ALBERT

Description: cAMP-mediated signaling potentiates glucocorticoid-mediated apoptosis in lymphoid cells, but an effective means by which to take advantage of this observation in the treatment of lymphoid malignancies has not been identified. The PDE4 enzyme family regulates the catabolism of cAMP to AMP in a wide range of tissues. PDE4 inhibitors have recently been submitted for approval for use in asthma and COPD. In leukemic samples from 11 B-CLL patients, rolipram and RO20-1724, two structurally unrelated PDE4 inhibitors, synergized with either hydrocortisone or dexamethasone in inducing B-CLL but not T cell apoptosis. Dose titration studies demonstrated that addition of a PDE4 inhibitor augmented B-CLL apoptosis even when maximally effective doses of either glucocorticoid were utilized. In five patients so analyzed, 10 uM rolipram augmented the induction of apoptosis by 100 uM hydrocortisone by 40 +/− 18%. Using transient transfection of a GRE-luciferase construct with an Amaxa nucleofector technique, we determined that treatment with PDE4 inhibitors augmented glucocorticoid receptor (GR)-mediated GRE transactivation in primary B-CLL cells. Strikingly, inhibition of PKA with the cAMP antagonist Rp-8Br-cAMPS inhibited glucocorticoid-induced apoptosis by 86 +/− 14% in 6 patients so tested and GRE transactivation by 83% in 8 patients so tested. Similarly, treatment with Ht31 peptide, a 23 residue peptide derived from an AKAP that binds with 4.0 nM dissociation constant to PKA RII subunits, also reduced hydrocortisone-induced transactivation. These studies suggest that PKA activity is required for both the ability of glucocorticoids to induce apoptosis and GRE transactivation in B-CLL cells. CCRF-CEM cells, a well-studied model of glucocorticoid and cAMP-induced apoptosis, differed from B-CLL cells in that stimulation of adenylyl cyclase with the diterpene forskolin was required to increase both glucocorticoid-mediated apoptosis and GRE activation, while PDE4 inhibition had no effect. We isolated both dexamethasone-sensitive and dexamethasone-resistant CCRF-CEM clones for these studies and demonstrated that forskolin induced glucocorticoid sensitivity even in the initially dexamethasone resistant clone. 1,9 dideoxyforskolin, a forskolin analogue that does not activate adenylyl cyclase, failed to augment glucocorticoid sensitivity in CCRF-CEM cells. Given the marked discrepancy in the sensitivity of B-CLL cells and CCRF-CEM cells to PDE4 inhibitor-induced augmentation of glucocorticoid apoptosis and GRE transactivation, we next examined the cAMP response and PDE4 isoforms in these two cell types. Inhibition of PDE4 induced cAMP elevation in B-CLL but not CCRF-CEM cells, while forskolin augmented cAMP levels in CCRF-CEM but not B-CLL cells. While rolipram but not forskolin treatment up-regulated 63 and 68 kDa forms of PDE4B (most likely PDE4B2) in B-CLL, forskolin but not rolipram treatment up-regulated 67 and 72 kDa forms of PDE4D (most likely PDE4D1/D2) in CCRF-CEM cells. These studies suggest that PKA is required for and enhances glucocorticoid-induced apoptosis in B-CLL by modulating GR signal transduction and that inhibition of PDE4 in the absence of exogenous adenylyl cyclase activation is a clinically tenable means by which to achieve such PKA activation. Clinical trials that examine whether PDE4 inhibitors enhance the efficacy of glucocorticoid-containing chemotherapy regimens in B-CLL and other lymphoid malignancies are indicated.

Description: We have performed 2,000+ Fluorodeoxyglucose PET (PET) scans for multiple myeloma (MM) staging and restaging at our facility since October 2001. While the usefulness of the PET scan for MM is reported by us and others elsewhere, we have reviewed our list of “incidental” but important findings, some of which are unique or occur more commonly with MM patients and some of which are common to many patients, the more common of which we present below: Occult infection occurs very commonly in patients with MM due to direct tumor effect on the immune system and to medication (especially high dose dexamethasone). Of the 2000+ PET scans for MM done at our facility, 300+ infections (about one half occult) have been detected by PET. These most commonly involve central lines (septic thrombophlebitis), diskitis, lung (either bacterial or fungal), and periodontal abscesses (a source of bacteremia in these patients), though non-catheter related spontaneous septic thrombophlebitis also occurs. While related to MM, extramedullary disease from MM (EMD) is seen more commonly with PET than MRI since PET has a wider field of view. In our series of 172 patients with both baseline PET and MRI, PET detected EMD in 11/11 patients versus MRI detection rate of 7/11. On follow-up, PET detected three times more EMD (29/31 sites) than MRI (9/31sites). Detection of EMD by PET at baseline is a profoundly negative prognostic factor in our series (12 month EFS 20% for EMD+ vs. 47% for EMD−, p = 0.002, and OS 42% for EMD+ vs. 70% for EMD−, p=0.005, n=48). Following tumor response in hypo- or non-secretory disease is difficult by standard prognostic factors (SPFs). FDG PET results were actively used for clinical management in 10 of 11 non-secretory patients at our facility. A major pitfall of PET scanning for MM comes from the use of steroids. Treatment with chemotherapy in general and steroids (i.e. prednisone or dexamethasone) in particular can result in a false negative PET scan by producing a profound but transient suppression of tumor metabolism. In addition, the hyperglycemic effect of the steroids produces competitive inhibition of FDG (a glucose analog) uptake, also causing suppression of FDG tumor uptake that can lead to an underestimation of disease. Second primary malignancies are frequently seen the MM age group. In our population, we have found unsuspected breast cancer, breast lymphoma, thyroid cancer, melanoma, colon cancer and lung cancer. In addition, we have found several functioning thyroid adenomas and premalignant colon polyps. Being a whole body imaging device for metabolism, FDG PET is a powerful though nonspecific tool for imaging that can not only help stage and restage the malignancy under question but which can yield a plethora of additional clinically useful information if used properly and with its limitations understood.

Description: This report of a phase 2 trial of thalidomide (THAL) (200 mg/d; 200 mg increment every 2 weeks to 800 mg) for 169 patients with advanced myeloma (MM) (abnormal cytogenetics (CG), 67%; prior autotransplant, 76%) extends earlier results in 84 patients. A 25% myeloma protein reduction was obtained in 37% of patients (50% reduction in 30% of patients; near-complete or complete remission in 14%) and was more frequent with low plasma cell labeling index (PCLI) (below 0.5%) and normal CG. Two-year event-free and overall survival rates were 20% ± 6% and 48% ± 6%, respectively, and these were superior with normal CG, PCLI of less than 0.5%, and β2-microglobulin of 3 mg/L. Response rates were higher and survival was longer especially in high-risk patients given more than 42 g THAL in 3 months (median cumulative dose) (landmark analysis); this supports a THAL dose-response effect in advanced MM.

Description: MGUS is a pre-malignant state, with a 1% annual risk of progression to overt myeloma (Kyle, NEJM346:564, 2004). Progression of MGUS to myeloma is preceded and can be predicted by increased rates of bone turnover (Betaille, JCI88:62, 1991). Progress to overt myeloma is associated with lytic bone disease in about 80% of patients. A simple, reliable test that identifies MGUS patients at risk of progression to symptomatic myeloma would elucidate biological mechanisms that govern the process and identify targets for early intervention. The intimate association of progression with changes in bone biology suggested that early recognition of changes in bone turnover would serve such a purpose. Therefore, we sought to identify protein biomarkers of bone disease in the sera of patients with myeloma that can be used as early predictors of progression. Given the limited reliability of individual markers of bone turnover to identify changes in bone metabolism, we elected to adopt a global proteomics approach. Sera from 62 untreated myeloma patients were collected and stored at −80°C for future analysis. 35 serum samples were from myeloma patients with 1–26 lytic bone lesions as identified on X-ray skeletal surveys and 27 serum samples were from patients without lytic lesions. The sera were analyzed by surface-enhanced laser desorption and ionization-time of flight mass spectroscopy (SELDI-TOF MS) using Ciphergen’s ProteinChip Biology System II (PBS II), to identify protein patterns associated with lytic bone disease. Each sample was applied in 4 replicates to randomly assigned spots on 12 IMAC30 ProteinChips placed in a bioprocessor and activated with Cu++ ions using a Biomek2000 robot. The chips were read on a PBS II reader. The mass spectra of proteins, generated using an average of 66 laser shots, were calibrated using peptide and protein standards and normalized to total ion current using CiphergenExpress 2.0 software. To develop a classification model that separates non-treated patients with focal lesions from those without focal lesions, we identified among the low molecular weight (1500–25000 kDa) proteins a set of 17 protein peaks that were differentially expressed between the two groups at a significance level of p

Description: Abstract 4603 Introduction: Successful transplantation of cryopreserved hematopoietic stem cell (HSC) can be regularly achieved provided sufficient numbers of cells are administered. However the optimal conditions for preparation, freezing and thawing remain to be defined. The duration of hematopoietic stem cell viability is unclear. The ultimate measure of viability has remained in vivo hematologic recovery following transplantation. Evidence of autologous repopulation in preclinical setting has been seen at 14 years after bone marrow transplant and after 12 years in the clinical setting after peripheral stem cell transplant. We report a successful transplantation in a patient in whom autologous cryopreserved marrow with cellular dose of 1.21 × 108/kg was infused 21 years following collection. Case: The patient is 43 year old man found to have follicular lymphoma with bone marrow involvement in 1989 at age 22. He achieved complete remission after treatment with two cycles of Chorambucil. Bone marrow procurement and cryopreservation was performed at that time for possible subsequent infusion. The procured marrow consisted of a total cell count of 1.21 × 10^8 cells/per kg body wt with a total volume of 354 ml. Equal parts of 20% DMSO were combined with marrow to a final concentration of 10% DMSO. The marrow was then stored in the liquid phase of nitrogen from date of collection until date of infusion 21 years later. Our patient relapsed in 1996, and eventually underwent treatment in 2006 with six cycles of Fludarabine and Rituximab, achieving a complete remission. He continued Rituximab therapy maintenance every 6 months for a total of 2 years. During Rituximab therapy, he was noted to have pancytopenia Work-up confirmed MDS with 5q- and translocation of long arm of chromosome 6q21 and short arm 17p13 in 20/20 cells by karyotype analysis. Patient elected to proceed with the cryopreserved marrow transplant. Assessment of cryopreserved marrow for dysplasia was undertaken showing no evidence of cytogenetic or histological changes. The patient was prepared with Busulfan IV at 0.8/kg q 6 hours × 4 days and Cyclophosphamide 60mg/kg IV × 2 days. The marrow was infused 21 years following its procurement. Samples from the infused marrow showed 65–75% viability by Tryptan blue assay. White cell engraftment occurred on day 17 and platelet reached 20,000 by day 30. Follow-up 2 months post transplant revealed persistent mild pancytopenia, WBC of 2.6 with ANC 1500, Hgb 9.8 and platelets of 43,000. FISH analysis for 5q performed showed 85/200 cells positive for 5q-. Bone marrow biopsy confirmed dysplastic features consistent with his pre-transplant bone marrow biopsy. Our case illustrates that even in the setting of marginal numbers of infused marrow components and after prolonged cryopreservation, repopulation can readily occur. However, inability to eliminate the malignant clone following stable engraftment brings to light both the necessity of adequate ablation in pretransplant conditioning, and the importance of graft-versus-tumor effect in marrow born malignancies. To our knowledge, this is the oldest successful cryopreserved autologous bone transplant at 21 years post preservation. As novel uses of stem cells advance, optimal storage of various cellular components is necessary. This should be further investigated on a larger scale in the future. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2957 Myeloma is intimately associated with osteolytic bone disease, resulting from myeloma cells' interactions with osteoclasts and osteoblasts and their progenitors, and is dependent on the changes it induces in bone metabolism for progression. Myeloma cell dependence on the bone marrow microenvironment is also evident experimentally, where interaction of primary myeloma plasma cells (MMPC) with osteoclasts (OC) and with mesenchymal stem cells (MSC) support the survival of primary myeloma cells. To understand the molecular mechanisms associated with the survival of MMPC, we used Acuity 4 software to analyze Affymetrix U133 Plus2 chip data and identify changes in gene expression in induced MMPC freshly isolated from 8 patients by interaction with OC and from 8 additional patients with MSC. Expression by MMPC of 675 genes was changed following interaction with OC; 552 genes were upregulated and 123 down regulated. Expression of 296 genes was changed in MSC co cultures (161 upregulated, 135 down regulated). Comparison of the genes whose expression was similarly changed in both co culture systems identified 72 probesets, representing 58 genes, that were commonly changed; 33 were upregulated and 25 down regulated. Ingenuity Pathway Analysis assigned 54 of the 58 genes to 4 distinguished networks of interrelated genes with high probability scores. We next tested the hypothesis that the expression of genes whose expression was commonly changed in the co culture systems has clinical significance. To accomplish this, we used gene expression data available on 127 relapsed patients who had been uniformly treated on our Total Therapy 2 protocol, and for whom gene expression (GEP) data at first relapse (RL) were available. 71 of these patients also had pre treatment (BL) GEP data; for these 71 patients we calculated change in expression of the 72 probesets as the ratio of RL/BL expression signal. We identified 7 genes whose expression changes were significantly (p≤0.05) associated with survival after relapse: These genes were, in order of significance: CCNE2, PECAM1, KLHL21, ICAM1, PLAU, ANPEP, and DUSP1, with p-values ranging from 0.017 to 0.05. Up regulation of PECAM1, ANPEP, DUSP1, and down regulation of CCNE2, KLHL21, ICAM1, and PLAU were associated with longer survival. We further determined whether expression level of these 72 probesets at relapse, defined by signal intensity, correlated with post relapse survival of the 127 patients; 18 genes were significantly (p

Description: Abstract 1815 The bone marrow (BM) microenvironment plays critical role in the progression of multiple myeloma (MM) and identification of dysregulated microenvironmental pathways is essential for development of effective interventions for this disease. We and others have shown that bone disease is both a consequence of and a necessity for MM progression and that restoring bone turnover helps control MM. Prooxidant environment has been shown to promote tumorigenesis and bone disease, while circulating levels of antioxidants are reduced in MM patients (Sharma et al., 2009). Heme oxygenase 1 (HMOX1) is an intracellular inducible antioxidant that acts in response to oxidative stress and mediates anti-inflammatory and wound healing processes. The aim of the study was to identify dysregulated microenvironmental factors in myelomatous bone using mesenchymal stem cell (MSC) cytotherapy as an approach previously shown by us to induce bone formation and inhibit osteolysis and MM progression (Li et al., 2011; 2012). Intrabone injection of fetal MSCs into myelomatous bones of SCID-hu mice engrafted with a patient's MM cells resulted in significant upregulation(≥2 folds) of 120 probe sets and downregulation of 44 probe sets 24 hours after MSC cytotherapy, assessed by global gene expression profile (GEP, 9 mice/group). HMOX1, one of top overexpressed genes by MSC cytotherapy (5.2 folds, p

Description: Multiple myeloma (MM) is a B-cell malignancy stratified in part by cytogenetic aberrations including the high-risk copy number alterations (CNAs) of 17p- and +1q21. CNAs of 1q21 are known to occur as a result of jumping translocations of 1q (JT1q12) which increase the copy number (CN) of 1q21, while at the same time causing collateral genomic damage including deletions and amplifications in some receptor chromosomes (RC). We have previously reported the decondensation of the 1q12 region and triradial chromosomes in the bone marrow specimens of patients with aggressive disease, and have speculated that hypomethylation of the pericentromeric region of 1q12 may play a role in the origin of 1q21 CNAs. To test the hypothesis that 1q12 hypomethylation induces CN gains of 1q21, we used the hypomethylating agent 5-azacytidine to treat blood lymphocyte cultures of five myeloma patients with balanced constitutional 1q12 aberrations as well as five normal control subjects. The patients with constitutional aberrations of 1q12 were selected based on their potential to show either novel CN gains of regions translocated distal to 1q12 or CN gains of 1q21 when 1q12 was translocated to a non-homologous chromosome. The constitutional aberrations included two patients with pericentromeric inversions of chromosome 1 inv(1)(p11q12), and one patient each with a balanced translocation of t(1;2)(q12;p11), t(1;2)(q12;q34), or t(1;9)(q12;q11). These particular aberrations should provide a unique model for testing the association between the hypomethylation of 1q12 and the origin of CN gains of 1q21. In vitro blood lymphocyte cultures of patients and controls were treated for 72 hours with 5-azacytidine at a final concentration of 10uM. 5-azacytidine is known to cause site-specific hypomethylation and chromatin decondensation in the 1q12 region. Utilizing FISH probes for 1q12 (satII/III) and 1q21(CKS1B), we analyzed 100 metaphase cells from each patient and each control for CN gains for 1q21 and any novel region translocated distal to 1q12. To fully characterize the induced rearrangements of 1q12, we also applied inverse G-banding and spectral karyotyping in selected cells. Remarkably, all patients with the balanced constitutional 1q12 rearrangements showed CN gains of chromosome regions on the derivative chromosomes distal to the inverted or translocated 1q12 regions. Recurring aberrations of 1q12 included pericentromeric decondensation and triradials of chromosome arms 1q, 1p, and 2p, in addition to localized pulverizations distal to 1q12 of these same arms. Specifically, in both patients with inv(1), whole-arm CN gains of 1p were identified in 2-3% of cells, while in the patient with t(1;2)(q12;p11), whole-arm gains of 2p (MYCN) were identified in 3% of cells. In these same three patients, on the derivative chromosomes where the 1q12 region was separated from 1q21 by an inversion or translocation, no CN gains of the 1q21 occurred. In the patients with 1q12 translocated to a non-homologous chromosome, 1q21 was amplified on the RC in 4% of the cells in the patient with t(1;2)(q12;q34), and in 3% in the patient with t(1;9)(q12;q11). Control subjects showed CN gains of 1q21on both chromosomes 1 in the range of 3-13%. Whole-arm JT1q12s to RC16q were identified in three patients and three controls. This JT1q12 results in a CN gain of 1q21, and a whole-arm CN loss in RC16q, identical to those found in the bone marrow specimens of MM patients. Interestingly, one control showed JT1q12s to two different RCs, 9q and 12q, causing whole-arm deletions in the long arms of both. The key structural aberration responsible for the generation of CN gains of 1q21 and subsequent JT1q12s is the formation of a triradial involving the 1q12 region. The novel finding in this study is the induction of triradials and CN gains for chromosome arms of 1p and 2p, which demonstrates that epigenetic modifications to 1q12 can amplify non-homologous chromosome regions juxtaposed to it. Furthermore, the CN gains induced here occurred only distal to 1q12 on all normal and derivative chromosomes and mimic the triradials and JT1q12s reported in the bone marrow of patients with aggressive disease. These findings demonstrate for the first time that the hypomethylation of the 1q12 region can potentially amplify any genomic region distal to it, and provide evidence for an epigenetic origin of the high-risk 1q21 CNAs found in the progression of MM. Disclosures No relevant conflicts of interest to declare.

Description: Families with 3 different syndromes characterized by autosomal dominant inheritance of low platelet count and giant platelets were studied. Fechtner syndrome is an autosomal-dominant variant of Alport syndrome manifested by nephritis, sensorineural hearing loss, and cataract formation in addition to macrothrombocytopenia and polymorphonuclear inclusion bodies. Sebastian platelet syndrome is an autosomal-dominant macrothrombocytopenia combined with neutrophil inclusions that differ from those found in May-Hegglin syndrome or Chediak-Higashi syndrome or the Dohle bodies described in patients with sepsis. These inclusions are, however, similar to those described in Fechtner syndrome. Other features of Alport syndrome, though, including deafness, cataracts, and nephritis, are absent in Sebastian platelet syndrome. Epstein syndrome is characterized by macrothrombocytopenia without neutrophil inclusions, in addition to the classical Alport manifestations—deafness, cataracts, and nephritis—and it is also inherited in an autosomal-dominant mode. We mapped the disease-causing gene to the long arm of chromosome 22 in an Italian family with Fechtner syndrome, 2 German families with the Sebastian platelet syndrome, and an American family with the Epstein syndrome. Four markers on chromosome 22q yielded an LOD score greater than 2.76. A maximal 2-point LOD score of 3.41 was obtained with the marker D22S683 at a recombination fraction of 0.00. Recombination analysis placed the disease-causing gene in a 3.37-Mb interval between the markers D22S284 and D22S693. The disease-causing gene interval in these 3 syndromes is similar to the interval described recently in an Israeli family with a slightly different Fechtner syndrome than the one described here. Recombination analysis of these 3 syndromes refines the interval containing the disease-causing gene from 5.5 Mb to 3.37 Mb. The clinical likeness and the similar interval containing the disease-causing gene suggest that the 3 different syndromes may arise from a similar genetic defect.

Description: Abstract 2915 Background: AMG is the most common plasma cell dyscrasia, currently classified as either monoclonal gammopathy of undetermined significance (MGUS) or asymptomatic multiple myeloma (AMM), based on the level of monoclonal immunoglobulin (M-protein), bone marrow plasmacytosis and other criteria defined by the International Myeloma Working Group. While information is available on the impact of clinical variables such as bone marrow plasmacytosis, free light chains, isotype and M-protein on the hazard of progression to symptomatic MM (MM), little is known about the value of the karyotype and DNA content, as determined by DNA/cIg flow cytometry, on the risk of progression from AMG to MM. Methods: Patients from the Myeloma Institute for Research and Therapy (MIRT) with AMG that were enrolled in a prospective observational clinical trial were evaluated. All patients underwent detailed clinical staging at entry and were followed at pre-specified intervals per protocol. Cox proportional hazards regression was used to model univariate and multivariate associations of baseline features with progression to MM. The number of distinct DNA stem lines in the flow cytometry assay, their percentages, respective DNA Indices (DI), cytoplasmic Immunoglobulin Indices (cIgI), and percent of cells in S phase were evaluated alone and in relation to the karyotype report at baseline. A DI between 0.99 and 1.01 referred to diploidy, lesser than 0.99 to hypodiploidy and more than 1.01 hyperdiploidy. Results: Data from 267 eligible MIRT patients with AMG were analyzed. Of these patients 99% (265/267) had performed DNA/cIg flow cytometry and had a karyotype report at diagnosis. Cytogenetic abnormalities were detected in 20 of the 265 patients from whom data were available. From the 265 patients from whom DNA/cIg flow cytometry data were available, no abnormal clones were identified in 14% (37/265), one clone was identified in 95 patients (36%), two clones in 122 patients (46%), three clones in 10 (4%), and in 1 patient 4 clones were identified. Most patients with abnormal DNA content had hyperdiploid clones (132/243 patients). The second most frequent finding was diploid DI, in 39% (104/243) of patients; 3% (7/243) had a hypodiploid DI. The median DI was 1.01 (0.9–2.02) and median cIg was 7 (1–50). Interestingly, the median cIgI value in AMG was more than twice that of its value (3.4, 1–22) in Total Therapy 3 MM patients (p=0.001). In univariate analysis of the parameters in this study, the presence of an abnormal karyotype (p=0.032, HR=2.62), the number of DNA/cIg clones (p=0.016, HR=1.69) and the percentage of the dominant clone (p=0.003, HR=1.03) were significantly related to progression to MM. Ploidy by DNA/cIg analysis, the S-phase fraction, and cIg did not reach statistical significance (p=0.863, p=0.132 and p=0.240, respectively). In multivariate analysis, only the number of abnormal clones (p=0.013, HR=1.78) retained statistical significance, while the percentage of the dominant clone neared significance (p=0.070, HR=1.02). Using running log rank tests we were able to identify optimal cut-points for the percentage of the dominant clone and the number of clones (12% and 2 clones respectively). From these, a risk score was obtained which identifies three distinct groups with 3-yr MM progression probabilities of 12%, 30% and 67% (p