells were isolated directly from consenting patients by negative selection. TLR7 mRNA expression by CLL cells was demonstrated by RT-PCR. CLL cells were then incubated with S28690 (a TLR7 agonist), or with a negative control for 24–72h. Expression of the costimulatory molecules CD80, 83, 86, and 54 was determined by flow cytometry pre and post-incubation. Experiments were repeated in the presence of a NFkB inhibitor (dexamethasone), a p38 MAPK inhibitor, and a protein kinase C agonist (PDB). The effects of S28690 on phosphorylated-IkB and phosphorylated-STAT3 levels were measured by immunoblotting. The capacity of S28690-incubated CLL cells to stimulate T cell proliferation and killing was determined in mixed lymphocyte responses. Results: All tested CLL samples (n=20) expressed TLR7 mRNA, while Jurkat cells (T cell origin) did not. After incubation with S28690, CD80, 83, 86, and 54 surface expression increased on all CLL samples tested. The relative increase varied from 4 to 9-fold and was positively correlated with CD38 expression. NF-kB and p38 inhibitors decreased the effects of S28690 on co-stimulatory molecule expression while PDB amplified the effect. After incubation with S28690, IkB and STAT3 phosphorylation increased in CLL cells. S28690-incubated-CLL cells were able to stimulate moderate T cell proliferation, but did not increase T cell mediated killing of CLL cells. However, CLL cells incubated with both S28690 and PDB (a PKC agonist), exhibited much lower amounts of phosphorylated STAT3, triggered marked T cell proliferation, and stimulated T cell mediated killing of CLL cells. Conclusions: S28690 (a TLR7 agonist) causes increased expression of co-stimulatory molecules by CLL cells in vitro and transforms CLL cells into moderate stimulators of T cell proliferation. The effects of S28690 are synergistic with PKC agonists, potentially as a result of S28690-mediated NFkB activation and concurrent PKC-mediated inhibition of STAT3. These findings may find clinical application in immunotherapeutic approaches to CLL.

Description: Introduction Low-molecular-weight heparin (LMWH) is used in pregnancy for venous thromboembolism (VTE) prophylaxis and for prevention of pregnancy complications, because of efficacy greater than or equal to unfractionated heparin, and a lower rate of side effects observed in non-pregnant patients. Due to the lack of data from large randomized controlled trials to guide physician’s practices, there is a need for increased data on safety and efficacy of LMWH for these indications. Our aim was to evaluate the safety and efficacy of using LMWH during pregnancy for preventing VTE and pregnancy complications by performing a systematic review of data from published literature. Methods Data from published studies on the use of LMWH during pregnancy as VTE prophylaxis or LMWH for prevention of pregnancy complications were identified by searching MEDLINE and EMBASE databases up to the end of 2003. The reference lists from identified articles were also hand searched. Data on the LMWH regime, incidence of VTE, pregnancy complications, clinical outcomes and side effects were extracted and entered into pre-piloted forms. Results Fifty studies reporting 2,322 pregnancies were included in this analysis. LMWH was received antenatally in 1883 (81%) of cases, and only peri or postpartum in 389 (17%) of cases. Dalteparin and enoxaparin were the most commonly used LMWH, but certoparin, nadroparin, rivaparin, and tinzaparin were also used. There were no maternal deaths. VTE was reported in 26 (1.1%) pregnancies. Severe maternal bleeding occurred in 46 (2%) pregnancies and was generally associated with obstetric causes. Thrombocytopenia occurred in 10 (0.4%) pregnancies and was not associated with thrombosis. Minor allergic skin reactions occurred in 23 (1%) pregnancies, and osteoporosis in two (0.09%) pregnancies. Conclusion Data from this systematic review of the literature suggest that LMWH is both safe and effective for use as VTE prophylaxis during pregnancy.

Description: Langerhans cell histiocytosis (LCH) can present in several clinical forms with expected response to therapy categorized by the organ systems involved. Low Risk patients (unifocal bone, skin, or lymph nodes disease) are nearly always cured of the disease, patients with multifocal bone involvement are more likely to have relapses, and High Risk patients (involving liver, lung, spleen, or bone marrow) have only a 50% chance of cure. The etiology of LCH is unknown and controversy exists whether this is purely an immunologic dysfunction or a neoplastic disease since the Langerhans cells have been identified as a clonal proliferation. Understanding interaction of Langerhans cells (LC) with T lymphocytes and macrophages in the lesions of Langerhans cell histiocytosis (LCH) is critical to elucidating LCH pathophysiology. We have used laser capture microdissection to isolate CD1a+ LC and CD3+ T cells from frozen LCH lesions. RNA from the dissected cells was enzymatically amplified, hybridized to a cytokine/growth factor array and analyzed for quantity of gene expression after normalizing to b-actin. A complex expression profile has emerged: Gene Expression in LCH Clinical Groups Low Risk Multifocal Bone High Risk * to ***= low to high expression of a gene, VEGF:vascular endothelial growth factor, FGF: fibroblast growth factor CD1a Cytokines IL-9**, -11***, -17**, Leptin*,TGF- β IL-10**, IL-11**,TGF- β IL-9*,11*,Leptin** CD1a Growth Factors FGF-6*, VGEF*** FGF-6**, VEGF** FGF-6***, VEGF* CD3 Cytokines IL-9**, Lymphotoxin- α IL-9*,-10 IL-9*** Lymphotoxin- α** CD3 Growth Factors GM-CSF* GM-CSF** FGF-5* GM-CSF*** FGF-5** Growth factor and cytokine interactions from these LCH patients with different clinical presentations illustrates the importance of both LC and T-cells as well as the known influence of macrophages on the type of LCH. Although previous studies have shown TNF-a is an important factor, our data illustrates that other cytokines and growth factors may play critical roles, because of their relative higher gene expression when compared with TNF-a. These data will facilitate development of in vitro systems to test our hypotheses that a combination of cytokines/growth factors may be important in varying LCH clinical presentations.

Description: Complete responses (CR) of up to 95% have been reported with both pentostatin and cladribine in patients with hairy cell leukemia (HCL). Although responses are durable, relapses occur even 10 years after treatment. No trial has compared pentostatin to cladribine for HCL. Furthermore, few series achieve sufficient maturity to answer the question: can these agents induce a cure in HCL? We reviewed retrospectively 219 patients with HCL (median follow up (FU) from diagnosis: 12.6 years) to compare pentostatin with cladribine in the treatment of HCL, and to assess the potential for cure in this disease. Diagnosis of HCL and exclusion of HCL-variant were confirmed by central review. Overall response to 1st line pentostatin (n=185) was 96% with CR of 81% and median FU of 10.8 (range 0.3–17.9) years. Response to cladribine was 100% with CR of 82%. No significant difference in response was seen with cladribine (n=34) at median FU of 7.2 (range 0.5–11.5) years. Median disease free survival (DFS) for both agents was 10 years. 38% of patients relapsed 4.9 (range 1–16) years and 4.4 (range 1–10) years after treatment with pentostatin and cladribine, respectively. Although responses were maintained for pentostatin and cladribine when used at 2nd (94% and 100% respectively) and 3rd line (100% and 100% respectively) treatment, CR decreased significantly with each sequential relapse through 70% to 45% (p≤0.01). Attainment of CR at 1st line treatment was significantly associated with increased DFS (p=0.000) as compared to those achieving only partial response (PR). Figure Figure A similar result was seen at 2nd line therapy (p=0.000). DFS also showed a significant decline with sequential treatment (p=0.001) mirroring the reduced likelihood of achieving CR with increasing number of courses. There was some crossover of patients. At 1st relapse, 20 patients received pentostatin of whom 17 had previously received this agent, and 53 patients received cladribine of whom 44 were previously treated with pentostatin. Patients relapsing after an initial CR showed no significant difference in ability to re-attain CR as opposed to PR or no response, whether retreated with the same agent, or switched to the other. However, for patients who initially failed to achieve CR, switching to the alternative agent was associated with an increased rate of CR although this difference did not achieve statistical significance. Known 2nd malignancies (excluding basal cell carcinoma) occurred in 20 patients (9 %). In a separate group of 7 patients who were never treated with either agent, 2 patients developed 2nd malignancies. We demonstrated equivalent efficacies of pentostatin and cladribine in the treatment of HCL. Median survival has not been reached for either agent. At 10 years FU, the survival is 83% (pentostatin) and 90% (cladribine). DFS curves show no plateau for either agent with relapses occurring up to 16 years after pentostatin treatment. True cure in this disease thus remains elusive, however, our results suggest that first line CR with purine analogue therapy should be the prime aim of HCL treatment.

Description: Background: The conventional anticoagulant management of patients with nonvalvular atrial fibrillation (AF) involves warfarin, administered to achieve a target international normalized ratio (INR) of 2.0–3.0. Ximelagatran is a novel oral direct thrombin inhibitor that, unlike warfarin, has a predictable anticoagulant effect and does not require dose adjustments based on the INR. In the SPORTIF III and V trials, ximelagatran was as effective as warfarin in preventing stroke and other thromboembolic complications in patients with nonvalvular AF. However, these studies were not designed to determine if ximelagatran was associated with a lower risk of major bleeding. Furthermore, these studies did not compare the case-fatality rate, time course, and anatomic sites of bleeding in ximelagatran- and warfarin-treated patients. Methods: We undertook a pooled analysis of the SPORTIF III and V trials that involved 7329 patients with AF who received oral ximelagatran, 36 mg twice daily, or warfarin, administered to achieve a target INR of 2.0–3.0. Patients had nonvalvular AF and 1 or more risk factors for stroke: hypertension; age ≥75 yrs; previous stroke, transient ischemic attack or systemic embolism; left ventricular dysfunction; age ≥65 yrs and coronary artery disease; or age ≥65 yrs and diabetes. Major exclusion criteria were: mitral stenosis; previous heart valve surgery; transient AF; and increased risk for bleeding. Bleeding event rates were compared using Fisher’s exact test and the log-rank test and were expressed as the number of bleeds per year. The case-fatality rate of major bleeding was defined as the number of fatal bleeds divided by fatal plus nonfatal bleeds. Results: The annual incidence of major bleeding was 2.0% in ximelagatran-treated patients and 2.7% in warfarin-treated patients (P = 0.029). The annual incidence of any (major or minor) bleeding was 31.7% in ximelagatran-treated patients and 38.8% in warfarin-treated patients (P 〈 0.0001). If episodes of intracranial hemorrhage were excluded, the annual incidence of major bleeding was 1.9% in ximelagatran-treated patients and 2.4% in warfarin-treated patients (P = 0.054). The case-fatality rate of major bleeding was 8.2% in ximelagatran-treated and 8.1% in warfarin-treated patients (P = 0.98). The time course and anatomic sites of major bleeding were not significantly different in ximelagatran-treated and warfarin-treated patients. Most bleeds involved the gastrointestinal tract, urinary tract or soft tissues. Conclusion: In patients with nonvalvular AF who require long-term anticoagulation, treatment with oral ximelagatran, 36 mg twice daily, is associated with a lower risk of bleeding complications than warfarin.

Description: The goal of the study was to evaluate long-term outcome of acute myeloid leukemia (AML) patients treated within the PALG 1999 DAC vs, DA Study. Between 1999–2002, 445 patients, aged 18–60 years, were randomized to the induction DAC-7: daunorubicin 60 mg/m2/d iv 1–3; cytarabine 200 mg/m2/d ci 1–7; cladribine 5 mg/m2 2h inf. iv d 1–5 or standard DA-7 regimen (the same regimen without cladribine). Patients achieving CR received two courses of subsequent intensive consolidation: 1) HAM (HD AraC, mitoxantrone) 2) HD AraC with or without cladribine in the DAC-7 or DA-7 arm, respectively. In case of PR after the first induction course the same regimen was repeated, NR patients received CLAG (2-CDA, HD-AraC, G-CSF), regardless the randomization arm. Post-consolidation therapy was in both arms comparable. As previously reported, a single course of DAC-7 induction resulted in 17% higher CR rate compared to the DA-7 treatment. The difference was particularly pronounced in a population of patients 〉40 years and for those with initial WBC 〉100x109/L. In the latter subgroup also the overall CR rate (achieved after 〉=1 induction course) was higher in the DAC-7 arm (71% vs. 43%). [Leukemia. 2004 May;18(5):989–97] In the present report we analyzed long-term outcome (median follow-up 3.2 years) in the whole study group and in pre-defined subgroups taking into account initial tumor burden, age, cytogenetics, FAB subtype, and preceding myelodysplasia. At five years the overall survival (OS) rate equaled 31% for DAC-7 and 25% for DA-7 arm (p=0.42) and leukemia free survivall (LFS) 32% vs. 29% (p=0.38), respectively. The subgroup analysis revealed higher probability of the OS in patients with initial WBC ≥100 G/l assigned to DAC-7 compared to DA-7 arm (39% vs. 11%, p=0.02). The LFS rate and the probability of relapse equaled 50% and 32% (p=NS) and 36% and 68% (p=0.0497), respectively. In patients aged 〉40 years, the therapy containing cladribine was associated with improved LFS (30% for DAC-7 vs. 20% for DA-7, p=0.01), and a tendency to improved OS (28% vs. 18%, p=0.09). In this subgroup DAC-7 therapy resulted in reduced relapse incidence (60% vs. 69%, p=0.04).We conclude that addition of cladribine to induction/consolidation therapy of AML may improve long-term outcome in higher age ranges patients and in those with high tumor burden. The improvement results mainly from reduced risk of relapse, and, in patients with high initial WBC, from higher CR rate.

Description: Chronic lymphocytic leukemia (CLL) is an incurable adult leukemia characterized by disrupted apoptosis. While the majority of patients with CLL are asymptomatic at diagnosis, most progress and require therapy. Identification of new targets and therapeutic agents is therefore a high priority for the treatment of CLL. Synthetic chemistry yielded derivatives of the COX-2 inhibitor, celecoxib, with increased ability to induce apoptosis in the 1–10 μ M range in prostate cancer cells, a similar proposed mechanism of action, and increased in vivo activity in a murine prostate cancer xenograft model. Based upon these data, a Rapid Access to Intervention Development (RAID) proposal is underway to generate OSU03012 for clinical studies in prostate cancer. In addition, we are examining the biologic effects of these new agents in primary CLL cells and lymphoblastic cell lines, showing a novel mechanism of cell killing independent of caspase activation and bcl-2 over-expression. To determine the in vitro activity against CLL cells, 11 CLL patient PBMCs were incubated in various concentrations of OSU03012. The LC50 at 24 hrs was 7.12μM and decreased to 5.45μM at 72 hrs. We show both early (annexin-V positive) and late (both annexin-V/PI positive) apoptosis concurrent with loss of mitochondrial membrane potential typical of apoptosis. These data suggest OSU03012 is highly cytotoxic toward CLL cells in vitro at doses well below those attainable without toxicity in a murine model. Additionally, we show that OSU03012 mediates apoptosis by activation of the intrinsic, mitochondrial pathway of apoptosis but also activates alternative caspase independent cell death pathways. CLL cells from 8 patients were incubated in 10μM OSU03012 for 24 hrs and assessed for caspase-3 and PARP. Immunoblots reveal a dose dependent increase in active caspase-3 concurrent with a decrease in the pro-form. This occurred concurrently with the appearance of the 85 kD cleaved product of PARP that is a known downstream target of caspase-3. In the same 8 patient lysates we saw no change in the inactive pro-form of caspase-8, but consistent processing of caspase-9. These data suggest that OSU03012 in part utilizes the intrinsic pathway of apoptosis to promote CLL cell death. Incubation of CLL cells with z-VAD-fmk and OSU03012 did not abrogate cell death, but eliminated processing of caspase-9, caspase-3 and PARP, suggesting that this agent also activates caspase independent mechanisms of cell death. Given the caspase dependent and independent pathways utilized by OSU03012, we assessed the dependence of cell death on bcl-2 expression. Here we show that bcl-2 over-expression in the 697 lymphoblastic cell line greatly diminishes the apoptosis observed with fludarabine, but potent apoptosis is equally observed with OSU03012 compared to the empty vector cell line. Furthermore, in the bcl-2 over-expressing cell line, caspase-3 and PARP cleavage was not observed despite equivalent apoptosis supporting further multiple mechanisms of cell killing induced by OSU03012. In summary, OSU03012 is an oral bioavailable therapeutic agent that has potent in vitro activity against primary CLL cells. This cytotoxicity is mediated by both caspase dependent and independent pathways and can overcome bcl-2 over-expression. These data provide support for further investigation of the mechanism of action of OSU03012 in CLL cells and performance of early Phase I studies in CLL as part of the RAID process.

Description: Flavopiridol has in vitro activity in CLL and promotes apoptosis independent of p53 function or prior fludarabine exposure. We sought to determine if flavopiridol administered using two different schedules has activity in CLL. Patients with previously treated CLL were enrolled on two sequentially performed phase II studies. Patients in the first trial received flavopiridol (50 mg/m2 daily) as a continuous infusion (CI) over 72-hours every 2 weeks. Patients in the second trial received flavopiridol 50 mg/m2 as a 1-hour intravenous bolus (IVB) daily for three days repeated every 3 weeks. Patients received up to 6 (CI cohort) or 8 (IVB cohort) cycles of therapy. Fifteen patients enrolled in the 72-hour CI phase II trial; 6 (40%) had intermediate (Rai stage I or II) and 9 (60%) high (Rai stage III and IV) risk stages. No responses were noted in this group with 27% having stable disease (SD) and 73% progressive disease (PD). Thirty-six patients enrolled in the IVB study, with 13 (36%) having intermediate and 23 (64%) having high-risk disease. Four patients (11%) had partial responses, 19 (53%) SD, and 13 (36%) PD. The progression-free survivals for responders in the IVB study were 2.9, 3.2, 8.7, and 19.3 months. The median progression-free survival was 2.1 months (95% confidence interval [CI] 1.8 – 3.8) for patients in the CI study and 3.2 months (95% CI [2.5 – 7.4]) for the IVB study. The median overall survival was 27 months (95% CI [20–42]) for the CI study and 24 months (95% CI [18–31]) for the IVB study. Toxicity was manageable and included mainly myelosuppression (granulocytopenia and thrombocytopenia), infections, diarrhea and fatigue. Grade 3 and 4 toxicities were 20% and 27%, respectively, on the CI study and 39% and 33% on the IVB study. One patient on the IVB study had tumor lysis syndrome that was managed medically and did not require dialysis. There was one on-study death following a myocardial infarction on the IVB study. We conclude that flavopiridol has modest, schedule-dependent clinical activity in relapsed CLL and warrants future investigation utilizing alternative schedules of administration.

Description: Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder of early childhood. Although allogeneic stem cell transplantation can induce long-term remissions, relapse rates remain high, and innovative approaches are needed. Since donor lymphocyte infusion in JMML is efficacious, T cell mediated immunotherapy may be effective, and appropriate antigenic targets must be identified. One candidate tumor-associated antigen for the immunotherapy of JMML is γ-globin, which is expressed at high levels in most JMML patients. Most clonogenic JMML cells constitutively express this onco-fetal protein, which is not necessary for the normal erythropoesis of children and adults. To determine whether γ-globin can serve as a target for immunotherapy in JMML, we sought to determine whether γ-globin is naturally processed and presented by the HLA complex. Using conventional bioinformatic techniques and the T2 binding assay to predict candidate epitopes, we identified 4 γ-globin derived peptides (g031, g071, g105, and g106) that were predicted to bind to the HLA-A2 molecule in vitro. Since this strategy provides no evidence for which predicted epitopes are processed and presented by tumor cells in vivo, we employed a biochemical strategy to determine which peptides are naturally processed and presented. This step is critical in certifying that a candidate peptide epitope is an appropriate target for immunotherapy treatments. Using our K562-derived artificial APC (aAPC), an APC that expresses A2 and no other HLA allele, we introduced the EGFP-γ-globin fusion gene. We then acid stripped peptides directly from the surface of one billion aAPC/EGFP-γ-globin cells without subjecting the cells to detergent mediated lysis. Peptides less than 5 kDa in size were fractionated by reverse phased HPLC analysis and analyzed by mass spectrometry. We identified two mass spectrometry peaks which corresponded to γ-globin derived peptides, g031 and g105. Of these, the identity of one peak, g105, was successfully confirmed by peptide sequencing, providing strong evidence that g105 is naturally processed and presented by aAPC/EGFP-γ-globin cells. Next, to confirm that g105 is processed and presented by primary JMML cells, we generated γ-globin specific CD8+ cytotoxic T cells (CTL) from A2 positive healthy donors using synthetic g105 peptide. γ-Globin specific CTL were able to specifically cytolyze A2+ γ-globin+ JMML cells but not A2+ γ-globin- JMML cells. Specific cytotoxicity was blocked by anti-A2 mAb but not isotype control. These results show for the first time that the γ-globin derived peptide, g105, can serve as a target epitope for the CTL directed immunotherapy of JMML. Furthermore, these results illustrate an innovative aAPC based strategy that can identify the antigenic peptide epitopes of putative tumor associated antigens that are naturally processed by tumor cells, presented via HLA class I, and can serve as targets for effective anti-cancer immunotherapy.

Description: An initial exposure to lipopolysaccharide (LPS, also known as endotoxin), which activates macrophages via Toll-like receptor 4 (TLR4), induces a transient state of hypo-responsiveness to a subsequent challenge with LPS. The mechanism underlying this phenomenon, termed endotoxin tolerance, remains poorly understood although the activation/upregulation of a number of negative regulators has been implicated. Related to this, we recently demonstrated that the SH2-containing inositol-5′-phosphatase, SHIP, which is a key negative regulator of the PI3K pathway in hematopoietic cells, is essential for endotoxin tolerance. Specifically, we found that SHIP−/− bone marrow derived macrophages (BMmΦs) do not display endotoxin tolerance. Moreover, an initial LPS treatment of wild-type BMmΦs increases the level of SHIP protein 10 fold and this increase is critical for the hypo-responsiveness to a subsequent LPS stimulation. Interestingly, this increase in SHIP protein is mediated by the LPS-induced production of autocrine-acting TGFβ and neutralizing antibodies to TGFβ, as well as antisense oligonucleotides to SHIP, block LPS-induced endotoxin tolerance. In vivo studies with SHIP+/+ and −/− mice confirm these in vitro findings and show a correlation between the duration of endotoxin tolerance and elevated SHIP levels (Sly et al., Immunity, in Press). We have now followed up on these studies by exploring the role of SHIP in the stimulation of other Toll-like receptors. Specifically, we asked whether SHIP plays a role in repressing CpG-induced pro-inflammatory cytokine production, which is mediated via the activation of TLR9. Our results, using SHIP+/+ and −/− BMmΦs, suggest that SHIP negatively regulates this TLR as well and is required for CpG-induced tolerance. Moreover, we found that CpG treated SHIP−/− BMmΦs show higher pro-inflammatory cytokine production compared to their SHIP+/+ counterparts when challenged with LPS. This indicates that SHIP plays a critical role in dampening down inflammatory responses in the innate immune system.

Description: Myelodysplastic Syndrome (MDS) results from ineffective hematopoiesis characterized by peripheral cytopenia(s) and hypercellular bone marrow. Both enhanced proliferation and accelerated apoptosis are hallmarks of the early phases of MDS, while the latter phases are characterized by diminished apoptosis and the accumulation of blasts. The genetic and biochemical basis of this clonal disorder is poorly understood; cell lines and mouse models are few and incomplete. We propose that patients with Severe Congenital Neutropenia who have a ten thousand-fold risk of developing MDS/AML provide a well-defined entity to study the signaling dysfunction in MDS. Almost all of these patients have a truncated G-CSF Receptor. We then compared Ba/F3 cells expressing either the full-length or the truncated G-CSF Receptor. Stimulation with G-CSF led to sustained Lyn tyrosine kinase activity. Altered PI 3-kinase signaling was evidenced by enhanced and sustained Akt serine kinase activity with diminished tyrosine phosphorylation of the adaptor molecule Gab2 and the inositol phosphatase SHIP-1. Furthermore, an in vitro lipid phosphatase assay showed decreased SHIP activity in cells expressing truncated G-CSF Receptor following G-CSF stimulation. Both Gab2 and SHIP-1 critically regulate PI 3-kinase activity in hematopoietic cells. We next sought to corroborate these findings in primary hematopoietic cells from adult patients with MDS. Biochemical analysis of CD3/CD19 depleted bone marrow mononuclear cells from patients with mid- to late-stage MDS revealed similar findings: increased tyrosine phosphorylation of the activated form of Lyn (of 13 specimens, 10 demonstrated 2 to 30-fold increase by western blotting when compared to CD34+ cells from umbilical cord blood), increased serine and threonine phosphorylation of Akt (8/10 specimens), and decreased tyrosine phosphorylation of Gab2 (6/6) and SHIP-1 (9/9). Importantly, decreased total protein levels of Gab2 and SHIP-1 accounted for their decreased tyrosine phosphorylation. Real-time PCR studies confirmed expression of Gab2 and SHIP-1 in MDS patients, but the level of SHIP-1 transcripts was ~40% less than that in CD34+ cells. Short-SHIP (SIP110), a 104 kDa form of SHIP-1 that lacks the SH2 domain and is expressed only in embryonic stem cells and hematopoietic stem cells, was not identified by RT-PCR in MDS mononuclear cells, and hence, it could not substitute for SHIP-1 deficiency. Ongoing studies are examining hypermethylation state of these genes and their sensitivity to azacytidine. Altogether, these findings suggest that enhanced proliferation and diminished apoptosis may be due to changes in PI 3-kinase regulation and activity in some patients with MDS, which is consistent with both cell lines expressing the truncated G-CSF Receptor and the SHIP−/ −, PTEN+/− mouse model for MDS (Blood103:4503, 2004).

Description: We investigated the antileukemic activity and molecular mechanisms of action of a newly synthesized ring-substituted diindolylmethane (DIM) derivative, named, 1,1-bis [3′-(5-methoxyindolyl)]-1-(p-t-butylphenyl) methane (DIM #34), in myeloid leukemic cells. DIM #34 inhibited leukemic cell growth via induction of apoptosis. DIM #34 inhibited clonogenic growth and induced apoptosis of AML CD34+ progenitor cells but spared normal progenitors. DIM #34 induced loss of mitochondrial membrane potential, which was accompanied by the release of cytochrome c into the cytosol and early cleavage of caspase-9 followed by the cleavage of caspases -8, and -3. Bcl-2 overexpression and caspase-9-deficient cells were partially protected against DIM #34-induced apoptosis, suggesting activation of the intrinsic apoptotic pathway. DIM #34 induced Bax cleavage, and Bax knockout cells were partially resistant to cell death. Furthermore, DIM #34 transiently inhibited the phosphorylation and the activity of the extracellular-regulated kinase (ERK) and abrogated Bcl-2 phosphorylation. Because other methylene substituted DIM analogs transactivate the nuclear receptor PPARγ, we studied the role of PPARγ in apoptosis induction. Although the co-treatment of cells with a selective PPARγ antagonist T007, and a low dose of DIM #34 partially diminished apoptosis, apoptosis was not inhibited at higher concentrations of DIM #34, suggesting the involvement of both, receptor-dependent and independent mechanisms. Co-treatment with RXR- and RAR-ligands enhanced DIM #34-induced cell death. Together, these findings showed that substituted DIMs represent a new class of compounds that selectively induce apoptosis in AML cells through interference with ERK and activation of PPARγ signaling pathways.

Description: The nucleoside analogue pentostatin has clinical activity in B-Cell Chronic Lymphocytic Leukemia (CLL) and has shown significant activity and minimal toxicity when combined with cyclophosphamide for previously treated CLL. Building on this, we initiated a trial in 2002 of combined pentostatin (2mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375mg/m2). Of the 33 enrolled eligible patients included in these analyses, seventeen patients were in the high Rai risk group, 25 were male, and median age for this cohort was 62 yrs (range: 40–79); 17 were non-mutated for the immunoglobulin heavy chain variable region gene, and the majority (67%) were CD38 negative. Only 5 patients had no detectable chromosomal abnormalities by FISH at baseline, 20 had a single FISH anomaly, and 8 had 2 or more FISH anomalies. Of all 28 pts with any anomaly, the following specific abnormalities were detected: 13q- (n=17), +12 (n=8), 11q- (n=7), 17p- (n=2), t(14;18) (n=1), 6q- (n=1), and MDM2 (n=1). Of the 33 patients, 22 had grade 3+ toxicity; 16 patients had non-hematologic toxicity wtih the most common symptoms being nausea (6) and vomiting (4). One patient died on study of grade 5 hypoxia as well as hypotension and this was deemed possibly related to treatment. Almost all patients (32/33; 97%) had a best response of PR or better. Including review of bone marrows done 2 months post-treatment, there were 11 CRs (complete response), 7 nPRs (nodal partial response) and 13 PRs. No differences were observed between type of response and mutation status or CD38+ status. Post-treatment FISH analyses were available on 27 patients; results for 25 patients became normal after treatment. Of the remaining 2, one patient was 13q- x1 and went from 94% to 27.5% abnormal nuclei after treatment; the other is the patient who died on study. To establish minimal residual disease (MRD) post-response, we used three color flow cytometry to detect CD5+/CD19+/CD79b− B cells. This approach found all patients had a reduction in CLL B cells with a median reduction of 91% (range: 5 – 100%). Of interest, all 3 response groups (CR vs. nPR vs. PR) had patients with significant reductions in CLL B cells (i.e., 〉90%). The nPR group was most variable in terms of MRD (median 47%; range: 5–93%) compared to the CR (median: 91%; range: 42–99.9%) and PR (median: 97%; range: 46–100%) groups. In conclusion this novel regimen of pentostatin, cyclophosphamide and rituximab has demonstrated significant clinical activity irrespective of risk stratification parameters with rapid induction of responses, achievement of minimal residual disease in some, and modest toxicity. Patients continue to be accrued to further explore correlative measures of response to treatment.

Description: The RUNX1/AML1 gene is essential for hematopoiesis and is most frequently involved in human leukemias. Loss-of-function of RUNX1 is the common underlying mechanism of RUNX leukemias: those carrying t(8;21), inv(16) and RUNX1 point mutations. As the weakest mode, haploinsufficiency of RUNX1/AML1 causes familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). Mice heterozygous for Runx1 null mutation, Runx1+/−, appear genetically equivalent to human FPD/AML patients, but showed no apparent defects and did not develop spontaneous leukemia, implying that additional genetic alterations are required for leukemia. To identify such abnormalities, retroviral insertional mutagenesis was employed by using BXH2 mice that have spontaneously transmittable ecotropic retrovirus. BXH2-Runx1+/− mice showed a shorter disease latency and more myeloid-specific phenotype than wild-type littermates. Therefore, BXH2-Runx1+/− mice appear to serve as the mouse model for FPD/AML to investigate myeloid leukemia. The genes selectively affected in BXH2-Runx1+/− mice by retroviral integration are likely to cooperate with Runx1+/−. c-Kit locus was involved twice in 24 BXH2-Runx1+/− but neither in 17 wild-type littermates nor in 135 inbred BXH2 mice reported in the RTCGD database, suggesting that c-KIT mutation cooperates with RUNX alteration in human leukemia. In fact, constitutively active mutants of c-KIT or functionally equivalent FLT3, were found in 10 out of 25 (40%) of human RUNX leukemias. Together, BXH2-Runx1+/− system appears useful to identify the genes cooperating with RUNX1 abnormalities.

Description: With the advent of reduced intensity conditioning regimens, new treatment options are available to patients who suffer relapse of Hodgkin’s lymphoma (HL) following autoSCT, but the efficacy of this procedure is not proven. We retrospectively compared the cohort of adult patients who received alloSCT for relapse of HL following autoSCT to those who did not at a single institution. 64 patients underwent autoSCT for HL between 7/87 and 5/02: 41 for relapsed disease, 16 for primary refractory disease and 7 unclassified. Thirty-five patients (55%) relapsed at a median 12.5 months (range 0.9 to 140) after autoSCT. Eleven patients underwent alloSCT a median of 182 (35 – 645) days after relapse; 7/11 received reduced intensity conditioning, 5/11 received SCT from an unrelated donor. Of 24 patients who had relapsed after autoSCT (and did not receive an alloSCT), 16 survived at least 6 months and were considered in this analysis. Acute graft-versus-host disease (GVHD) 〉 grade 2 occurred in only one patient, 5 /9 evaluable patients developed chronic GVHD (2 extensive). Five recipients of alloSCT have died: 2 with relapsed disease, 2 with disseminated fungal infections and 1 with interstitial pneumonitis. 6 patients remain alive, 5 remain free of disease. Median event free survival is12.5 months for recipients of alloSCT with median follow up of 3.1 years. Median overall survival is 4.1 years (64 days – 12.5+ years) and estimated overall survival is 71% at 3 years. This compares favorably with a median overall survival of 1.4 years in patients who survived at least 6 months, but did not receive alloSCT after relapse. Improved survival in the alloSCT cohort demonstrates that potent graft-versus-lymphoma activity can be achieved against HL without significant GVHD.

Description: DC play an essential role in the initiation of (allo)immune responses. Downregulation of NF-kB activity has been shown to prevent the differentiation and maturation of DC. PS-341 a novel antimyeloma terapeutic agent, promotes the degradation of NF-kB. In this study we have investigated whether PS-341 affects DC differentiation and/or maturation. DC were generated by culturing immunomagnetically purified CD14+ monocytes for 1–5 days with GM-CSF (50 ng/ml) and IL-4 (800 U/ml) in the presence or absence of PS-341 at 0.1 to 100 ng/ml. Addition of PS-341 at doses as low as 10 ng/ml significantly reduced the recovery of viable cells, as determined by trypan blue exclusion assay, both after 24h and 48h of culture (3±7% vs 35±25% in control cultures and 3±7% vs 32±25%, respectively, at 24 and 48 hours) (n=5 experiment, p

Description: We have previously reported that CD45 functions as a positive regulator for myeloma cell proliferation in the downstream of interleukin-6 (IL-6) by regulating activation of Lyn (Blood99: 2172, 2002). It remains to be clarified how CD45 regulates IL-6-induced proliferation in myeloma cells. In this study, we aim to explore the regulation and isoform-specific difference on the mechanism of CD45 translocation induced by IL-6 in human myeloma cells. We used myeloma cell lines, U266 and ILKM2, which express CD45RO and CD45RB but not CD45RA in this study. We showed that methyl-beta-cyclodextrin, a lipid rafts inhibitor, significantly inhibited IL-6 induced proliferation, diminished phosphorylation of Lyn and signal transducer and activator of transcription 3 (STAT3), and abolished nuclear localization of STAT3, suggesting the significant role of lipid rafts on IL-6 signaling. Using sucrose density gradient fractionation method, we observed the dramatic change of CD45 distribution by IL-6 stimulation. Without IL-6 stimulation, CD45RO and RB molecules were excluded from lipid rafts. But, IL-6 stimulation results in significant translocation of both isoforms to lipid rafts. On the other hand, IL-6R alpha, gp130, Lyn and a raft integral protein, flotillin-2, were always localized inside lipid rafts. Furthermore, it was also confirmed that IL-6 induced CD45RO and CD45RB translocation to lipid rafts by confocal laser scanning microscopy, while IL-6R alpha always remained inside rafts. These observations have also been confirmed by the exogenous expression of CD45RO, RB tagged with green fluorescent protein in CD45 negative U266 cells. As negative control, Epstein-Barr Virus transformed B cells, C066 or KUS, which express CD45RA and CD45RB, but not CD45RO, were used. BCR cross-linking using anti-IgM stimulation induced IgM and Ig beta translocation to lipid rafts, but both CD45RB and RA showed no marked translocation. And we couldn’t observe marked translocation of CD45RB nor CD45RA to rafts after IL-6 stimulation. As the level of IL-6R alpha was very low in these cells, we introduced IL-6R alpha gene into KUS or C066 cells. Then, translocation of CD45RB to lipid rafts was observed while CD45RA did not in response to IL-6 stimulation. In addition, we confirmed IL-6 induced complex formation of CD45 with IL-6R alpha, Lyn on lipid rafts. We also checked the phosphorylation state of two tyrosine residues (Tyr396 and Tyr507) of Lyn. In CD45 positive cells, IL-6 induced dephosphorylation of the negative regulatory tyrosine, Tyr507, after that, Tyr396, the positive regulatory site of kinase activity, was phosphorylated. While, in CD45 negative cells, Tyr507 dephosphorylation and significant phosphorylation of Tyr396 were not observed. And after IL-6 stimulation, we also observed PAG dephosphorylation and Csk release from lipid rafts, which might finally lead to the dephosphorylation of Tyr507 of Lyn. Considering these results, we conclude that CD45RO and RB translocation to lipid rafts is significant for myeloma cells to proliferate in response to IL-6.

Description: Multiple Myeloma (MM) is a malignancy of monoclonal plasma cell proliferation in the bone marrow, which accounts for 10% of all hematologic cancers. This slow proliferating B-cell malignancy accumulates apoptosis-resistant and replication-quiescent cell populations, posing a challenge for current chemotherapeutics, which target rapidly replicating cells. MM remains an incurable disease in need of new therapeutic approaches. We have previously shown that the nucleoside analog, 8-chloro-adenosine (8-Cl-Ado), has significant activity for MM and that its sister compound, 8-amino-adenosine (8-NH2-Ado), appears to be even more potent, exhibiting greater activity in pre-clinical studies. 8-NH2-Ado is cytotoxic to MM cell lines both sensitive and resistant to conventional chemotherapies. This cytotoxic effect requires phosphorylation of 8-NH2-Ado to its tri-phosphate form, 8-NH2-ATP, and accumulation of 8-NH2-ATP results in a concomitant loss of endogenous ATP levels. 8-NH2-Ado induces apoptosis in MM cells as measured by an increase in Annexin V binding, a decrease in mitochondrial membrane potential, an increase in caspase activity, cleavage of caspase substrates, and an increase in cells with a subG1 DNA content. Here we demonstrate the novel effect of 8-NH2-Ado on the phosphorylation status of key cellular signaling molecules. MM cells treated with 8-NH2-Ado exhibit a dramatic loss of phosphorylation of several important signaling proteins, including ERK1/2, p38 MAPK and Akt kinase, within 30–120 minutes. Cells depleted of ATP by antimycin A or 2-deoxyglucose treatments to mimic 8-NH2-Ado-induced ATP depletion do not exhibit the same decrease in phosphorylation of vital cellular proteins. Therefore, the significant shifts in endogenous ATP pools caused by 8-NH2-Ado treatment cannot account for the changes in phosphorylation levels. Instead, 8-NH2-Ado may be affecting the activity of protein phosphatases involved in the negative regulation of these signaling molecules. Based on studies using the phosphatase inhibitor okadaic acid, it appears that PP2A may play a role in the 8-NH2-Ado-induced decrease in phosphorylation of p38. The distinctive effect of 8-NH2-Ado on the phosphorylation status of cellular proteins is a novel phenomenon for a nucleoside analog drug and appears to be unique to 8-NH2-Ado among this class of drugs. The kinetics of 8-NH2-Ado-mediated changes in phosphorylation levels of critical pro-survival and apoptosis-regulating proteins suggest that the modulation of these proteins by de-phosphorylation at early time-points may be an important mechanistic step in 8-NH2-Ado-induced programmed cell death.

Description: Treatment of Waldenström’s macroglobulinemia (WM) is based on the use of oral alkylating agents, even though recently, more expensive drugs have been proposed for its treatment.The aim of this study was to report the results obtained combining oral melphalan, cyclophosphamide and prednisone (MCP) in 72 WM patients, and to compare the results and related costs with those observed using more aggressive and expensive protocols. Between July 1973 and April 2002, we have observed 100 newly diagnosed WM, of whom 72 were symptomatic and therefore requiring a treatment. The antineoplastic treatment consisted of melphalan (6 mg/m2, days 1–7), cyclofosfamide (125 mg/m2, days 1–7) and prednisone (40 mg/m2, days 1–7) administered orally for a total of 12 courses. Thereafter, responding and stable patients were maintained with chlorambucil (3 mg/m2) and prednisone ( 6 mg/m2) until progression. Levels ≥1.5 g/dL of IgM immunoglobulin associated to 30% of lymphoplasmocytic cells in the bone marrow aspirate were required for diagnosing WM. Among these patients, 46 (63%) were men and 26 (47%) were women. Median age, IgM Immunoglobulin and Hb levels were at diagnosis: 61 years (range 42–87 years), 3.8 gr/dl ( range 1.5–6.5) and 12 gr/dl ( range 6.5 – 16) respectively. Of these patients, we evaluated response rate, overall survival (OS), response duration (RD), freedom from progression (FFP), event free survival (EFS) duration, toxicities and costs/course in € of the drugs utilized.Of the 72 treated patients,71 (98.6%) were evaluable and 55 (77.5%) achieved a response, 7 (9.9%) showed a disease stabilization and 9 (12.6%) disease progression. After a median follow-up of 72 months (range 3–195 months), median OS, RD, FFP and EFS were: 66, 64, 55 and 47 months, respectively. No grade III or IV WHO toxicities were observed and toxicity was limited to transient nausea, vomiting and mild neutropenia. The cost/course for MCP was € 13, similar to that of standard protocols based on chlorambucil, and much less expensive compared to the costs of more aggressive protocols proposed for the treatment WM, ranging from € 74 to € 3260.90.In conclusion,the MCP is, as chlorambucil based protocols, a cost effective and safe option for treating patients with WM. Moreover, the obtained results do not appear inferior to those so far reported with more expensive, aggressive and toxic intravenous protocols.

Description: Less than 60% of patients with B-cell non-Hodgkin’s lymphoma (B-NHL) can be cured with contemporary therapy. Using artificial receptors it is possible to redirect the specificity of immune cells to tumor-associated antigens, a strategy that holds great potential as a novel cancer therapy. Since B-NHL cells invariably express CD19, we transduced human peripheral blood T lymphocytes with a recently developed receptor (anti-CD19-BB-ζ), which consists of the single-chain variable domain (scFv) of an anti-CD19 monoclonal antibody, the hinge and transmembrane domains of CD8α, and the signaling domains of CD3ζ and 4-1BB. CD3ζ delivers the primary stimulus upon receptor engagement, while 4-1BB delivers co-stimulatory signals that are crucial for T-cell cytotoxicity. It has been shown that elicitation of 4-1BB signaling enhances the immune response to tumors in vivo, even when an immune response cannot be induced by CD28 stimulation. Retroviral transduction led to anti-CD19-BB-ζ expression in T cells with high efficiency: median percent of transduced cells was 60.3% (range, 25.7%–83.4%; n = 9). T lymphocytes expressing anti-CD19-BB-ζ expanded more vigorously that T cells transduced with receptors lacking 4-1BB and exerted powerful cytotoxicity against the CD19+ B-NHL cell lines Raji, Daudi, RL, and HT in vitro: at a 0.5: 1 effector: target ratio, mean (± SD) cell specific lymphoma cell killing was 96.6% ± 4.6% after 5–7 days of culture (4 experiments in each cell line). Transduced T cells were also effective against freshly isolated cells from patients with diffuse large, follicular large, Burkitt, and mantle cell lymphoma cultured on bone marrow-derived mesenchymal cells: in 10 samples, cell killing was 93.6% ± 5.7% at a 0.5: 1 ratio after 5–7 days of culture. Sensitivity to anti-CD19-BB-ζ-mediated killing was observed regardless of high Bcl-2 expression. T cells expressing anti-CD19-BB-ζ were also effective in a xenograft model of NHL, in which NOD/SCID mice were inoculated subcutaneously with lymphoma cells (1 x 107). Subsequent inoculation of T cells (2 x 106) transduced with anti-CD19-BB-ζ receptors significantly suppressed tumor growth, whereas inoculation of T cells transduced with empty control vector had no effect (3 mice for each treatment). These results provide a rationale for clinical testing of autologous T cells modified with anti-CD19-BB-ζ receptors in patients with aggressive or relapsed B-NHLs refractory to conventional therapy.

Description: Introduction: few studies have explored the usefulness of a prognostic index specifically devised for patients with localized DLBCL. The IIL has performed a retrospective analysis of a large group of patients with limited stage DLBCL and developed a new prognostic model. Results: 1,252 patients with localized (Ann Arbor stage I-II) aggressive B-cell lymphoma (IWF: G or H, WHO:DLBCL) diagnosed from 1988 to 2002 and without CNS involvement, treated by 4 cooperative groups and 2 single institutions, are the subject of this analysis. Patient’s median age was 57 years (range, 17–91) and M/F ratio was 1.26. Clinical stage was I in 239 (19%), IE in 303 (24%), II in 356 (28%) and IIE in 354 (28%) patients, respectively. Supradiaphragmatic disease was present in 56% of patients, 13% had 〉 3 nodal sites, 53% had extranodal involvement, and 7% had 〉1 extranodal site. Bulky disease (≥10 cm) was present in 26% of patients, ECOG-PS 〉1 in 12% and B symptoms in 14%. Abnormal biochemical data included: elevated LDH (28%), β2-microglobulin (B2M;19%) and ESR (38%) and reduced albumin (〈 3.5 g/dL) in 21% of the cases. Patients were treated with anthracyclin-containing regimens ± IF-RT. After a median follow-up of 62 months for alive patients (range 1–183 months), 3 and 5-year OS rates were 73% and 71%, respectively. By univariate analysis the following 11 variables were found to be predictive of a short survival: age ≥65 yrs (P=0,0001), stage II nodal (P=0,0001), number of nodal sites (P

Description: Outside-in signaling is triggered by αIIbβ3 interaction with fibrinogen or von Willebrand factor and is required for effective platelet thrombus formation on damaged vascular surfaces. It requires ligand-induced αIIbβ3 clustering, which results in the activation of several Src family tyrosine kinases that are directly associated with αIIbβ3. However, the mechanism of Src activation by αIIbβ3 is incompletely understood. Here we demonstrate that the protein tyrosine phosphatase, PTP-1B, is required for integrin activation of c-Src and for outside-in signaling in platelets. In human or mouse platelets, c-Src was constitutively-associated with αIIbβ3 through an interaction that involved the β3 cytoplasmic domain and the c-Src SH3 domain. In resting platelets, the catalytic activity of this pool of c-Src was relatively low due to auto-inhibitory constraints imposed, in part, by an intramolecular interaction between the SH2 domain and the C-terminus of c-Src. This was promoted by phosphorylation of the C-terminal tyrosine of c-Src, tyrosine 529, by the Csk kinase. When platelets became adherent to fibrinogen or bound soluble fibrinogen in response to MnCl2, PTP-1B became associated with αIIbβ3 and c-Src, as determined by co-immunoprecipitation. At the same time, Csk dissociated from the integrin complex, c-Src tyrosine 529 became dephosphorylated, and c-Src became activated, as measured by phosphorylation of activation loop tyrosine 418. In marked contrast, gene-targeted mouse platelets deficient in PTP-1B (PTP-1B−/−) exhibited no dephosphorylation of c-Src tyrosine 529 and no activation of c-Src in response to fibrinogen binding. Furthermore, unlike wild-type platelets, PTP-1B−/− platelets exhibited little or no fibrinogen-dependent tyrosine phosphorylation of c-Src substrates, and they failed to undergo cytoskeletal reorganization or to spread on fibrinogen. These abnormalities of PTP-1B−/− platelets were due to defective outside-in signaling because surface expression of αIIbβ3 was normal, and fibrinogen bound normally in response to platelet stimulation with ADP or PAR-4 receptor agonists. When Fura-2-loaded platelets were infused and visualized by real time intravital microscopy during arterial thrombus development following laser injury to the cremaster microcirculation of a living mouse, PTP-1B−/− platelets exhibited significantly reduced intracellular free Ca2+ responses and formed smaller, less stable thrombi compared to wild-type platelets. Altogether, these studies establish that PTP-1B is a necessary, positive regulator of αIIbβ3-associated c-Src during the initiation phase of outside-in signaling in platelets. Consequently, disruption of specific interactions between αIIbβ3, c-Src and PTP-1B, or abnormalities in the activation of integrin-associated c-Src or PTP-1B, may cause defects in primary hemostasis. Moreover, these interactions provide potential targets for pharmacological blockade of outside-in signaling during arterial thrombosis.

Description: Introduction: The function of tumor-infiltrating T cells in lymphoma is unknown. Previous studies have shown that an increased percentage of tumor-infiltrating CD4+ T cells was associated with a better overall survival in diffuse large B-cell lymphoma (Ansell S. et al. JCO 2001; Xu et al., British Journal of Haematology 2001). Furthermore, the CD4+ T cells observed in one study was associated with a CD3+/HLA-DR + phenotype, suggesting that they were activated T helper cells. Recently, a molecular predictor model for follicular lymphoma was reported based on microarray analysis of gene expression within the cell populations of biopsy specimens. Three groups of “predictor genes” were identified: one representing B-cell differentiation and two groups representing genes expressed by T cells and macrophages, respectively (ASH abstract, 2003). This raised the question of whether it was simply the presence of tumor-infiltrating T cells that correlated with clinical outcome as opposed to specific activation pathways within those cells. Purpose: The purpose of this study was to examine whether the percentage of tumor-infiltrating T cells is correlated with prognosis of follicular lymphoma. Patients and Methods: We identified 293 follicular lymphoma patients with clinical outcomes available within the Stanford lymphoma database, who had undergone an excisional biopsy, and whose tumor biopsy specimens had been analyzed by flow cytometry. These patients were diagnosed between 1977 and 2003. Among these patients, 167 (57%) had follicular small cleaved lymphoma, 112 (38%) had follicular mixed lymphoma, and 14 (5%) had follicular large cell lymphoma. A total of 66 deaths were documented. The overall survival was estimated from the time of diagnosis to the time of death or last follow-up. Biopsies had been analyzed for cell surface marker expression by single parameter flow cytometry. The percentage of CD3+, CD4+, CD8+, CD20+, and HLA−DR+ cells was determined directly. We also estimated activated T cells, defined by HLA−DR+ T cells, by subtracting the value for total B cells from the value for HLA + cells. Cell population percentages were treated as continuous variables and related to overall survival, each in a univariate analysis. Results: We found no statistically significant correlation between the percentage of any cell population and overall survival. For each group (CD3+, CD4+, CD8+, and estimated HLA−DR+ T cells), we used the percentage of the cell population to divide patients into 3 subgroups of equal number of patients: low-, medium-, and high-percentage. We then compared Kaplan-Meier curves of the 3 subgroups for each cell population. We found no significant differences in survival among the 3 subgroups for each of the cell populations, CD3+, CD4+, CD8+, and the estimated activated T cells, after correcting for multiple hypotheses testing. Conclusion: The percentage of tumor-infiltrating T cells or T cell subsets was not correlated with survival outcome in patients with follicular lymphoma. This finding provides the basis for building molecular predictive models based on gene expression analysis, which represents not only the presence of certain cells but their physiologic state as well.

Description: Unmethylated cytosine-phosphorothioate-guanine nucleotides (CpG) are immuostimulators that can activate both innate and adaptive immunity. By binding to its receptor, TLR9, CpG can activate dendritic cells and B cells by inducing the secretion of cytokines, and increasing the expression of costimulatory molecules, such as CD80 and CD86. In this study, a B type CpG, CpG1826, was given intratumorally in an A20 lymphoma animal model. CpG alone can delay the growth of tumor in this model, however, the majority of the treated tumor-bearing mice still die, and it has very little effect on systemic tumor cells. Here we propose a new therapeutic strategy, a combination of cyclophosphomide (CTX), followed by subsequent intratumoral injection of CpG. BALB/c mice were inoculated s.c. with 107 A20 cells in the lower back, and treatment started on day 17 when tumor reached approximately 1.0 cm in diameter. CTX (100mg/Kg) was given i.p. on day 17 and 18, and for the groups that received CpG, CpG was injected intratumorally on day 19,20,21,24, and 26 with 100μg/dose. As shown in Fig.1, the combination of CTX with CpG could cure well-established large size (〉1.2cm in diameter) tumor, and mice remained tumor-free 5–6 months after the initial treatment. Conversely, the majority of mice that received CTX or CpG alone died of lymphoma. Tumor-free mice from this combined maneuver are protected from a lethal dose of A20 challenge even 5 months after the initial treatment. Our studies also showed that this combination is site dependent, as CTX combined with intratumoral injection of CpG had a much better therapeutic effect (100% survival), as compared to injection of CpG into a distant normal site (20% survival). Our studies also indicated that this combined maneuver is CD8+T cell dependent, as no therapeutic effect was observed when we repeated this experiment in a CD8+ T cell knockout model. We also demonstrated the systemic effect of this combination strategy in mice bearing tumors at two different sites. In combination with CTX, injection of one site with CpG controlled the growth of the second site tumor as opposed to a transient effect from chemotherapy or CpG alone. This led to significant prolongation of overall survival (Fig.2). In summary, we have shown that this combined chemoimmunotherapy strategy can cure both local and systemic tumor, which may provide a powerful antitumor therapy in the clinic. Figure Figure

Description: Transforming growth factor-β1 (TGF-β1), an immunosuppressive cytokine, potently suppressed cytotoxic T lymphocyte (CTL) differentiation of human cord blood naïve CD8+ T cells as determined by reduced induction of characteristic phenotypes of effector cells and cytotoxic activity. In contrast, 4-1BB (CD137), a costimulatory molecule in the TNF receptor family, promoted effector and memory CTL differentiation. We present evidence that the suppressive TGF-β1 effects can be effectively reversed by anti-4-1BB costimulnation during in vitro CTL differentiation driven by IL-15 from exclusively naïve cord blood CD8+ T cells. 4-1BB may counteract TGF-β1 effects at the level of TGF-β1 signaling by inhibiting Smad2 phosphorylation induced by TGF-β1. In contrast to 4-1BB, recent studies show that CD30, another member of the TNF receptor family, may play an important role in down-modulating effector or memory CTL responses. Thus, we hypothesized that CD30 may interact with 4-1BB in responding to the TGF-β1 suppression. TGF-β1 inhibits 4-1BB expression on CTLs. Conversely, TGF-β1 greatly up-regulates CD30 expression. IL-4 augments the TGF-β1-mediated up-regulation of CD30 expression resulting in further increase of CD30 expression. In contrast, IL-12 abrogates TGF-β1-induced CD30 expression. Such IL-4 and IL-12 effects on CD30 expression appear to be exactly opposite for 4-1BB expression: down- and up-regulating 4-1BB expression, respectively. Co-engagement of CD30 abrogates 4-1BB expression as well as its activity for reversing TGF-β1-mediated suppression of CTL proliferation particularly in the presence of IL-4. Expression of NKG2D, an important stimulatory receptor for NK and CTL cytotoxic activity is inhibited by TGF-β1. 4-1BB stimulation restores the NKG2D expression inhibited by TGF-β1. Again, co-engagement of CD30 significantly inhibits the 4-1BB activity for restoration of NKG2D expression. Taken together, our data suggest that 4-1BB promotes effector CTL differentiation and CD30 acts as a negative feed-back molecule, promoting TGF-β1-mediated CTL suppression and counteracting 4-1BB activity. In conclusion, we have delineated a novel link between CD30 expression and TGF-β1 which is pivotal to the down-regulation of 4-1BB-induced CTL effector differentiation.

Description: In erythroid cells iron uptake from transferrin (Tf) is utilized largely for heme synthesis. Here we provide evidence that Tf receptor (TfR) expression and cellular uptake of iron from Tf is stimulated by enhanced heme synthesis. Incubation of murine erythroleukemia (MEL) cells with 5-aminolevulinic acid (ALA) resulted in an increase in TfR expression accompanied by enhanced uptake of iron from Tf and incorporation of iron into heme. ALA-mediated enhancement of TfR mRNA expression was completely prevented by succinylacetone, an inhibitor of ALA dehydratase, and N-methylprotoporphyrin, an inhibitor of ferrochelatase, indicating that the effect of ALA required its metabolism to heme. Treatment of cells with ALA was associated with enhanced iron regulatory protein-2 (IRP-2) binding activity, which could be blocked by inhibitors of heme synthesis and supplementation of the culture medium with a permeable iron chelate or Tf. In all cases, IRP-2 activities were correlated exactly with TfR mRNA levels. Thus, in addition to the previously characterized transcriptional up-regulation of TfR expression in differentiating erythroid cells, increased TfR expression mediated by enhanced heme biosynthesis may ensure sufficient iron availability for optimal heme synthesis and prevent possible protoporphyrin accumulation under conditions of inadequate iron supply.

Description: We have shown that high IL-7 transgene (Tg) over-expression (39-fold at day 1 in thymic tissue) under the T cell specific, proximal lck promoter had a dose effect on TCRαβ that was accompanied by active B cell development in the thymus. To further characterize these affects in the thymus of IL-7 transgenic mice, we analyzed thymi from day 18 embryos and newborn Tg mice, as well as fetal thymic organ culture (FTOC) derived from using day 16 embryos. We show that arrested T-cell and increased B-cell thymic development is initiated during fetal development. Using mixed bone marrow chimeras and anti-IL-7 monoclonal antibody injection, we further demonstrate that abnormalities in thymic T and B cell development are non-cell autonomous and are due to IL-7 over-expression. Recently, it was shown that only the early thymocyte progenitor (ETP, c-kit+IL-7R−/lo) fraction within the DN1 subpopulation had a T-cell proliferative potential in contrast to the c-kit−IL-7R+ DN1 subset. Here we show that in Tg mice the ETPs were decreased, while the c-kit−IL-7R+ cells are increased in both percentage and absolute count when compared to normal controls. In order to explore the T vs. B ETP potential, we seeded re-aggregate thymic organ cultures with sorted lin−CD44+CD25−c-kit+IL-7R+ cells. While ETPs derived from normal controls were able to proliferate and produce 83% of DP thymocytes, ETPs sorted from Tg mice developed poorly (10-fold less) into DP cells (30%) and produced 14% of B220+ cells vs. 6% in controls. Moreover, sorted Pro/Pre B derived thymic B cells from Tg mice, but not BM-derived Pro/Pre B cells had the TCRβD-J rearrangement, suggesting a T-specific origin. Since the B-cell differentiation pathway in normal mice is selectively inhibited by thymic presentation of Notch ligands, we hypothesized that IL-7 down-regulates Notch signaling. To test this hypothesis, we analyzed thymocyte progenitors (DN1-DN4) in normal and Tg mice for the intra-cytoplasmic part of Notch, that is cleaved upon Notch/Notch-ligand activation. Notch staining was decreased in the lin−CD44+CD25inter representing the only DN2 population present in these Tg mice. These data favour a decrease of Notch signalling in mice with high IL-7 Tg over-expression, inducing a block in TCRαβ development, and skewing of thymic B cell development by T vs. B lineage subversion. These conclusions may have implications for IL-7 in the clinical setting.

Description: NKG2D is an NK cell activating receptor that is also expressed on CD8+ T cells and γδ T cells where it up-regulates activation through TCR co-stimulation. NKG2D is triggered by ligands that are structurally related to MHC1 and expressed either by normal tissues early in ontogeny or by stress, infection or malignant transformation. The function of NKG2D on NK cells and CD8+ T cells is mediated by two distinct pathways of signaling through its association with two adapter proteins, DAP10 and DAP12. Two NKG2D isoforms that preferentially bind to either DAP 10 or DAP12 have been described in murine species. In evaluating human NKG2D gene expression by RT-PCR in cytokine activated CD8+T cells derived from 7 different donors, we consistently observed two distinct PCR bands differing by 218bp. Cloning and sequencing of both PCR amplicons revealed a previously described alternative spliced NKG2D variant resulting from inclusion of intron 6. The intron 6 inclusion introduces a stop codon almost immediately after the start of the intron 6 sequences resulting in a predicted protein product truncated without an extracellular domain. To determine the functional consequences of truncated NKG2D expression we isolated and cloned both full length NKG2D and truncated NKG2D into pRJ3 retroviral vector for expression as NKG2D full length with dsRed and NKG2D truncated EGFP fusion proteins. Activated human CD8+ T cells were trandused and FACS-sorted for dsRed or GFP+ cells and used against human cell line targets in Cr51 release cytotoxicity assays. We found that expression of the full length NKG2D dsRed resulted in ~15% increase in cytotoxicity while expression of the truncated NKG2D GFP resulted in ~70% (p.0009) reduction in cytotoxicity. Results were controlled for cytotoxicity obtained against the same tumor targets using non treated CD8+ T-cells as well as CD8+ T-cells transduced with an empty vector. To further elucidate the role of each NKG2D isoform, we co-expressed both full length NKG2D and truncated NKG2D-EGFP with either DAP-10 or DAP-12. A similar cloning strategy was employed to generate a retroviral expression vector for DAP10-dsRed, DAP10-gfp, DAP12-dsRed and DAP12-gfp fusion proteins. Co expression of NKG2D with dsRed with either DAP10-gfp or DAP12-gfp resulted in comparable 20–25% increase in cytotoxicity (p=.0001) verses NKG2D alone. Co expression of the full length and truncated form also resulted in significant reduction in cytotoxicity of up to 65% (p.0001). Coexpression of the truncated NKG2D-gfp isoform with DAP10-dsRed or DAP12 dsRed did not alter the reduction in cytotoxicity observed with truncated NKG2D alone. We also performed similar experiments using murine CD8+ T cells with human cell line targets RPMI and U266. erexpression of full length NKG2D resulted in 〉32% increase in cytotoxicity while co-expression of full length NKG2D and DAP10 resulted in 42% increase in cytotoxicity against these human target cell lines. Coexpressing truncated NKG2D and DAP12 resulted in increased cytotoxicity slightly above the background and always less than full length NKG2D. Addition of DAP 10 and DAP 12 to the truncated NKG2D had a minimal impact on cytotoxicity. These results show that the NKG2D isform lacking the extracellular domain inhibits cytotoxicity of activated human CD8+ T-cells. Studies are ongoing to distinguish whether cytotoxocity inhibition is caused by hetrodimerization of the full length and truncated NKG2D incapable of ligand binding, or constitutive and competitive binding by truncated NKG2D of adaptor signaling.

Description: We have demonstrated that co-culture of hESCs with OP9 bone-marrow stromal cells induced efficient hESC differentiation into hematopoietic colony-forming cells (CFCs). During hESC/OP9 co-culture, the first CFCs were detectable 1–2 days after emergence of CD34+ cells (day 4–5 of co-culture) and 3–4 days before generation of CD45+ cells, indicating that early CFCs arose in the CD45- population. To define phenotype of early hematopoietic progenitors, we examined the expression of CD41a, CD43, CD61 and glycophorin A (CD235a) on the CD34+ cells in the course of hESC/OP9 co-culture. The first detectable CD34+ cells expressed a high level of VEGF-R2 (KDR) and were CD41a, CD43, CD61 and CD235a negative. Appearance of CFCs was accompanied with down-regulation of KDR and almost simultaneous expression of CD41a, CD43 and CD235a on the CD34+ cells. CD43+ cells gradually increased thereafter, whereas the expression of CD41a was retained on the subset of CD43+ cells that co-express CD235a and CD61. CD45+ cells emerged within CD43+ population and the most of CD45+ cells did not express CD235a, CD41a or CD61. The expression of CD34 was decreased progressively on CD43+CD41a+CD235a+CD61+ cells, but retained by CD43+CD45+ cells. To assay hematopoietic potential of defined subsets, CD34+ cells were isolated by magnetic sorting and further separated into CD34+ CD43+ and CD34+CD43− cell fractions. In addition, CD34−CD43+ cells were isolated from CD34− fraction. CD34+ selection markedly enriched all types CFCs except of E-CFCs. All GEMM-CFCs were found within CD34+ population and none within CD34−. However, CD34− cell fraction contained E-CFCs and minimal numbers of M-CFCs and GM-CFCs. CD34+ cells were also heterogenous in morphologic appearance and contained endothelial cells that were capable to endocytose Ac-LDL. Positive selection of CD43+ cells resulted in total recovery of all types of CFCs. E-CFCs, which consistently detected in the CD34- fraction, were also recovered by CD43 selection. Morphologically, CD43+ cells comprised a homogenous blast-like population and were lack of endothelial-like cells. Thus, expression of CD43 molecule (leukosialin/sialophorin) is detected on the earliest clonogenic hematopoietic progenitors before expression of CD45, persists on developing hematopoietic cells after their lineage specification and most likely defines the divergence of hematopoiesis from endothelial cells during early development in humans.

Description: Divalent metal ion transporter 1 (DMT1) functions in iron absorption at the apical surface of the duodenal enterocyte and in iron transport out of the endosomal compartment of the erythroblast, however, DMT1 is also expressed in cells which have no apparent role in iron metabolism. At least 4 isoforms of DMT1 mRNA have been identified that contain alternative 5′ (1A, 1B) and 3′ (IRE+, IRE-) exons. Recently we reported the first human mutation of the DMT1 gene in a patient with severe hypochromic/microcytic anemia. This patient was homozygous for a mutation in the ultimate nucleotide of exon 12 which resulted in preferential skipping of exon 12 during processing of pre-mRNA. Amplification of DMT1 mRNA from peripheral blood cells of this patient revealed multiple products; hematopoietic cells from normal controls had a similar, but not identical, pattern. No such additional PCR products were seen when DMT1 mRNA from other tissues or cultured cells was evaluated. To further investigate this observation, we amplified full length (1700–1800 bp) DMT1 transcripts (1A/IRE+, 1A/IRE-, 1B/IRE+ and 1B/IRE-) from a variety of tissues types, cell lines, and from hematopoietic cells. Expression of the 4 isoforms differed between the different cell types. EBV lymphocytes expressed primarily 1B/IRE+ and 1B/IRE- isoforms. Notably, EBV lymphocytes derived from the mutant patient expressed DMT1 mRNA which was 120 bp smaller than DMT1 PCR products from controls, corresponding to skipping of exon 12. Platelets, granulocytes and reticulocytes expressed primarily the 1B/IRE+ isoform, however, unlike the other cell types examined, more than one mRNA species was present (see figure). Full length high molecular weight PCR products derived from these cells were isolated and subcloned. Plasmid DNA was isolated, subjected to digestion with EcoR1, and the DMT1 inserts were sequenced. All inserts began with exon 1B and ended with exon 16 (IRE+); however, none contained “normal” full length DMT1 sequence. The majority contained either truncated portions of exon 3 or inserted portions of intron 3. Utilization of cryptic splice sites within exon 3 or within intron 3 resulted in early stop codons. We propose that alternative splicing of DMT1 mRNA either via skipping of entire exons or via the use of cryptic splice sites may provide a manner for regulating DMT1 activity in hematopoietic cells. Further studies are underway to elucidate this possibility and to evaluate the signals for alternative splicing. Figure Figure

Description: Ribavirin(RBV)is a water soluble synthetic guanosine analog that exerts antiviral activity against DNA and RNA viruses after intracellular phosphorylation. Current studies indicate that combination therapy with RBV and Interferon a (IFNa) is associated with higher rates of sustained virological and biochemical response compared to IFN a monotherapy for chronic hepatitis type C (CHC). Hemolytic anemia is the major side effect of the therapy, with 67% of treated patients developing anemia. The exact mechanism for RBV-related anemia is unknown, but it is thought to be decreasing deformability of eythrocytes resulting from accumulation of phosphorylated RBV in erythrocytes. Highly purified eicosapentaenoic acid (EPA) is widely used for the treatment of hyperlipidemia and atherosclerosis in Japan. We have investigated that EPA has a beneficial effect in patients with RBV-related anemia (Int J Mol Med11:729–732, 2003). Our aim of this study is to establish the safer treatment of RBV/IFNa using EPA for CHC patients and elucidate the mechanism of EPA action for improving anemia. [Patients and methods] Thirty three patients with CHC were randomized to RBV/IFNa therapy (800mg/6MU/day) with or without EPA (1800mg/day) for 12 weeks (14:+EPA; 19:-EPA). Erythrocyte filterability (Nickel mesh filtration method; Jpn J Physiol53: 481-486, 2003) was measured before and after 12 weeks of treatment in 8+EPA and 9 -EPA patients. Statistical analysis was performed by Wilcoxon and ANOVA test. [Result] As shown in Fig. 1, hemoglobin (Hb) levels decreased in both groups by the treatment. However, decrease of Hb levels significantly attenuated by the addition of EPA at 4 and 8 weeks after initiation of the treatment. This effect continued for 12 weeks. Initial mean erythrocyte filterability (%) at study entry was 65.3% and 68.1% in the + and -EPA group, respectively. After 12 weeks treatment, improvement of erythrocyte filterability occurred in 6/8 patients in the +EPA group. In contrast, 7/9 patients resulted in the decrease of erythrocyte filterability in the -EPA group. As shown in Fig 2, the mean erythrocyte filterability (%) for the +EPA group was 71.0%, against 51.4% in the -EPA group (P=0.01) after 12 weeks. [Discussion and Conclusion] EPA significantly attenuated the anemic effect of RBV/IFNa therapy for CHC patients. Erythrocyte filterability decreased in the -EPA group. On the contrary, decrease of erythrocyte filterability was not found in the +EPA group. Therefore, EPA improved the anemic side effect of RBV/IFNa via the stabilizing action of the membrane by amelioration of erythrocyte filterability. This study would be useful for clinical application.

Description: Jens Pedersen-Bjergaard, Debes H. Christiansen, Mette K. Andersen. Dept. Clinical Genetics, University Hospital Rigshospitalet, Copenhagen, Denmark. 140 unselected patients with t-MDS (n=90) or overt t-AML (n=50) were studied for chromosome aberrations, for mutations of 6 different genes and 69 patients were examined for methylation of the p15 promotor. Cytogenetically, 5q−/ −5: n=34; 7q−/ −7 but normal chromosomes 5: n=39; balanced translocations: n=23; normal karyotype: n=24; other abnormalities: n=20. Mutations of p53: n=34; AML1: n=22; FLT3: n=11; c-KIT: n=2; K- or N-RAS: n=14; BRAF: n=2; methylation of the p15 promotor: 55/69 cases. Abnormalities of chromosomes 5 and 7 were significantly associated with t-MDS (p=0.001), whereas balanced translocations and a normal karyotype were significantly associated with overt t-AML, (p=0.0001 and p=0.006 respectively). Mutations of p53 were significantly associated with 5q−/ −5 (p=0.001), with 17p−/−17 (p=0.0004), with a complex karyotype (p=0.001), and with MLL and AML1 amplifications. Mutations of AML1 were significantly associated with 7q−/−7 )(p=0.001) and with t-MDS progressing to t-AML (p=0.0001). Mutations of FLT3 were significantly associated with presentation as overt t-AML (p=0.0002), with a normal karyotype (p=0.004) and with previously radiotherapy only (p=0.03). RAS mutations were significantly associated with t-MDS progressing to overt t-AML (p=0.008) and borderline with a normal karyotype (p=0.07). Methylation of p15 was significantly associated with 7q−/−7 (p=0.0006). Mutations of FLT3, RAS and c-KIT were mutually exclusive, and a synergism between AML1 and RAS mutations (22/5/14, p=0.046) and between p53 and AML1 mutations in patients with 7q−/−7 is suggested. Based on these findings a revised model of the genetic pathways of t-MDS and t-AML (Blood2002;99: 1909–1912) is discussed.

Description: Introduction. B cell chronic lymphocytic leukaemia (B-CLL) is a heterogeneous disease as shown by differential expression of a variety of surface and cytoplasmic markers. In a search for markers that could define biological activity of different B-CLL subsets, we have studied the surface expression of the Toll-like receptor (TLR) family member CD180 in relation to other surface markers and mutation status of IgVh genes. Methods. Seventy eight B-CLL patients (68 untreated and 10 treated) and 15 age-matched controls were studied in three different clinics. CD19+ B cells were stained using indirect immuno fluorescence for CD180, surface IgM (sIgM), CD79b and CD38, analysed by flow cytometry and data expressed as the relative antibody binding sites (RBS)/cell for each marker. Monoclonal anti-CD5 antibodies were also used with anti CD180 to determine levels of expression of CD180 in control CD5+ cells. IgVh mutation was determined for 47 patients. Results B-CLL cells had variable levels of CD180 expression, but this was always less (1036 ± 935 RBS/cell) than that expressed by normal blood B cells (5548 ± 2271 RBS/cell) and was stable for up to 18 months. Significantly higher levels of CD180 were expressed by B-CLL cells with mutated (M) compared with those using unmutated (UM) IgVh genes. This was in contrast to the higher levels of expression of sIgM by B-CLL cells using UM than M IgVh genes (Figure). Conclusions. CD180 is expressed at higher levels on B-CLL cells using M than those using UM IgVh genes and is in contrast to the level of expression of sIgM which is higher on B-CLL cells using UM versus M genes. This differential expression of CD180 supports the notion that B-CLL cells using UM IgVh genes represent a population of cells actively responding to signals (perhaps to self antigens) via their surface IgM. Figure Figure

Description: Non-Hodgkin’s Lymphoma (NHL) causes many deaths world wide, and is one of the few cancers that have a continual increase in incidence and mortality rates over the last few decades. Diffused large B cell lymphoma (DLBCL) is an aggressive B cell lymphoma that accounts approximately 40% of all NHL-B cells. We have previously shown that dysregulated CD40 ligand (CD40L/CD154) expression in DLBCL maintains lymphoma cell growth and cell survival. CD40 ligand is a member of the TNF superfamily of proteins that has a wide variety effects throughout the immune system and is critical for both cellular and humoral immunity. It was originally thought that CD154 expression was restricted to activated CD4+ T lymphocytes. However, expression of CD154 has also been demonstrated in non-lymphocytic leukocytes such as mast cells, basophils, eosinophils, dendritic cells and macrophages. More importantly, dysregulated CD154 expression has been noted in a number of diseases, including systemic lupus erythematosus (SLE), Alzheimer’s, and in B cell lymphomas. Although the functional analysis of CD154 has been extensive, little is known about the mechanisms controlling CD154 expression in activated T cells, let alone in normal and malignant B cells. In this study, we show that constitutive NF-kB and NFAT activation in aggressive lymphoma B cells directly interact and synergistically regulate CD154 gene expression. Immunoprecipitation and confocal analyses indicate that NFATc1 and NF-kB (p65 and c-rel) complex and colocalize in lymphoma B cells. Chromatin Immunoprecipitation (CHIP) assays analysis demonstrate that NFATc1, p65 and c-rel bind to the CD154 promoter, that is enhanced by HDAC inhibitor (TSA or SAHA) treatments. HDAC inhibitors also enhance CD154 promoter activity, providing evidence that CD154 transcriptional regulation involves histone acetylation. Over-expression studies show a synergistic effect by NFATc1 and NF-kB (c-rel) on the CD154 promoter. Promoter deletion and site direct mutagenesis studies on the CD154 promoter reveal that the transcriptional regulation of CD154 requires two sites, the distal kB site at −1180 and a proximal NFAT site at −250. These sites bind to both, NFAT and NF-kB proteins, as wells as the DNA architectural protein HMG-1, indicating that the CD154 promoter DNA may loop and form an enhanceosome-like complex. Studies using siRNA to p65, c-rel, or NFATc1 repress CD154 promoter activity, indicating that these transcription factors are necessary and sufficient for CD154 gene transcription. These findings strongly suggest a novel control mechanism for CD154 gene transcription, therefore, providing potential treatment modalities for lymphoma B cells as well as other disorders involving dysredulated CD154 expression.

Description: Chronic Myeloid Leukemia (CML) results from transformation of hematopoietic stem cells by the BCR/ABL oncoprotein. CML can be effectively treated with the targeted ABL kinase inhibitor Imatinib (STI571; Gleevec), but patients rarely achieve molecular remission by sensitive PCR methods, and drug resistance limits the durability of drug response. A significant minority of patients treated with Interferon (IFN) alpha achieve complete cytogenetic remission, and response is correlated with immune reactivity against leukemic clones. This suggests that IFN-induced immunotherapy might be an important adjunct to Gleevec, thus prompting us to investigate immunoprotective mechanisms. We have shown previously that ectopic expression of Interferon Regulatory factor 8 (IRF8, also called ICSBP) can stimulate a potent CD8+ cytotoxic anti-leukemia response in a murine model of BCR/ABL-induced leukemia (Deng and Daley, Blood 97;3491, 2001). In transcriptional profiling experiments with cells that overexpress IRF8/ICSBP, we detected increased expression of IFN alpha and beta. To test whether IFN alpha or beta can mediate the same protective immune-response as IRF8/ICSBP, we co-injected BCR/ABL-transformed BaF3 cells together with non-transformed BaF3 cells engineered to overexpress IFN alpha or beta. Expression of IFN alpha or beta in trans protected mice from developing leukemia. Control constructs in which IFN alpha or beta were cloned in the anti-sense orientation failed to confer protection, confirming the importance of IFN alpha or beta expression. Importantly, IRF8/ICSBP expression is protective against cells transformed by Gleevec-resistant mutant BCR/ABL proteins (T315I and L248R). Our data suggest that IFNs are functionally equivalent to IRF8/ICSBP in conferring a vaccine-like anti-leukemic immune effect in our model, and may act through an autoregulatory loop involving IRF8/ICSBP. Defining the precise epitopes targeted by the IFN/ICSBP-induced immune response may enable immunotherapy in human CML patients.

Description: Epipodophyllotoxins, anthracyclines and other chemotherapeutic DNA topoisomerase II poisons are associated with MLL-rearranged leukemias and, less often, leukemias with different balanced translocations. Every MLL translocation identified so far in patients has been associated with leukemia and, where tested, murine models have established that the der(11) protein products are leukemogenic. We here describe a highly novel t(4;11)(p12;q23) that emerged in the bone marrow that confers a proliferative advantage but not a leukemia phenotype in a patient with stage 4 neuroblastoma (NB) following N8 therapy, which consisted of 3 cycles of cyclophosphamide/doxorubicin/vincristine, 2 cycles of cisplatin (P)/etoposide (VP), surgery, local radiation, anti-GD2 monoclonal antibody (3F8), myeloablative therapy (thiotepa, carboplatin, topotecan) plus autologous PBSC transplant (harvested after 1 cycle of PVP), followed by 7 more cycles of 3F8 plus GM-CSF, 4 cycles of oral VP, and 5 cycles of accutane. Cumulative doses of doxorubicin and VP were 225 mg/m2 and 5.4 g/m2 (4.2 g/m2 orally), respectively. Seventeen months after NB diagnosis (11.5 months after transplant) at completion of oral VP, surveillance FISH showed t(4;11)(p12;q23) in 10.5% of marrow cells. The patient is now 32 months from NB diagnosis and 1 year off all therapy. Although both karyotype and FISH showed t(4;11) in 82–100% cells in 7 serial subsequent marrows, all had normal morphology and there is no clinical evidence of leukemia. Molecular analyses of sequential bone marrow samples were undertaken to characterize the translocation. Southern blot analysis of the MLL bcr in BamHI and BglII digested DNAs showed two rearrangements consistent with both derivative chromosomes. A new BglII-based reverse panhandle PCR identified the der(4) breakpoint junction sequence in a 3.2 kb BglII genomic rearrangement and provided the sequence to isolate the der(11) genomic breakpoint junction by clonotypic PCR. The partner DNA was a novel gene localized to chromosome band 4p12. The translocation fused intron 8 of MLL with intron 8 in the new partner gene, and occurred with a loss of 8–13 bases from MLL and 31–36 bases from the partner gene. Both breakpoint junctions contained short homologous sequences suggesting NHEJ. By clonotypic nested PCR (sensitivity 1 cell in 104–105 cells) the translocation was absent at NB diagnosis, and was first detectable at the completion of oral VP (16 months after neuroblastoma diagnosis). RT-PCR revealed in-frame der(11) and der(4) fusion transcripts. Real-time PCR analysis of known MLL target genes and differentially expressed genes in MLL-rearranged leukemias revealed low levels of HOXA4, A5, A7, A9, FLT3, MEIS1, and PBX3 compared to other treatment-related leukemias. Temporal emergence of this translocation after the high total VP dose is of interest because others recently found that cumulative doses of VP 〉6 g/m2 carry an especially high risk of leukemia, raising concern about the role of the oral VP in the genesis of the translocation. This study indicates that the different partner genes of MLL do not confer the same leukemia potential. Because the t(4;11)(p12;q23) confers a proliferative advantage but is insufficient for leukemia, we designated the new partner gene MIFL (MLL Insufficient for Leukemia), however it remains to be determined whether any relevant secondary alteration will result in leukemic transformation.

Description: Various theories can explain the expansion of glycosyl phosphatidyl inositol anchored protein (GPI-AP)-deficient clones in PNH. The differential growth advantage of PIG-A mutant stem cell scould be due to immune escape in the context of an autoimmune attack on hematopoietic targets explaining the close clinical relationship to aplastic anemia. The causes of evolution of the PNH clone may be related to a less efficient immune recognition of PNH cells, their relative resistance to apoptosis or slower senescense. These changes may be related to additional mutations or genetic damage present in either normal or GPI-AP deficient hematopoietic clone. Using traditional karyotyping, chromosomal defects are not common in PNH but the limited resolution of this method may preclude detection of smaller aberrations. Due to these limitations, novel technologies which improve resolution and sensitivity are under development. In array-based comparative genomic hybridization (A-CGH), differentially labeled test and reference DNA samples are hybridized to genomic microarrays. Differences in sequence copy number between the samples are reflected in a shift of the fluorescence spectrum. The principle of A-CGH allows for detection of unbalanced chromosomal changes of the whole genome and its resolution is limited solely by the number of clones. In preliminary studies we found significantly decreased telomerase activity in normal vs. CD59−CD55− CD34+ cells derived from patients with PNH suggesting: 1) more accelerated telomere shortening in phenotypically normal hematopoietic cells derived from PNH patients, or 2) the possibility of chromosomal instability and acquisition of karyotypic defects should a critical telomere length be reached. We hypothesized that higher resolution analysis of the karyotypic changes in PNH using CGH may result in detection of cryptic karyotypic abnormalities present in either the normal or GPI-AP deficient clone. We have analyzed a cohort of patients (N=6) with hemolytic PNH and major expansion of a GPI-AP deficient clone (50–95% of PNH granulocytes). All patients had normal cytogenetics by metaphase karyotyping. We have separated GPI-AP-deficient myeloid peripheral blood cells using CD55 and CD59 as well as CD2 and CD19 staining to exclude lymphocytes. Separated CD55−CD59− and CD55+CD59+ non lymphoid cells were sorted and, following DNA extraction, subjected to A-CGH analysis (Vysis microarrays containing 287 probes). In one patient, PNH cells appeared normal by A-CGH but the corresponding “normal” cells contained deletions of telomeric region of several chromosomes (2, 5, 16, 18, 19, 20qtel as well as 5, 11,19ptel). However, in the remaining patients telomeric deletions were present in both normal and PNH cells. There was no difference in the numbers of chromosomes affected and diverse chromosomal telomeric deletions were present. No other lesions were found. By comparison when A-CGH analysis was performed in 8 normal individuals telomeric deletions were only rarely found and only occasional gains of contiguous clones on chromosomes were detected. Deletion of the telomeric portions of multiple chromosomes is compatible with accelerated global telomere loss in PNH. However, it appears that this phenomenon was not restricted to either normal or PNH cells. Our results are in agreement with previously described telomere shortening measured by FISH, flow cytometry or Southern blot analysis in PNH, a change not consistently restricted to normal cells.

Description: The RHD gene encoding the Rhesus blood group antigen D is flanked by two highly homologous DNA segments of about 9,000 bp, the upstream and downstream Rhesus boxes. In haplotypes with an RHD deletion, the fusion of the two Rhesus boxes within the 1,463 bp identity region generates the single hybrid Rhesus box. Its detection has been applied to determine RHD zygosity. For instance, testing for RHD heterozygosity is of clinical interest, because all children of an RHD homozygous father will be D positive and at risk of hemolytic disease of the newborn, if the mother carries an anti-D. Aberrant Rhesus boxes can confound this application and appear to be frequent among Africans. A comprehensive, systematic analysis was lacking. We sequenced about 5,850 bp of the upstream and downstream Rhesus boxes in 18 samples that were representative for all four D clusters and of the hybrid Rhesus box in 4 samples that were mistyped in assays for the hybrid Rhesus box. The known differences between upstream and downstream Rhesus boxes were in part restricted to subsets of RHD alleles. We detected 46 additional polymorphism, which represented single nucleotide substitutions, short insertions or deletions. Gene conversions were found in the upstream Rhesus boxes of RHDψ, DAU-1 and DAU-3 and in the downstream Rhesus boxes of Ccdes, weak D type 4.1, type 4.2 and DAU-0. All 6 different gene conversions started or ended in or near the identity region, which may have been instrumental for generating these gene conversions. Recombinations between haplotypes were likely in several alleles like DIII type 4. Four non-standard hybrid Rhesus boxes were suggestive of multiple independent RHD deletion events. We found considerable variation of Rhesus box sequences, which explained the limitations for RHD heterozygosity testing that were previously observed in several laboratories. The general topology of the genomic Rhesus locus was however similar in all cases and consistent with our initial description. There was evidence for 5 probably independent RHD deletion events. Testing RHD heterozygosity in Africans is best based on quantitative PCR or amplification of the full length hybrid Rhesus box. Because in Europeans who harbored RHD alleles belonging to African D clusters the Rhesus box variation was also substantial, use of two complementary methods for hybrid Rhesus box detection may even be advisable in whites. We conclude that a long range PCR spanning the whole hybrid Rhesus box, while technically demanding, is not influenced by the kinds of aberrations observed in this study and may hence remain the standard for research purposes and quality assurance.

Description: Recently, among myelodysplastic syndromes with IPSS low/intermediate 1 risk classes, promising results have been obtained in the treatment of anemia by high dosage recombinant human erythropoietin (rHuEPO) treatment, in the cases with low endogenous erythropoietin (EPO). We have reproduced these clinical results in the patients treated at our Institution. Based on these premises, in order to evaluate the molecular basis of the rHuEPO response in MDS patients, we investigated the differential gene expression in the bone marrow erythroid cells expressing glycophorin (Gly+) of EPO-responders (ER) and EPO-non responders (ENR) MDS patients, using commercially available macroarrays (Clontech), comparing gene level of expression with a baseline represented by a pool of erythoid cells isolated from normal bone marrow donors. We analysed 4 ER and 4 ENR MDS patients (RA and RARS). BM erythroid cells, after Ficoll density gradient, were separated by positive selection using an immunomagnetic procedure (MACS, glycophorin isolation kit; Miltenyi Biotech, Auburn, CA). The purity of Gly+ cells after magnetic immunosorting was tested by flow-cytometry and was superior to 97%, in all the cases. RNA extraction, cDNA synthesis and macroarrays hybridization and analysis were performed following manufacturer instructions (Clontech Laboratories, Palo Alto, CA). The data were validated on 4 differentially expressed genes by real time RT-PCR TaqMan technology, obtaining results that were superimposable to the macroarrays ones. Using a cut off of an at least two fold difference in gene expression, we obtained a differential pattern of expression in ER and ENR patients, BM Gly+ erythroid cells, with respect to normal donors. In particular, ER patients presented the up-regulation of interleukin-1 beta, apolipoprotein E precursor, CREB2, high mobility group protein (HMG-I), transducin beta-1 subunit and the down regulation of protein kinase MLK-3, cAMP-dependent phosphodiesterase (PDE43), IL-5R-alpha, caspase-9. On the other hand, in ENR group, we found a down regulation of proliferating cyclic nuclear antigen (PCNA), B-myb, prothymosin alpha, phenylethanolamine N-methyltransferase, thymosin beta-10 and an up regulation of homeobox protein HOX-A5, monocarboxylate transporter 1, interleukin-6, deoxyribonuclease II, sterol regulatory element-binding protein, metalloproteinase inhibitor 1 precursor, rap1 GTPase activating protein 1 (RAP1GAP). In conclusion, ER and ENR MDS patients erythroid cells present a different pattern of gene expression with respect to normal. These results may provide the basis for early testing of the genes differentially expressed in ER and ENR patients in order to elucidate their role in the prognostication of rHuEPO response in MDS patients with low endogenous EPO levels.

Description: Introduction: Intensive therapy (IT) including autologous stem cell transplantation (ASCT) is considered superior to conventional therapy in newly diagnosed myeloma pts