Bortezomib and Vorinostat Therapy as Maintenance Therapy after Autologous Transplant for Multiple Myeloma.

BACKGROUND: In multiple myeloma (MM), relapse is a frequent complication after autologous hematopoietic stem cell transplant (ASCT). To reduce the risk of relapse, additional therapy has been added post-ASCT. In a nontransplant relapse setting, the combination of intravenous bortezomib and oral vorinostat (BV) was studied and showed efficacy. Therefore, it was reasonable to study this combination therapy post-ASCT. PATIENTS AND METHODS: We report on BV given post-ASCT. All 30 patients underwent conditioning for ASCT with high-dose melphalan. After recovery from the acute transplant-related toxicity, BV therapy was started and given for a total of 12 (28-day) cycles. RESULTS: The most common toxicities were hematological, gastrointestinal (diarrhea and nausea), fatigue, and peripheral neuropathy. The median follow-up for BV patients is 7.8 (range: 6.12-9.03) years. After BV therapy, 18 patients (60%) are alive, and 9 (30%) are alive without disease progression. CONCLUSIONS: BV can be given post-ASCT with an acceptable toxicity profile and produces reasonable disease-free and overall survival rates. A randomized study comparing the BV regimen to single-agent lenalidomide or bortezomib is needed.

Four amino acid exchanges located in the alpha-2 domain specify the novel HLA-B*50:20 allele.

HLA-B*50:20 contains seven nucleotide substitutions leading to four amino acid changes in the alpha-2 domain.

The novel alleles HLA-B*44:101 and HLA-B*57:48 of Caucasian origin are characterized by amino acid substitutions in the alpha 2 domain.

HLA-B*44:101 and HLA-B*57:48 are characterized by amino acid substitutions in the alpha 2 domain.

The novel HLA-C*12:92 allele is characterized by one amino acid exchange located in the T-cell receptor binding region of the alpha 2 domain.

HLA-C*12:92 contains one amino acid exchange in the T-cell receptor binding region.

Two amino acid changes located in the alpha 1 domain specify the novel HLA-B*27:67 allele affecting the peptide-binding-site characteristics.

The novel HLA-B*27:67 contains three nucleotide substitutions in exon 2 leading to two amino acid changes.

One amino acid change located in the conserved region of the alpha 1 domain specifies the novel HLA-C*07:147 allele.

The novel HLA-C allele HLA-C*07:147 contains one nucleotide substitution in exon 2 leading to an amino acid change in the alpha 1 domain from phenylalanine to leucine.

Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector.

One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34(+) cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor, and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC showed increased colony formation compared with 21% oxygen without NAC (P<0.03), showed increased resistance to mitomycin C compared with green fluorescent protein (GFP) vector-transduced controls (P<0.007), and increased survival. Thus, combining short transduction and reducing oxidative stress may enhance the viability and engraftment of gene-corrected cells in patients with FANCA.

Abnormal oxidant sensitivity and beta-chain structure of spectrin in hereditary spherocytosis associated with defective spectrin-protein 4.1 binding.

Hereditary spherocytosis (HS) is an inherited disorder of erythrocyte shape associated with spectrin deficiency and hemolytic anemia. In a subset of patients with the autosomal dominant form of HS, spectrin displays a reduced capacity to bind protein 4.1 and, therefore, actin; both functions that are critical to the membrane skeleton. A specific structural defect has not been identified in the spectrin from these patients. Chymotryptic digestion of the isolated spectrin chains shows impaired cleavage of the distal peptide of the beta subunit, the beta IV domain. In previous work, we have shown that mild oxidation markedly diminishes the binding capacity of normal spectrin for protein 4.1. Here we observe that chemical reduction of freshly isolated, untreated HS spectrin dramatically improves its function. Thus, a primary structural defect in the beta subunit of spectrin in this subtype of HS may lead to oxidant sensitivity, and secondarily, to a functional defect in the binding of spectrin to protein 4.1 and actin.

Beta spectrin kissimmee: a spectrin variant associated with autosomal dominant hereditary spherocytosis and defective binding to protein 4.1.

We analyzed the DNA sequence of the cDNA encoding the NH2 terminal region of beta spectrin from members of a kindred with autosomal dominant hereditary spherocytosis associated with defective protein 4.1 binding. We found a point mutation at codon 202 within the 272 amino acid NH2-terminal region of beta spectrin. TGG was changed to CGG, resulting in the replacement of tryptophan by arginine. The base change eliminates a normally occurring PvuII restriction site and creates a new MspI site. This finding enabled rapid detection or exclusion of the mutation at the DNA level among the family members, including one member for whom this analysis was performed prenatally. The mutation was found only in the affected family members and occurred as a de novo mutation in the proband. It has not been found in 20 other kindreds. The recombinant peptide derived from the normal cDNA retains the capacity to sediment with protein 4.1 and F-actin. The mutant peptide spontaneously degrades. This variant represents both the first point mutation and the first beta spectrin mutation demonstrated in autosomal dominant hereditary spherocytosis. Furthermore, the mutation is located within a conserved sequence among spectrinlike proteins and may define an amino acid critical for protein 4.1 binding activity.

Characterization of the novel HLA-B*49:39 allele identified in a German leukaemia patient.

HLA-B*49:39 allele is characterised by 1 amino acid substitution in the alpha 1 domain.

Mitoxantrone, etoposide and cytarabine following epigenetic priming with decitabine in adults with relapsed/refractory acute myeloid leukemia or other high-grade myeloid neoplasms: a phase 1/2 study.

DNA methyltransferase inhibitors sensitize leukemia cells to chemotherapeutics. We therefore conducted a phase 1/2 study of mitoxantrone, etoposide and cytarabine following 'priming' with 5-10 days of decitabine (dec/MEC) in 52 adults (median age 55 (range: 19-72) years) with relapsed/refractory acute myeloid leukemia (AML) or other high-grade myeloid neoplasms. During dose escalation in cohorts of 6-12 patients, all dose levels were well tolerated. As response rates appeared similar with 7 and 10 days of decitabine, a 7-day course was defined as the recommended phase 2 dose (RP2D). Among 46 patients treated at/above the RP2D, 10 (22%) achieved a complete remission (CR), 8 without measurable residual disease; five additional patients achieved CR with incomplete platelet recovery, for an overall response rate of 33%. Seven patients (15%) died within 28 days of treatment initiation. Infection/neutropenic fever, nausea and mucositis were the most common adverse events. While the CR rate compared favorably to a matched historic control population (observed/expected CR ratio=1.77), CR rate and survival were similar to two contemporary salvage regimens used at our institution (G-CLAC (granulocyte colony-stimulating factor (G-CSF); clofarabine; cytarabine) and G-CLAM (G-CSF; cladribine; cytarabine; mitoxantrone)). Thus, while meeting the prespecified efficacy goal, we found no evidence that dec/MEC is substantially better than other cytarabine-based regimens currently used for relapsed/refractory AML.

A multicenter trial of myeloablative clofarabine and busulfan conditioning for relapsed or primary induction failure AML not in remission at the time of allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic cell transplantation (HCT) may produce long-term survival in AML after relapse or primary induction failure (PIF). However, outcomes of HCT performed for AML not in remission are historically poor given high relapse rates and transplant-related mortality. Preliminary studies suggest conditioning with clofarabine and myeloablative busulfan (CloBu4) may exert significant anti-leukemic effects without excessive toxicity in refractory hematologic malignancies. A prospective multicenter phase II trial was conducted to determine the efficacy of CloBu4 for patients proceeding directly to HCT with AML not in remission. Seventy-one patients (median age: 56 years) received CloBu4. At day 30 after HCT, 90% achieved morphologic remission. The incidence of non-relapse mortality and relapse at 2 years was 25% and 55%, respectively. The 2-year overall survival (OS) and event-free survival (EFS) were 26% and 20%, respectively. Patients entering HCT in PIF had significantly greater EFS than those in relapse (34% vs 8%; P<0.01). Multivariate analysis comparing CloBu4 with a contemporaneous cohort (Center for International Blood and Marrow Transplantation Research) of AML not in remission receiving other myeloablative conditioning (n=105) demonstrated similar OS (HR: 1.33, 95% confidence interval: 0.92-1.92; P=0.12). HCT with myeloablative CloBu4 is associated with high early response rates and may produce durable remissions in select patients with AML not in remission.

The novel HLA-C*15:103 is characterised by an amino acid substitution in the alpha 2 domain.

HLA-C*15:103 allele is characterised by one amino acid substitution in the alpha 2 domain.

A randomized study of melphalan 200 mg/m(2) vs 280 mg/m(2) as a preparative regimen for patients with multiple myeloma undergoing auto-SCT.

We aimed to examine whether doses of melphalan higher than 200 mg/m(2) improve response rates when used as conditioning before autologous transplant (ASCT) in multiple myeloma (MM) patients. Patients with MM, n=131, were randomized to 200 mg/m(2) (mel200) vs 280 mg/m(2) (mel280) using amifostine pretreatment. The primary end point was the proportion of patients achieving near complete response (⩾nCR). No treatment-related deaths occurred in this study. Responses following ASCT were for mel200 vs mel280, respectively, ⩾nCR 22 vs 39%, P=0.03, ⩾PR 57 vs 74%, P=0.04. The hazard of mortality was not statistically significantly different between groups (mel200 vs mel280; hazard ratio (HR)=1.15 (95% confidence interval (CI), 0.62-2.13, P=0.66)) nor was the rate of progression/mortality (HR=0.81 (0.52-1.27, P=0.36)). The estimated PFS at 1 and 3 years were 83 and 46%, respectively, for mel200 and 78 and 54%, respectively, for mel280. Amifostine and mel280 were well tolerated, with no grade 4 regimen-related toxicities and only one grade 3 mucositis (none with mel200) and three grade 3 gastrointestinal (GI) toxicities (two in mel200). Hospitalization rates were more frequent in the mel280 group (59 vs 43%, P=0.08). Mel280 resulted in a higher major response rate (CR+nCR) and should be evaluated in larger studies.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) priming with successive concomitant low-dose Ara-C for elderly patients with secondary/refractory acute myeloid leukemia or advanced myelodysplastic syndrome.

Patients with advanced MDS and secondary AML respond poorly to chemotherapy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) can stimulate proliferation of leukemic blasts and sensitize these cells to the cytotoxic effects of S-phase-specific drugs. This is the first report of safety and efficacy of GM-CSF prior to and during cytarabine in a low-dose, intermittent regimen for elderly patients with poor risk acute myelogenous leukemia or myelodysplastic syndrome. Twenty patients, age 68 to 86 years, each received 250 microg/m2 of GM-CSF (Sargramostatin; Immunex, Seattle, WA, USA) subcutaneously (s.c.) or intravenously (i.v.) for 3 days followed by GM-CSF at the same dose and cytarabine 100 mg/m2 i.v. for 3 days. GM-CSF and cytarabine were both administered for 3 days during weeks 2 and 3 followed by a 3-week rest period. Rates of CR and PR were 20% and 40%, respectively. These included clinically significant resolution of cytopenias and transfusion requirements. Many of the responding patients had been heavily pretreated prior to enrollment. One- and 2-year survival estimates are 44% and 19%, respectively. Myelosuppression was the most significant toxicity. Our findings suggest that this novel combination of GM-CSF with sequential and concomitant low-dose cytarabine can benefit patients with poor risk myeloid malignancies.

The molecular basis for the cytokine-induced defect in homing and engraftment of hematopoietic stem cells.

OBJECTIVE: Hematopoietic stem cell homing and engraftment is dramatically altered by cytokine exposure. These studies address the molecular mechanisms responsible for the observed changes in transplantation biology. METHODS: Primitive murine hematopoietic stem cells were isolated by fluorescence-activated cell sorting of lineage depleted (Lin(-)) cells exhibiting low staining of Hoechst 33342 and rhodamine 123 dyes or Lin(-) cells bearing Sca. Adhesion receptor expression was examined by immunofluorescence and reverse transcriptase polymerase chain reaction. In vitro adhesion assays were employed to define binding interactions between stem cells and stroma or extracellular matrix proteins. RESULTS: Adhesion of Lin(-)Sca+ cells to Dexter stroma could be blocked by about 90% with antibodies to PECAM-1, alphaa(4), or beta(1), and partially blocked by antibodies to alpha(5), CD44, or L-selectin. By immunofluorescence, about 30% of purified Lin(-)Ho(lo)Rho(lo) cells expressed alpha(4), alpha(5), beta(1), and L-selectin, about 15% expressed alpha(L) and alpha(6), half expressed PECAM-1, and none expressed alpha(1) or alpha(2). After 48 hours in expansion cytokines, only 9% of the cells expressed alpha(4) and none expressed beta(1), whereas alpha(L) expression was fully restored, PECAM-1 and L-selectin partially restored, CD44 expression was newly induced, and adhesion to both fibronectin and laminin was reduced. Adhesion to purified collagen, fibronectin, or laminin enhanced expression of beta(1) integrins. CONCLUSION: Expansion cytokines that move quiescent primitive hematopoietic stem cells into S phase markedly altered adhesion receptor expression and reduced their functional binding to extracellular matrix, which could reduce engraftment after transplant.

Gene transfer to ankyrin-deficient bone marrow corrects spherocytosis in vitro.

OBJECTIVE: The goal of this study was to transfer by retroviral vector the cDNA for ankyrin to progenitors from normal bone marrow and from the nb/nb spherocytosis mutant deficient in expression of full-length ankyrin to achieve erythroid expression of functional ankyrin protein. MATERIALS AND METHODS: A minigene composed of the human ankyrin promoter, murine ankyrin cDNA, and the 3' human domain corresponding to the ankyrin 2.2 isoform was assembled in the retroviral vector, pG1. Murine erythroleukemia (MEL) cells, normal murine bone marrow cells, 3T3 fibroblasts, and nb/nb mutant bone marrow and spleen cells were transduced with the retroviral supernatant. Transduced mutant cells were induced to differentiate in liquid culture. Gene transfer was assessed by colony polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR, immunofluorescence, and Southern, Northern, and Western blot analysis. RESULTS: MEL cells, normal bone marrow progenitors, and nb/nb cells were all successfully transduced and expressed ankyrin by RT-PCR and Western blot. Transduced murine 3T3 fibroblasts and MEL cells exhibited cell membrane staining by immunofluorescence. Colony RT-PCR demonstrated dependence of expression on erythropoietin. In vitro, the transduced nb/nb cells matured to polychromatophils, whereas nontransduced nb/nb cells matured to microspherocytes. CONCLUSION: Retroviral transfer of ankyrin corrected the defect leading to formation of microspherocytes in erythroid differentiation cultures from the nb/nb mutant. The human ankyrin promoter conferred erythropoietin-dependent expression in normal and mutant erythroid progenitors, which could have implications for the gene therapy of human hemolytic anemias.

Adhesion receptor expression by hematopoietic cell lines and murine progenitors: modulation by cytokines and cell cycle status.

Hematopoietic progenitor cells are incubated with cytokine combinations for in vitro expansion of stem cells and to enhance retrovirus-mediated gene transfer. Optimization of the engraftment of these treated cells would be critical to the success of stem cell transplantation or gene therapy. Previous studies demonstrated that a 48-hour incubation of donor BALB/c bone marrow with a mixture of four cytokines (IL-3, IL-6, IL-11, and SCF), resulted in expansion of primitive progenitor/stem cells but a loss of long-term engraftment in nonmyeloablated or myeloablated recipients. We have established the expression pattern for a number of adhesion receptors by normal hematopoietic progenitors and cell lines and the modulation in expression induced by cytokines or cell cycle progression to ascertain the molecular basis for such defective engraftment. Northern blot analysis demonstrated that the cytokine combination of IL-3, IL-6, IL-11, and SCF dramatically down-regulated alpha 4 integrin receptor expression in HL-60 cells. Synchronized FDC-P1 cells exhibited modulation of alpha 4 expression through cell cycle progression, both by quantitative RT-PCR and flow cytometry. Normal murine bone marrow lineage-depleted, Sca+ cells expressed a number of adhesion receptors, including alpha L, alpha 1, alpha 3, alpha 4, alpha 5, alpha 6, beta 1, L-selectin, CD44, and PECAM as assessed by flow cytometry, immunofluorescence, and RT-PCR. There was modulation of the expression of several of these receptors after incubation in the four cytokines for 24 and/or 48 hours: the proportion of cells expressing alpha L, alpha 5, alpha 6, and PECAM increased, whereas the proportion of cells expressing alpha 4 and beta 1 decreased, after cytokine incubation. There was a demonstrable concomitant decline in adhesion of these cells to fibronectin after the cytokine incubation, a finding that correlates with the decrease in expression of alpha 4. These changes in adhesion receptor expression and function with cytokines and during cell cycle transit may be critical to stem cell homing and engraftment after transplantation, as multiple receptors could be involved in the process of rolling, attachment to endothelium, endothelial transmigration, and migration within the marrow space.

Mesenchymal stromal cells derived from acute myeloid leukemia bone marrow exhibit aberrant cytogenetics and cytokine elaboration.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) play a fundamental role in the BM microenvironment (BME) and abnormalities of these cells may contribute to acute myeloid leukemia (AML) pathogenesis. The aim of the study was to characterize the cytokine and gene expression profile, immunophenotype and cytogenetics of BM-MSCs from AML patients compared to normal BM-MSCs from healthy donors. AML BM-MSCs showed decreased monocyte chemoattractant protein-1 levels compared to normal BM-MSCs. AML BM-MSCs expressed similar ß1 integrin, CD44, CD73, CD90 and E-cadherin compared to normal BM-MSCs. Cytogenetic analysis revealed chromosomal aberrations in AML BM-MSCs, some overlapping with and others distinct from their corresponding AML blasts. No significant difference in gene expression was detected between AML BM-MSCs compared to normal BM-MSCs; however, comparing the differences between AML and MSCs from AML patients with the differences between normal hematopoietic cells and normal MSCs by Ingenuity pathway analysis showed key distinctions of the AML setting: (1) upstream gene regulation by transforming growth factor beta 1, tumor necrosis factor, tissue transglutaminase 2, CCAAT/enhancer binding protein alpha and SWItch/Sucrose NonFermentable related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4; (2) integrin and interleukin 8 signaling as overrepresented canonical pathways; and (3) upregulation of transcription factors FBJ murine osteosarcoma viral oncogene homolog and v-myb avian myeloblastosis viral oncogene homolog. Thus, phenotypic abnormalities of AML BM-MSCs highlight a dysfunctional BME that may impact AML survival and proliferation.

Dural scrofula.

A middle-aged woman presenting with multiple cranial neuropathies, hemiparesis, and CSF pleocytosis had tuberculous infection of the cranial dura mater at autopsy. This is the first description of dural scrofula in modern medical literature.