ALBERT

Description: Abstract 4013 Background: In recent years, azacytidine (AZA) has become the standard of treatment for high risk myelodysplasia (MDS). The approved schedule of AZA uses a 75mg/m2/d s.c. regimen for 7 days based on the CALGB-9221 (Silverman, JCO 2002) and the AZA-001 studies (Fenaux, Lancet Oncol 2009). The clinical response rates after AZA have been extensively presented but there is only limited data on the rates of cytogenetic response (CyR). Based on the review of the literature, there are no specific cytogenetic data published on prospective trials. Methods: We based our analysis on a randomized phase 2 study from the US Leukemia Intergroup (E1905 study, NCT00313586) testing 10 days of AZA (50mg/m2/d s.c.) vs 10 days of AZA+ the histone deacetylase inhibitor entinostat (4 mg/m2/d PO days 3 and day 10). MDS, CMML, and AML with myelodysplasia-related changes were included. This analysis includes all patients with cytogenetic abnormalities (at baseline or acquired following treatment) with available cytogenetic follow-up (cycle 6). Of 150 patients, 70 demonstrated baseline cytogenetic abnormalities. To date, forty patients (27 MDS and 13 AML) were evaluable for both time points. Karyotypes were performed at local laboratories, and reviewed centrally (RPK and GH). Cytogenetic response was assessed using IWG 2000 (Cheson et al, Blood 2000) criteria. Results: The clinical response rate (CR+PR+ trilineage HI) according to IPSS cytogenetic risk stratification were of 20%, 33%, and 35% for favorable, intermediate and poor cytogenetic risk groups respectively (p=NS). Patients with Chr 7 abnormalities (i.e. -7 or -7q, n=18) had a response rate of 28% including 17% CR. Of patients with complete cytogenetic data, the rate of overall CyR was 52% (n=21): 22% (n=9) complete CyR, 30% (n=12) partial CyR. This represents a complete CyR of 13% and a partial CyR of 23% as a proportion of all treated patients with initial cytogenetic abnormalities (including those who did not receive six cycles of therapy). To date, confirmatory FISH analyses were available for 4 patients with CyR (2 CCyR and 2 PCyR). All four had complete clearance of their cytogenetic clone. Among the cytogenetic responders, 15 had MDS and 6 had AML (p=NS). CyR did not differ between the two treatment arms. CyR and clinical response were highly correlated (p

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: 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: AIMS Computerised Tomography (CT) scanning is the primary modality for assessment of therapeutic response in DLBCL. We evaluated whether functional imaging utilising FDG-PET may provide additional prognostic information in response assessment prior to completion of chemotherapy for patients with DLBCL. METHODS We performed a retrospective, single-centre study of patients with DLBCL who received anthracycline-based chemotherapy +/− radiotherapy (RT) and had PET prior to completion of therapy. RESULTS From 1996–2004 there were 45 eligible patients. Median age was 59 years (range 26–82) with disease stage I–II (n=25) or III–IV (n=20). Median IPI was 2[IPI 0–2(n=27), IPI 3–4(n=13)] in 40 evaluable patients. Therapy included full-course CHOP/CHOP-like therapy (n=26); HyperCVAD (n=6) and limited (3–4 cycles) CHOP/CHOP-like chemotherapy with RT (n=13). Rituximab was used concurrently in 18 patients. Planned RT was administered in 23 patients. 13 (29%) patients were PET positive after a median of 3 chemotherapy cycles (range 1–5). Of these, 7 (54%) progressed a median of 7.2 months following completion of therapy. Of the 6 progression-free, 4 demonstrated residual low-grade activity at sites of prior bone involvement (median follow-up 50.8 months), while 2 patients (stage I and IV disease) had limited follow-up (

Description: Aims. RIT with an anti-CD20 monoclonal antibody conjugated to a beta-emitting radioisotope may overcome rituximab resistance in B-cell NHL, delivering radiation not only to tumor cells that bind the antibody but also, due to a cross-fire effect, to neighboring unbound tumor cells. PET has emerged as a major imaging modality for response assessment of aggressive NHL, and recent data suggests PET has excellent sensitivity and specificity in indolent NHL. In follicular NHL, high molecular response rates for BCL-2 re-arrangements occurs following first-line treatment with RIT. Outcomes following molecular remission, and the impact of PET response post RIT at relapse, are unknown. We present the prospectively collected data for patients (pts)at a single institution enrolled in a multi-center phase II trial of 131-I labelled rituximab treatment of patients with relapsed or refractory indolent B-cell NHL. Methods. All pts were required to have histologically proven relapsed or refractory indolent CD20 positive B-cell NHL. The administered activity of 131I-rituximab was estimated to deliver a whole-body radiation absorbed dose of 0.75 Gy. Post treatment surveillance PET-CT scans were performed at baseline, 3, 6, and 12 months after RIT. In patients with follicular NHL, multiplex nested PCR with a sensitivity of 1 in 10−4 for the major and minor breakpoint regions of the Bcl-2 rearrangement were performed on BM and PB prior to treatment. If positive this was followed by repeat BM and PB at 3, 6 and 12 months, and then only at suspected relapse. The influence of metabolic and PCR responses to 131I-rituximab on event free survival (EFS) and overall survival (OS) were then evaluated. Relapses after 1 year were established with a dedicated CT and PET when clinically suspected. Results. 29 pts [follicular (24), small lymphocytic (2), marginal zone (3)] had a response rate of 83%. At 3-months, 15/27 (56%) evaluable pts achieved a complete metabolic response (CMR). The 3-month PET status predicted outcome with median time to progression (TTP) of 15 months for CMR (n = 15), 11 months for PMR (n =7) and three months for non-responders (n=5) (P=0.001). CT response status at 3-months did not display as clear a separation of categories, with a median TTP of 24 months for CR (n=9), 12 months for PR (n=10) and six months for non-responders (n=9) (P=0.0023). 5/11 evaluable pts with Bcl-2 re-arrangements achieved a complete molecular response (CMolR) with a median TTP of 24 months, and six remained positive with a median TTP of 6 months (P=0.0025). Conclusions. 131I-rituximab is able to induce metabolic and molecular responses in pts with relapsed indolent NHL. Although CT CR predicts TTP, the lack of a PET response conveys the most robust prediction of lack of treatment benefit. PET provides statistically superior prognostic stratification, although both modalities are able to identify good responders from poor-responders. Patients with Bcl-2 re-arrangements at salvage RIT who become PCR negative have a significantly prolonged TTP.

Description: Deficiency of folate or vitamin B12 (cobalamin) causes megaloblastic anemia, a disease characterized by pancytopenia due to the excessive apoptosis of hematopoietic progenitor cells. Clinical and experimental studies of megaloblastic anemia have demonstrated an impairment of DNA synthesis and repair in hematopoietic cells that is manifested by an increased percentage of cells in the DNA synthesis phase (S phase) of the cell cycle, compared with normal hematopoietic cells. Both folate and cobalamin are required for normal de novo synthesis of thymidylate and purines. However, previous studies of impaired DNA synthesis and repair in megaloblastic anemia have concerned mainly the decreased intracellular levels of thymidylate and its effects on nucleotide pools and misincorporation of uracil into DNA. An in vitro model of folate-deficient erythropoiesis was used to study the relationship between the S-phase accumulation and apoptosis in megaloblastic anemia. The results indicate that folate-deficient erythroblasts accumulate in and undergo apoptosis in the S phase when compared with control erythroblasts. Both the S-phase accumulation and the apoptosis were induced by folate deficiency in erythroblasts fromp53 null mice. The complete reversal of the S-phase accumulation and apoptosis in folate-deficient erythroblasts required the exogenous provision of specific purines or purine nucleosides as well as thymidine. These results indicate that decreased de novo synthesis of purines plays as important a role as decreased de novo synthesis of thymidylate in the pathogenesis of megaloblastic anemia.

Description: In healthy carriers of human cytomegalovirus (HCMV), the virus-specific memory CD8+ T-cell population is often dominated by CD28− CD45RAhi cells that exhibit direct ex vivo cytotoxicity but whose capacity for proliferation and generation of further memory cells has been questioned. We show that when highly purified CD28− CD45RAhi CD8+ T cells are stimulated with viral peptide presented by autologous monocytes, the virus-specific T cells show early up-regulation of CD137 (4–1BB) and CD278 (ICOS), re-express CD28, and proliferate with similarly high cloning efficiency in limiting dilution analysis as CD28+ CD45ROhi cells or CD28− CD45ROhi cells. Using peptide-pulsed autologous fibroblasts transfected with individual costimulatory ligands as antigen presenting cells, we showed CD137L to be a key costimulatory ligand for proliferation of CD28− CD45RAhi CD8+ T cells and not CD80, CD86, or CD275 (ICOSL). Therefore, CD28− CD45RAhi CD8+ T cells were not terminally differentiated but required a specific costimulatory signal for proliferation.

Description: Hodgkin lymphoma (HL) occurs in HIV-infected individuals more frequently than in the HIV-negative population and the incidence is rising. Patients (pts) with non-Hodgkin’s lymphoma in HIV appear to have improved outcomes if they receive HAART with chemotherapy (CT). ABVD is standard CT for HL and is frequently administered with HAART in pts with HIV-HL. However, some components of the ABVD regimen may interact with antiretroviral (ARV) medications to alter metabolism and increase toxicity. In particular, vinblastine (VBL) is metabolized by CYP3A4 and protease inhibitors, particularly ritonavir (RTV), appear to inhibit this cytochrome potentially leading to higher VBL exposure. Little definitive information is available regarding how interactions might affect clinical outcome. We conducted a retrospective review of 36 pts with HIV-related HL to identify the frequency of neurotoxicity (NT), hematologic toxicity (HT), and lung toxicity (LT), to identify risk factors for severe (grade III–IV) toxicity, and to determine its clinical significance. Clinical data were collected from the CFE database and by chart review from 3 centers. The median age at HL diagnosis (dx) was 41 (range 29–66) years and 34 (94%) were male. HL was advanced stage in 28 (78%). Hasenclever score could be calculated in 23 pts and was 0–4 and ≥5 in 15 and 8 pts respectively. ECOG PS was 0–1 in 13 and ≥2 in 8 (n=21). HIV risk factor was: sexual, n=21 and other, n=5 (n=26). Median CD4 count at HL dx was 210 (2–660) cells/ul (n=31). Median HIV viral load (VL) was undetectable (

Description: Introduction: Nearly one in five cancer survivors report limitations in ability to work following diagnosis, with poor work-related outcomes particularly noted in the hematologic cancers. Although much is known about the efficacy, toxicity and direct costs of treatment for follicular lymphoma, there is no data assessing the impact of this diagnosis on productivity of affected individuals. Methods: We conducted a consecutive cross-sectional study of patients attending a malignant hematology clinic at a large multi-disciplinary cancer centre. Patients with a diagnosis of FL or other indolent NHL were asked to complete questionnaires assessing demographics, health status (EQ-5D), and work productivity and activity impairment (WPAI questionnaire). Results: Eighty-four patients completed the survey study (〉95% response rate). Mean age was 58.7 (+/−13.8 SD) and 55% were male. Diagnoses included FL (55%), CLL (25%), and other indolent NHL (20%). The majority of patients presented in advanced stage (stage III–IV; 65%) and had received some therapy, although 29% were still being observed without having received therapy by the time of survey administration. The median disclosed income was $40,000–$59,000; 76% had pursued post-secondary education. Over 61% were working full-time prior to diagnosis while 14% were retired. Patients reported a minimal impact on their work productivity (1.9+/−3.2 on a scale of 0 to 10; 0=no effect and 10=complete impairment of activity) and on their daily activities (2.4+/−3.1) attributable to their cancer diagnosis. However, following diagnosis of NHL (and at the time of survey completion), only 33% were able to continue full-time work, 7% were working part-time, 10% required disability, and 37% were retired. Of those still working, a mean of 2.1 days (+/−6.9) were missed due to illness in the preceding 4 weeks, with a mean of 16 days (+/−8.7) worked in that period. Only 6% received paid assistance, while 17% required unpaid care from a partner/spouse, relative, or friend. Unpaid caregivers missed a mean of 11.3 days (+/−16.2) of work and provided a mean of 9.8 days (+/−13.4) of care. There was a significant inverse correlation between daily activity scores (high values=complete impairment) and health status ratings (high values=excellent health status/utilities) ascertained by the EQ-5D instrument (Spearman correlation coefficient −0.69; p5) was predicted by poor self-rated health status (OR 32.1; 95% CI 5.9–174.2; p

Description: Background: Patients with relapsed or refractory aggressive B-cell lymphoma, or transformed indolent lymphoma can achieve long-term survival with high dose therapy and autologous stem cell transplant (HDT/ASCT), provided their disease is sensitive to salvage chemotherapy. Unfortunately, approximately 50% of patients are insensitive to standard salvage regimens. Objectives: This trial investigated whether adding Rituximab to ESHAP (etoposide, solumedrol, cytosine arabinoside, cisplatin) induction improved chemosensitivity. The primary outcome was overall response rate (CR + CRu + PR) to R-ESHAP. Secondary outcomes were toxicity, ability to undergo ASCT, progression free survival (PFS) and overall survival (OS). Methods: The protocol was approved by the local ethics review board and all patients provided informed consent. Eligible patients received ESHAP every 28 days with GCSF support until 〈 15% bone marrow involvement was achieved (2–4 cycles). Rituximab was given weekly x 8 weeks concurrent with the first 2 cycles of ESHAP. GCSF mobilized stems cells were collected on day 10–11 of cycle 1 or 2. Results: The trial was stopped early after the complete response (CR) rate at a planned interim analysis exceeded 40% (a pre-specified criteria for stopping the trial). Final results of 26 patients are presented. Median age was 55.5 years (range 42–64). Twelve patients had relapsed aggressive lymphoma, 2 had refractory disease and 12 had transformed indolent lymphoma. Twenty-two of 26 patients were stage III/IV. The overall response rate to R-ESHAP was 92% (95% CI 82% to 100%). Twelve patients (46%; 95% CI 27% to 65%) had a CR or unconfirmed CR. Grade 3–4 thrombocytopenia, neutropenia, and anemia occurred in 57%, 40%, and 15% of R-ESHAP cycles respectively. Grade 3–4 infections complicated 7% of cycles. Median follow-up was 17 months (range 2.9 to 43.2) from enrollment. Twenty-three of 26 patients (88%) were transplanted. Notable post-transplant toxicity included 5 cases of herpes zoster, 2 cases of bacterial pneumonia, 1 case of pulmonary aspergillosis, and 1 fatal case of pneumocystis carnii pneumonia (PCP). Three patients did not proceed to HDT/ASCT; 2 were refractory to R-ESHAP and 1 died of a myocardial infarction after induction chemotherapy but prior to ASCT. Fifteen of 23 patients who received ASCT remain in remission, 6 have relapsed. Seven patients have died, 4 of progressive disease, 1 of myocardial infarction, 1 of PCP, and 1 of accelerated Parkinson’s Disease. Median PFS and median OS have not yet been reached. Conclusions: In this single-arm, phase II study of relapsed or refractory aggressive B-cell lymphoma and transformed indolent B-cell lymphoma, R-ESHAP induction therapy resulted in a very high ORR (92%) and enabled a large percentage of patients (88%) to proceed to HDT/ASCT. Toxicity of the R-ESHAP regimen was acceptable, and its efficacy compared favorably with other salvage regimens reported in the literature, including R-ICE.