ALBERT

Description: A pilot study was undertaken to assess the safety, activity, and immunogenicity of a polyvalent Wilms tumor gene 1 (WT1) peptide vaccine in patients with acute myeloid leukemia in complete remission but with molecular evidence of WT1 transcript. Patients received 6 vaccinations with 4 WT1 peptides (200 μg each) plus immune adjuvants over 12 weeks. Immune responses were evaluated by delayed-type hypersensitivity, CD4+ T-cell proliferation, CD3+ T-cell interferon-γ release, and WT1 peptide tetramer staining. Of the 9 evaluable patients, 7 completed 6 vaccinations and WT1-specific T-cell responses were noted in 7 of 8 patients. Three patients who were HLA-A0201-positive showed significant increase in interferon-γ–secreting cells and frequency of WT1 tetramer-positive CD8+ T cells. Three patients developed a delayed hypersensitivity reaction after vaccination. Definite related toxicities were minimal. With a mean follow-up of 30 plus or minus 8 months after diagnosis, median disease-free survival has not been reached. These preliminary data suggest that this polyvalent WT1 peptide vaccine can be administered safely to patients with a resulting immune response. Further studies are needed to establish the role of vaccination as viable postremission therapy for acute myeloid leukemia. This study was registered at www.clinicaltrials.gov as #NCT00398138.

Description: Despite advances in therapy, many patients with systemic light-chain amyloidosis (AL) die within 3 years from diagnosis. The humanized 2B6 monoclonal antibody (MoAb) is specific for the low-affinity IgG Fc receptor CD32B and effective in a human CD32B+ B-cell lymphoma murine xenograft model. Because MoAb therapy could improve outcomes in AL, we studied CD32B expression by clonal plasma cells obtained from 48 patients with AL. Transcript profiling showed that expression of CD32B was significantly higher than expression of all other Fc receptor family members. Reverse-transcriptase polymerase chain reaction (RT-PCR) using double-enriched CD138+ plasma cells showed uniform expression of the stable cell surface CD32B1 isoform at diagnosis and relapse, and flow cytometry showed intense CD32B cell surface staining on 99% of CD138+ plasma cells at diagnosis and relapse. These data provide a rationale for the novel therapeutic targeting of CD32B using the humanized 2B6 MoAb in patients with systemic AL-amyloidosis.

Description: Cdc7 is a heterodimeric serine/threonine protein kinase that is a key regulator in the process of initiation of DNA replication and the G1 to S phase transition. Both the kinase and its known substrates are over-expressed in the majority of human cancers. As a result of the recent progress in the areas of pharmacogenetics and high throughput screening technology, identifying specific small molecule inhibitors of cell cycle regulated protein kinases has provided a means not only to study these signal transduction pathways but also to identify potential novel therapeutic agents. To this end, we have developed an assay for Cdc7 kinase inhibitory activity using a highthroughput screening (HTS) approach, screening over 250,000 natural and synthetic small molecules. As a result, we have identified and confirmed seventeen compounds, representing nine different chemical scaffolds, with Cdc7 kinase inhibitory activity. Based on potency, we selected the lead compound (CKI-7) which was further characterized using kinase profiling, microarray experiments, and standard cell based cytotoxicity assays. These latter studies demonstrated that CKI-7 induced cytotoxicity of established leukemia and lymphoma cell lines in culture with inhibitory concentrations (IC50s) in the low nanomolar range. Significantly, CKI-7 likewise induced cytotoxicity of MDR1 overexpressing cell lines with similar IC50s, demonstrating that this novel compound can overcome a major mechanism of chemotherapy resistence in human tumor cells. We additonally demonstrate that CKI-7 induces cytotoxicity of patient-derived primary acute leukemia tumor cells (both chemotherapy naïve and relapsed/refractory samples) in vitro at similarly low nanomolar concentrations. In vivo dose-dependent anti-tumor activity of CKI-7 was subsequently demonstrated in a SCID-Beige mouse systemic tumor model utilzing a recently isolated Philadelphia chromosome positive acute lymphoblastic leukemia cell line (PhALL3.1). Standard cell cycle synchronization studies established that exposure to CKI-7 results in cell cycle dependent caspase 3 activation and apoptotic cell death. This cell death is the direct result of Cdc7 kinase inhibition by CKI-7 as demonstrated using a substrate biomarker assay. In conclusion, our data confirm that Cdc7 is a new promising target for cancer therapy, and that CKI-7, a selective small molecule inhibitor of this enzyme, is an equally promising novel cancer therapeutic agent.

Description: Literature in CLL has emphasized the importance of patient evaluation with measures of minimal residual disease (MRD) by flow cytometry, FISH, or RT-PCR after complete remission (CR). However, the widely used National Cancer Institute-Working Group (NCI-WG) criteria for response in CLL do not mandate the use of CT scanning or ultrasound (US) for the evaluation of patients with abdominal lymphadenopathy. Recently, the German CLL Study Group reported that the CR rate in chemonaïve patients treated with fludarabine (Flu) alone or in combination with cyclophosphamide (Flu/Cy) was reduced by almost one-third when patients were routinely scanned using CT/US (Eichhorst et al, Blood 2006). We recently completed a randomized trial in patients with relapsed/refractory CLL, which afforded an opportunity to examine the impact of CT/US on residual disease in this setting. Methods: Patients were randomized to treatment with Flu/Cy with or without oblimersen. The primary objective was to determine the proportion of patients who achieved CR or nodular partial response (nPR) using NCI-WG criteria. Response and progression were independently assessed by blinded expert review of clinical data, radiology, and bone marrow histopathology. The protocol required that patients with abnormalities on CT/US at study entry must have a repeat CT/US in order to document CR/nPR. CT/US followup was not required in pts who did not respond by other clinical criteria. Results: At entry, 92 of 120 pts (77%) on the oblimersen arm and 97 of 121 pts (80%) on the Flu/Cy arm had baseline CT/US. These baseline scans were abnormal in 88 (73%) and 90 (74%) of pts in each group, respectively. Followup of patients with an initially abnormal scan showed that repeat assessments (i.e., one or more followup CT/US) were similar in each group: 71 pts (59%) in the oblimersen group and 66 (55%) of pts in the Flu/Cy group. The number of followup CT/US (mean = 3; range = 1–14 for both groups), as well as the time between followup scans was also balanced in pts receiving oblimersen (mean = 113 days; median = 97, range = 9–324) or Flu/Cy (mean = 125 days; median = 121, range 5–327). The addition of oblimersen to Flu/Cy significantly increased the proportion of pts who achieved a confirmed CR/nPR (17% vs. 7%) when CT/US was required. To estimate the incidence of CR/nPR when CT was omitted, we assumed that marrow biopsy of pts with a histologically unconfirmed CR/nPR would have shown CR/nPR. With that assumption, the estimated incidence of CR/nPR (ie, absent the requirement for CT/US) increased to 25% and 14% for pts treated with oblimersen and Flu/Cy, respectively. Conclusion: CT or US appear to be widely used by clinicians who treat CLL. Our results confirm and extend prior data that suggest the routine use of CT or US reduces the CR or CR/nPR rates by approximately 30% in both treatment-naïve and previously treated pts with CLL. These data argue that lack of mandatory abdominal CT/US routinely results in an overestimation of CR/nPR incidence, and that evaluation of MRD is potentially meaningful only in the context of confirmed elimination of gross residual disease.

Description: Abstract 2482 Despite the advent of prognostic factors such as cytogenetics and IGH mutational status, the natural history of chronic lymphocytic leukemia (CLL) remains heterogenous and difficult to predict in individual patients. We used the carbocyanine dye, JC-1, to investigate mitochondrial membrane potential in patients with untreated, predominantly early stage CLL. We found that JC-1 staining separated CD5+/CD19+ CLL cells into two discrete populations, Low Red (R)/Green (G) and High R/G, which mainly differed with regard to JC-1 green fluorescence (530 nm) in all patients (Fig. 1). In evaluating CLL patients, we found substantial variation in the proportion of cells in the two populations, with a range of 1.1% to 96.7% (median=79.9%) in the High R/G population. CD19+ B-cells from normal individuals typically had few cells with high JC-1 red fluorescence and had a JC-1 staining pattern that was distinct from CLL patients. Electron microscopy revealed disorganized mitochondrial christae, particularly in patients with elevated High R/G cell populations, compared with mononuclear cells from normal donors. Consistent with the morphologic disorganization, we found that the two populations of CLL cells had differential survival in culture with pyruvate and glucose as primary energy sources; cells in the High R/G population had inferior survival (p=0.016), while cells in the Low R/G population had equal survival in pyruvate compared with glucose (Fig. 2). This result indicates that the CLL clonal cell population can be subdivided into cells able to use oxidative phosphorylation and those dependent on aerobic glycolysis fro metabolic energy needs. Interestingly, patient samples with High R/G cell populations 〉90% had better cell survival in glucose-containing medium than samples derived from patients with 〈 75% High R/G cells (p=0.0085). We found that the mononuclear cells from patients with CLL expressed the M2 isoform of pyruvate kinase, which has been linked to the regulated utilization of aerobic glycolysis. We assessed the outcome of 100 patients with untreated CLL with more than 1 month of follow up (median 36 months) after measuring the proportion of cells in the mononuclear fraction of the peripheral blood with in the High R/G population. The clinical characteristics of the patients at the time of JC-1 staining are characteristic of patients with early stage, untreated CLL. The population was divided into approximate quartiles based on the percentage of mononuclear cells in the High R/G population, and progression-free survival (PFS) was estimated within each quartile. Patients with 50% or fewer cells in the High R/G gate appeared to have an indolent natural history; an estimated 88% (95% CI: 73%, 99%) of the patients had no evidence of significant clinical progression at two years. In contrast, patients with more than 90% of cells in the gate with High R/G cells had rapid progression with an estimated PFS of 50% (95% CI: 31%–82%) at two years (Fig. 3). Patients with 50–90% cells in the High R/G gate had an intermediate disease course. There was strong evidence of a difference in survival distributions across the four JC-1 categories (log-rank p-value 〈 0.001). The percentage of cells in the High R/G gate provided prognostic information that was independent of good risk or poor risk cytogenetic changes. In summary, we found that the clonal, neoplastic cells in patients with CLL comprise discrete populations separated on their metabolic dependence on aerobic glycolysis and aerobic glycolysis. We also show that the percentage of cells utilizing glycolysis has a significant adverse impact on the natural history of patients with CLL. Aerobic glycolysis, the so-called Warburg effect, is a common and incompletely understood aspect of cancer biology. These data tie the acquisition of aerobic glycolysis to more aggressive cancer biology, identify an important new prognostic measure for CLL and could lead to the development of therapies that target metabolic differences in cancer cells. Disclosures: No relevant conflicts of interest to declare.

Description: Combination therapy with purine analogs, alkylators, and monoclonal antibodies has transformed the treatment paradigm in patients with CLL by dramatically enhancing both the quality and frequency of responses that can be achieved in these patients. However, combinations utilizing fludarabine as the purine analog have augmented myelosuppression and immunosuppression requiring careful attention to dosing and schedule in order to minimize these complications. Even with these precautions many patients are unable to complete the entire treatment program at full dose and for the planned number of cycles. Comparative experience with pentostatin indicates that it is less myelosuppressive than either fludarabine or cladribine. We previously reported our experience with pentostatin and cyclophosphamide. Subsequently, we have added rituximab to this active combination (PCR regimen) and treated a second cohort of 46 patients with previously treated CLL (32 patients) and other low grade lymphoid neoplasms (14 patients). The PCR regimen consists of pentostatin 4mg/m2, cyclophosphamide 600mg/m2, and rituximab 375mg/m2 all given on a single day with anti-emetics, hydration, and careful monitoring of renal function. The treatment was administered every 3 weeks for a total of 6 treatments. Rituximab was not given during the first cycle to reduce the frequency and severity of infusion reactions. Filgrastim, sulfamethoxazole/trimethoprim, and acyclovir were administered prophylactically. The median age of the patients treated was 62 (range 44–80) and the median number of prior regimens was 2 (range 1–7). The overall frequency of response was 75% with 25% achieving a complete response, 3% a nodular response, and 47% a partial response. We have compared these results to the recently reported MD Anderson FCR regimen. In terms of pre-treatment characteristics the patient groups in both studies appear comparable with the exception of a higher proportion of high-risk patients treated with PCR (78%) compared to FCR (50%) (P=0.003). The response frequencies are virtually identical in both studies with responses seen 75% of PCR treated patients and 73% of FCR treated patients and CR achieved in 25% in both studies. In terms of toxicity, however, PCR compares favorably to FCR in the following categories: Grade 3/4 neutropenia PCR 53% vs FCR 81% (P=0.0007), thrombocytopenia PCR 16% vs FCR 34% (P=0.04), anemia PCR 9% vs FCR 24% (P=0.06), and grade 3/4 infections (including fever of unknown origin) PCR 28% vs FCR 47% (P=0.05). PCR also appeared to be better tolerated than FCR as indicated by the fraction of patients completing all planned cycles of chemotherapy at full dose 72% vs 38% (P=0.0004). An important caveat in these comparisons is that myeloid growth factor was routinely administered to patients on the PCR study but was not routinely administered to patients treated with FCR. In conclusion, PCR appears to be equivalent in activity to FCR but may be better tolerated and less toxic. These results indicate that a prospective randomized comparison is warranted.

Description: Despite stem cell transplant and new therapies, nearly all patients with AL and MM die of disease or complications of treatment. Novel approaches that selectively kill clonal plasma cells are needed. CD32B, the inhibitory Fcγ receptor IIB, is a member of the Fc receptor (FcR) family on chromosome 1q. B-cells and some monocytes and dendritic cell subtypes express cell-surface CD32B, which has a cytoplasmic inhibitory motif important in regulating immune responses. Unlike the CD32B2 isoform on myeloid cells, CD32B1 on B-cells is not internalized, making it a suitable target for monoclonal antibody (MAb) therapy (Blood 2006Jun6 Epub). CD32B has not previously been found on AL and MM plasma cells. We used purified CD138+ marrow and blood cells from AL, MM and plasma cell leukemia (PCL) patients, and 5 human MM cell lines, to evaluate CD32B gene and cell-surface expression with gene expression profiles (GEP) (Affymetrix U133 PLUS 2.0), RT-PCR for CD32B1 and B2, and flow-cytometry with the 2B6 MAb for CD32B. In AL, GEP showed that CD32B expression was significantly higher than other FcR genes (p98% of all AL plasma cells. In MM, public GEP data (http://lambertlab.uams) showed that CD32B expression was significantly higher than other FcR genes while RT-PCR showed CD32B1 message in CD138+ MM and PCL specimens but not in the RPMI 8226 cell line. Cytogenetic analysis then showed that 8226 cells lack t(4;14) but have 4 copies of 1q, implicating segmental uniparental tetrasomy of a mutant allele in the lack of CD32B message. Flow cytometry showed median 96% CD32B expression on CD138+ marrow cells from patients with normal or hyperdiploid cytogenetics, but significantly lower expression (median 69%, p=0.01) in patients with unfavorable cytogenetics (del 13, t(4;14)). CD32B expression was lower still on PCL specimens (median 10%) and nil on MM cell lines. We then investigated the GEP of sorted CD138+/CD32B+ and CD32B− fractions from MM patients. In CD138+/CD32B+ cells, CD45C and CXCR4 were overexpressed. In CD138+/CD32B− cells, genes on chromosome 1q were overexpressed, including those for cancer testis antigens and others possibly associated with biologic aggressivity (Cancer Cell2006;9:313). Notably, the CD32B-specific MAb 2B6 directs human mononuclear cell cytotoxicity against CD32B+ cell lines, reduces tumor growth and improves tumor-free survival in a mouse xenograft model. Moreover, an Fc-engineered humanized variant of 2B6 elicits ADCC-mediated specific cytotoxicity against a low-level CD32B+ MM cell line in vitro. In sum, these data show that CD32B is important in AL and MM; that CD32B on AL and the majority of MM plasma cells provides a target for MAb therapy; and that, given the results in MM, PCL and MM cell lines, CD32B expression is inversely related to biologic aggressivity. Genetic instability of chromosome 1q may provide a direct basis for this relationship thereby mechanistically linking CD32B expression in MM to prognosis. These data support further evaluation of CD32B and clinical studies of anti-CD32B MAb therapy in AL and MM.

Description: Background: Immunophenotyping is an important method to define hematopoietic malignancies, but used alone, its ability to provide functional information about the malignant cells is limited. JC-1 is a fluorescent dye used to measure mitochondrial membrane potential in cells. We have developed a flow cytometric assay using JC-1 in conjunction with standard cell surface markers to characterize clinical samples from patients with hematologic malignancies. Methods: Ficoll-purified cells derived from the blood or marrow were stained with JC-1, and analyzed at 530 nm and 590nm to assess mitochondrial mass and mitochondrial membrane potential, respectively. Malignant and normal cell populations were assigned and gated by analysis of forward scatter and side scatter. Gating in this manner identified energetically discrete cell populations confirmed by back gating analysis. Results: To date we have measured the mitochondrial membrane potential from the bone marrow or peripheral blood of 3 normal individuals and 22 patients with acute and chronic leukemias and lymphoma involving the marrow. Samples have been analyzed from AML (6), ALL (3), CML- chronic phase (2), CLL (5), mantle cell lymphoma (3), diffuse large B-cell lymphoma (1) and acute biphenotypic leukemia (2). Compared with samples derived from normal individuals, malignant cells have a different energetic signature, and in nearly all instances, a substantially higher mitochondrial membrane potential. The mitochondrial membrane potential of malignant cells was 5.5 times higher (range 2.15–12) than normal cells within the same samples in 19 of 20 specimens (given the overlap in cell populations, CML samples were excluded and 1 CLL sample had a mitochondrial membrane potential that was the same as the normal population). Mitochondrial membrane potential is also higher in malignant cells compared with peripheral blood and marrow cells from normal individuals. While cells derived from patients with acute leukemias appear to be energetically homogeneous, cells from both patients with CML and 4 of 5 patients with CLL had at least 2 energetically discrete subpopulations despite being cytogenetically homogeneous (in the case of the CML samples) or immunophenotypically homogenous (in the case of the CLL samples). Conclusions: JC-1 staining can be performed reliably in concert with immunocytochemistry and provides insight into the metabolism of malignant hematopoietic cells. Preliminary data indicate that malignant cells from patients with leukemia and lymphoma have a high mitochondrial membrane potential which may reflect altered oxidative metabolism. This may serve as a metabolic signature for monitoring minimal residual disease or assessing the effect of therapeutic agents on functional status of malignant populations.

Description: Chromium (51Cr) release assays have long been a standard technique for measuring the cytolytic activity of various immune effector cells. Although relatively easy to perform, these assays suffer from problems with sensitivity and require special radiation precautions limiting their routine use in clinical laboratories. Several flow cytometric techniques measuring cytotoxicity have been introduced and used as a measure of immune response. These assays are more rapid than the 51Cr based assay and have the added advantage of freeing the laboratory from the precautions required when working with radioactivity. Such testing has however largely been restricted to the research setting. In an attempt to extend these benefits to a clinical laboratory, we attempted to validate two commercially available flow cytometry kits (CyToxiLux Plus® & GranToxiLux®, OncoImmunin, Gathersburg, MD) measuring either caspase or granzyme B activity inside K562 target cells induced by interaction with NK cells. These assays were then compared with a standard NK cytotoxicity assay using 51Cr release technique. With K562 as a target, we parallel tested three CD56+CD3-KIR- NK clones; both the 51Cr and caspase based cytotoxicity values were highly positive. With WH autologous EBV cell line as target, we parallel tested five NK clones and five normal control donors as effectors. As expected, all cytotoxicity values for both assays were