ALBERT

Description: We conducted a phase II randomized trial of recombinant granculocyte-macrophage colony-stimulating factor (GM-CSF) administered before topotecan chemotherapy to determine whether it could prevent myelosuppression and to determine the antitumor activity of this topoisomerase I inhibitor in 53 patients with metastatic malignant melanoma and renal cell cancer. All patients received GM-CSF after topotecan at a dose of 250 μg/m2 daily for at least 8 days. Patients randomly assigned to receive GM-CSF priming were treated with GM-CSF at 250 μg/m2 twice daily for 5 days before treatment. Twenty-five patients were randomly assigned to receive GM-CSF priming and 28 to receive topotecan without priming. The primary analysis was restricted to the protective effects seen during the first cycle of therapy. Grade 4 neutropenia occurred in 8 of 23 patients (35%) and grade 3 neutropenia in 5 of 23 patients (22%) randomized to GM-CSF priming, whereas 18 of 26 (69%) and 5 of 26 (19%) patients experienced grade 4 or 3 neutropenia, respectively, without GM-CSF priming (P = .0074). The mean duration of neutropenia was reduced by GM-CSF priming: grade 3 neutropenia from 5.2 ± 0.7 to 2.8 ± 0.7 days (P = .0232) and grade 4 neutropenia from 2.7 ± 0.6 to 1.1 ± 0.4 days (P = 0.0332). The protective effects of GM-CSF extended to the second cycle of treatment. The incidence of febrile neutropenia was also reduced. Chemotherapy-induced anemia and thrombocytopenia were similar in both groups. One partial response was seen in a patient with melanoma, and one patient with renal cell cancer had complete regression of pulmonary metastases and was rendered disease-free by nephrectomy.

Description: Introduction: Mantle cell lymphoma (MCL) is a CD20+ malignancy comprising up 5% of non-Hodgkin’s lymphomas, and has a poor prognosis under standard chemotherapy. The HyperCVAD-M/A regimen (fractionated high-dose cyclophosphamide, vincristine, doxorubicin and prednisolone alternated with methotraxate and cytarabine) has yielded encouraging results when combined with autologous stem cell transplantation (ASCT) in MCL, with 5-year failure-free survival of 54% and overall survival 72%. In an effort to improve these results further, we have combined rituximab in vivo purging and post-transplant consolidation with HyperCVAD-M/A plus ASCT. Methods: Patients aged

Description: Factor V Leiden, (FVL) is the most common known inherited thrombotic risk factor and is present in approximately 5% of most Western populations and 25–50% of patients presenting with venous thrombosis. However, FVL is incompletely penetrant, with only approximately 10% of FVL carriers developing thrombosis in their lifetimes. Though interactions between FVL and other known prothrombotic mutations have been documented in a few cases, the genetic factors responsible for the incomplete penetrance of FVL remain largely unknown. We previously reported a remarkable synthetic lethality in mice carrying the FVL mutation and partial deficiency of a key coagulation component, tissue factor pathway inhibitor (TFPI). Complete TFPI deficiency in mice is embryonic lethal, whereas heterozygosity is compatible with normal survival. However, homozygosity for FVL (FvQ/Q) in the context of heterozygosity for TFPI (Tfpi+/−) is uniformly lethal due to disseminated perinatal thrombosis. In order to identify potential modifier genes contributing to FVL penetrance, we have utilized this lethal genetic interaction as a phenotyping tool for a sensitized ENU mutagenesis screen in laboratory mice. We hypothesize that dominant mutations in key components of the coagulation system will improve hemostatic balance and allow survival in mice carrying the lethal FvQ/Q Tfpi+/− genotype combination. As an example, we propose that loss of one tissue factor allele might compensate for reduced TFPI and rescue FvQ/Q Tfpi+/− . To test this hypothesis, we bred tissue factor heterozygous mice (Tf+/−) with FvQ/Q Tfpi+/− mice and observed complete rescue, with normal survival and the expected number (8 of 57) of FvQ/Q Tfpi+/− Tf+/− mice from a FvQ/+ Tfpi+/− Tf+/−x FvQ/Q cross. In order to identify candidate modifier genes, we performed a whole genome mutagenesis screen. In this screen, male FvQ/Q mice were mutagenized with ENU and bred to FvQ/+ Tfpi+/− double heterozygous females. DNAs from surviving offspring were PCR assayed to identify rescued mice with the FvQ/Q Tfpi+/− genotype. Analysis of 2250 offspring, corresponding to approximately half genome coverage, has identified 15 mice that survived to weaning. Heritability was demonstrated for the 5 mutant lines subjected to progeny testing to date. Genetic crosses are in progress to map the mutant genes in 3 of the 5 progeny tested lines. These preliminary results demonstrate the feasibility of this sensitized screen for the identification of dominant suppressors of thrombosis. Based on our data, we estimate that there are likely 10–20 mammalian genes for which a

Description: Alternative pre-mRNA splicing switches provide an important mechanism for regulating gene expression during differentiation. In differentiating erythroblasts, stage-specific activation of protein 4.1R exon 16 splicing promotes the synthesis of protein isoforms with high affinity for spectrin and actin, which is important for mechanical stabilization of the red cell membrane skeleton. Regulation of this alternative splicing switch is mediated by the interaction of multiple trans-acting splicing factor proteins with cis RNA elements in the pre-mRNA. We previously identified an intron splicing enhancer downstream of exon 16 that is essential for optimal inclusion of exon 16 in functional splicing assays. This enhancer contains three repeats of the sequence UGCAUG, a highly specific binding site for the newly described Fox-1 family of splicing activator proteins. Our results implicate the highly homologous Fox-2 protein as a candidate splicing activator for exon 16 inclusion during erythroid differentiation, based on the observations that Fox-2 is expressed in mouse erythroblasts; and that it both binds to the intron 16 enhancer, and enhances exon 16 splicing, in a UGCAUG-dependent manner. Here we also report genome sequence analyses showing that UGCAUG is evolutionarily conserved in the proximal downstream intron sequence near many tissue-specific alternative exons. First, examination of the 4.1R genes revealed that three repeats of the UGCAUG motif were conserved in introns of all mammals tested (human, chimp, mouse, rat and dog) as well as in chicken; two repeats were found in intron 16 of the frog; and one repeat in the zebrafish. These data suggest that Fox-2 may be important for regulating exon 16 splicing from fish to man. Next, to test whether a similar mechanism might regulate other tissue-specific exons, we analyzed a larger group of some 27 alternative exons with predominant expression in the brain. Remarkably, 80–90% of these exons possessed one or more UGCAUG motifs within 1kb of flanking intron sequence. The majority of these elements were concentrated in the intron 10–400nt downstream of the regulated exons. Phylogenetic analysis of individual exons revealed that the number and position of intronic UGCAUG elements were highly conserved among mammalian species and in the chicken, but more divergent in fish. In marked contrast, control datasets of constitutively spliced exons, and alternatively spliced exons not known to be regulated in tissue-specific patterns, exhibited a low incidence of UGCAUG elements in the flanking introns. These findings are exactly as expected for a functionally important regulatory element. We propose that the high binding specificity of Fox proteins, and the unique localization of UGCAUG enhancers near regulated alternative exons, is advantageous for splicing switch mechanism(s) designed to activate a limited repertoire of splicing events in specific cell types including erythroblasts. We speculate that erythroid-specific splicing is accomplished by coordination between Fox-2 and other as yet unknown splicing factors.

Description: Defects in a triad of organelles (melanosomes, platelet granules, and lysosomes) result in albinism, prolonged bleeding, and lysosome abnormalities in Hermansky-Pudlak syndrome (HPS). Defects in HPS1, a protein of unknown function, and in components of the AP-3 complex cause some, but not all, cases of HPS in humans. There have been 15 inherited models of HPS described in the mouse, underscoring its marked genetic heterogeneity. Here we characterize a new spontaneous mutation in the mouse, cappuccino (cno), that maps to mouse chromosome 5 in a region conserved with human 4p15-p16. Melanosomes ofcno/cno mice are immature and dramatically decreased in number in the eye and skin, resulting in severe oculocutaneous albinism. Platelet dense body contents (adenosine triphosphate, serotonin) are markedly deficient, leading to defective aggregation and prolonged bleeding. Lysosomal enzyme concentrations are significantly elevated in the kidney and liver. Genetic, immunofluorescence microscopy, and lysosomal protein trafficking studies indicate that the AP-3 complex is intact in cno/cno mice. It was concluded that the cappuccino gene encodes a product involved in an AP-3–independent mechanism critical to the biogenesis of lysosome-related organelles.

Description: The prevalence and significance of genetic abnormalities in older patients with acute myeloid leukemia (AML) are unknown. Polymerase chain reactions and single-stranded conformational polymorphism analyses were used to examine 140 elderly AML patients enrolled in the Southwest Oncology Group study 9031 for FLT3, RAS, and TP53 mutations, which were found in 34%, 19%, and 9% of patients, respectively. All but one of the FLT3 (46 of 47) mutations were internal tandem duplications (ITDs) within exons 11 and 12. In the remaining case, a novel internal tandem triplication was found in exon 11. FLT3 ITDs were associated with higher white blood cell counts, higher peripheral blast percentages, normal cytogenetics, and less disease resistance. All RAS mutations (28 of 28) were missense point mutations in codons 12, 13, or 61. RASmutations were associated with lower peripheral blast and bone marrow blast percentages. Only 2 of 47 patients with FLT3 ITDs also had a RAS mutation, indicating a significant negative association between FLT3 and RAS mutations (P = .0013). Most TP53 mutations (11 of 12) were missense point mutations in exons 5 to 8 and were associated with abnormal cytogenetics, especially abnormalities in both chromosomes 5 and 7. FLT3 and RAS mutations were not associated with inferior clinical outcomes, but TP53mutations were associated with a worse overall survival (median 1 versus 8 months, P = .0007). These results indicate that mutations in FLT3, RAS, or TP53 are common in older patients with AML and are associated with specific AML phenotypes as defined by laboratory values, cytogenetics, and clinical outcomes.

Description: Thymic-deficient hosts rely primarily on antigen-driven expansion to restore the peripheral T-cell compartment following T-cell depletion (TCD). The degree to which this thymic-independent pathway can restore immune competence remains poorly understood but has important implications for a number of clinical conditions including stem cell transplantation and human immunodeficiency virus (HIV) infection. A model of HY-mediated skin graft rejection by athymic, TCD mice was used to show that restoration of naive and recall responses via peripheral expansion requires transfer of only 25 × 106 lymph node (LN) cells representing approximately 10% of the T-cell repertoire. Consitutive expression of bcl-2 in the expanding inocula restored recall responses to HY at a substantially lower LN cell dose (1 × 106), which is normally insufficient to induce HY-mediated graft rejection in athymic hosts. Interestingly, bcl-2 had no effect on primary responses. Interleukin-7 (IL-7) potently enhanced thymic-independent peripheral expansion and led to HY graft rejection using an LN cell dose of 1 × 106 in both primary and recall models. The restoration of immune competence by IL-7 appeared to be mediated through a combination of programmed cell death inhibition, improved costimulation, and modulation of antigen-presenting cell (APC) function. These results show that immune competence for even stringent antigens such as HY can be restored in the absence of thymic function and identify IL-7 as a potent modulator of thymic-independent T-cell regeneration.

Description: INTERCEPT Plasma (I-FFP) uses amotosalen (S-59, 150μM ) and UVA light (3 J/cm2 to inactivate pathogens in single plasma units. TPE is the mainstay therapy for TTP. Efficacy and safety of I-FFP for TPE of TTP were evaluated in a double-blinded, randomized, controlled trial. Eligible patients (pts) with TTP unrelated to cancer, cancer therapy, transplantation, AIDS, HUS, or SLE received daily TPE (1.0 to 1.5 blood volumes) with either I-FFP or conventional plasma (C-FFP) for up to 2 TPE cycles, each with a maximum of 35 days (d) of TPE. Treated pts were followed for 7 d after the last TPE to detect overall adverse events (AE) and for 60 d after achieving remission to detect relapse and serious adverse events (SAE). The primary endpoint was the proportion of pts in remission (platelet count ≥ 150,000/μL for 2 consecutive d without neurologic progression) within 30 d after the 1st TPE. Secondary endpoints were: time to 1st remission, volume of plasma used, number of TPEs, relapse rates after remission, vWF cleaving protease (vWF-CP) activity and inhibitor levels, antibodies to potential S-59 neoantigens, and safety. 35 pts (17 I-FFP, 18 C-FFP), mean age of 40 yr, 80% female were treated. TPE with I-FFP was comparable to C-FFP in remission rates (82% I-FFP, 89% C-FFP:p=0.66), median time to remission (6 d I-FFP, 6 d C-FFP:p=0.58), mean total volume of plasma exchanged (40.6 L I-FFP, 41.3 L C-FFP:p=0.86), mean number TPEs (11.3 I-FFP, 10.3 C-FFP:p=0.68), and relapse rates (36% I-FFP, 38% C-FFP:p=1.00). Baseline and post TPE vWF-CP activity and inhibitor levels were similar between groups. Overall AEs, serious AEs, deaths (1 I-FFP and 1 C-FFP: neither attributed to TPE), rates of discontinuation from study, laboratory abnormalities, and vital signs (VS) during TPE were comparable between groups. 1 C-FFP pt had TRALI. Five I-FFP pts had AEs coded to the cardiac system organ class compared to no C-FFP pts (p=0.02). In 4 of the I-FFP pts, these AEs were non-serious and did not interrupt TPE. One I-FFP pt with relapsing TTP had a serious AE after 12d of TPE, recovered without sequelae, was withdrawn from study, and continued on non-study FFP TPE with recurrent relapse. No antibodies to potential S-59 neoantigens were detected in any pts. In this trial, efficacy endpoints and safety profiles of TTP pts treated with I-FFP TPE were similar to those for TTP pts treated with C-FFP TPE.

Description: New allogeneic transplant protocols with non myeloablative conditioning regimens for treatment of multiple myeloma (MM) have been developed in the attempt to reduce the transplant related toxicity associated with myeloablation. Preliminary data have been encouraging with remarkable clinical response rates (Maloney et al, Blood 2003). However, data on the achievement of molecular remission, prerequisite for eventual cure, are still lacking. We implemented a tandem transplant approach consisting of high dose melphalan (200 mg/sqm) with autografting followed by non myeloablative low dose (2.0 Gy) total body irradiation and G-CSF mobilized PBSC infusion from HLA-identical siblings. The curative potential relies exclusively upon a potent graft versus myeloma (GVM) effect through donor T cells. At diagnosis, patient specific clonal markers were generated based upon the rearrangement of the immunoglobulin heavy chain (IgH) genes and used for nested polymerase chain reaction (PCR) detection of minimal residual disease after transplant. Molecular remission was defined as the disappearance of the molecular marker post transplant in both bone marrow and blood. The sensitivity of the nested PCR-based assay was 1 in 100000 cells. A patient specific marker was generated in 11/15 (73%) patients who entered the study. After a median follow up of 16 months (range 5–50), molecular follow up post transplant showed that 3/11 (27%) reached molecular remission at 1, 3 and 7 months post allografting, respectively. Of the remaining 8 patients, 3/8 and 5/8 reached clinical complete remission, defined as the disappearance of the monoclonal paraprotein by immunofixation, and partial remission, respectively. However, minimal residual disease by nested PCR could be detected at all timepoints. The molecular remissions have been durable at 7, 30, and 48 months post transplant, respectively. In 1 case the remission was achieved and sustained in the absence of graft versus host disease (GVHD) which is consistent with the notion that GVHD is not essential for GVM. Furthermore, in 4/11 patients real-time quantification of IgH rearrangements was performed on genomic DNA samples using tumor specific primers and consensus probes. All patients showed a considerable tumor burden reduction post autografting. Samples from two patients became negative by real time PCR at 3 months post allografting, but became PCR-negative by nested PCR at 3 and 7 months, respectively. This discrepancy is explained by the greater sensitivity of nested PCR and the larger amount of IgH copies which are expected in cDNA compared to genomic DNA. The remaining two patients only obtained a clinical partial response throughout the study period. This report indicates that the tandem auto-allo transplant approach can lead to molecular remission in MM. Prospective quantitative monitoring of disease response may be helpful to design individual additional immunotherapeutic manoeuvres, such as donor lymphocyte infusions, to enhance GVM. Longer follow up on a larger series of patients is needed to determine the frequency and durability of molecular remissions.