Paul M. Armistead

Expression of receptor tyrosine kinases and apoptotic molecules in rhabdomyosarcoma: Correlation with overall survival in 105 patients

Rhabdomyosarcoma (RMS) is a rare mesenchymal tumor with few treatment options after the failure of first‐line therapy. Understanding the expression of kinases and apoptotic molecules in RMS tumors may lead to elucidation of mechanisms of resistance to chemotherapy and development of new therapies.

Multicenter validation study of a transplantation-specific cytogenetics grouping scheme for patients with myelodysplastic syndromes

Cytogenetics is an important prognostic factor for patients with myelodysplastic syndromes (MDS). However, existing cytogenetics grouping schemes are based on patients treated with supportive care, and may not be optimal for patients undergoing allo-SCT. We proposed earlier an SCT-specific cytogenetics grouping scheme for patients with MDS and AML arising from MDS, based on an analysis of patients transplanted at the Dana-Farber Cancer Institute/Brigham and Womens Hospital. Under this scheme, abnormalities of chromosome 7 and complex karyotype are considered adverse risk, whereas all others are considered standard risk. In this retrospective study, we validated this scheme on an independent multicenter cohort of 546 patients. Adverse cytogenetics was the strongest prognostic factor for outcome in this cohort. The 4-year relapse-free survival and OS were 42 and 46%, respectively, in the standard-risk group, vs 21 and 23% in the adverse group (P<0.0001 for both comparisons). This grouping scheme retained its prognostic significance irrespective of patient age, disease type, earlier leukemogenic therapy and conditioning intensity. Therapy-related disease was not associated with increased mortality in this cohort, after taking cytogenetics into account. We propose that this SCT-specific cytogenetics grouping scheme be used for patients with MDS or AML arising from MDS who are considering or undergoing SCT.

Quantifying the survival benefit for allogeneic hematopoietic stem cell transplantation in relapsed acute myelogenous leukemia

Allogeneic hematopoietic stem cell transplantation (HSCT) is the recommended therapy for patients with relapsed acute myelogenous leukemia (AML), despite little evidence showing a survival benefit in patients who undergo HSCT versus chemotherapy alone. Because a prospective randomized trial addressing this issue is unlikely, we retrospectively reviewed all patients receiving initial salvage therapy for AML at M.D. Anderson Cancer Center between 1995 and 2004, focusing on patients undergoing HSCT or chemotherapy without HSCT as second salvage after first salvage failed to produce complete remission (CR) (group A) and patients in first salvage-induced CR (group B). Median survival was 5.1 months for HSCT (n=84) versus 2.3 months for chemotherapy (n = 200; P = .004) in group A and 11.7 months for HSCT (n = 46) versus 5.6 months for chemotherapy (n = 66; P < . 001) in group B. HSCT was associated with a survival benefit in each of 8 subgroups defined by age < or > or = 50, high-risk cytogenetics or not, and treatment in first salvage-induced CR or second salvage, and also in 5 of 6 subgroups defined by age < or > or = 50 years and duration of first CR (CR1) (primary refractory, CR1 < or = 36 weeks, CR1 > 36 weeks). Our data suggest that HSCT is preferable to chemotherapy alone in these patients with poor prognoses, with particular benefits noted in patients under age 50 years.

Automated Capillary Electrophoresis System for Fast Single-Cell Analysis

Capillary electrophoresis (CE) is a promising technique for single-cell analysis, but its use in biological studies has been limited by low throughput. This paper presents an automated platform employing microfabricated cell traps and a three-channel system for rapid buffer exchange for fast single-cell CE. Cells loaded with fluorescein and Oregon green were analyzed at a throughput of 3.5 cells/min with a resolution of 2.3 ± 0.6 for the fluorescein and Oregon green. Cellular protein kinase B (PKB) activity, as measured by immunofluorescence staining of phospho-PKB, was not altered, suggesting that this stress-activated kinase was not upregulated during the CE experiments and that basal cell physiology was not perturbed prior to cell lysis. The activity of sphingosine kinase (SK), which is often upregulated in cancer, was measured in leukemic cells by loading a sphingosine-fluorescein substrate into cells. Sphingosine fluorescein (SF), sphingosine-1-phosphate fluorescein (S1PF), and a third fluorescent species were identified in single cells. A single-cell throughput of 2.1 cells/min was achieved for 219 total cells. Eighty-eight percent of cells possessed upregulated SK activity, although subpopulations of cells with markedly different SK activity relative to that of the population average were readily identified. This system was capable of stable and reproducible separations of biological compounds in hundreds of adherent and nonadherent cells, enabling measurements of previously uncharacterized biological phenomena.

Kernel Machine SNP-set Testing under Multiple Candidate Kernels

Joint testing for the cumulative effect of multiple single‐nucleotide polymorphisms grouped on the basis of prior biological knowledge has become a popular and powerful strategy for the analysis of large‐scale genetic association studies. The kernel machine (KM)‐testing framework is a useful approach that has been proposed for testing associations between multiple genetic variants and many different types of complex traits by comparing pairwise similarity in phenotype between subjects to pairwise similarity in genotype, with similarity in genotype defined via a kernel function. An advantage of the KM framework is its flexibility: choosing different kernel functions allows for different assumptions concerning the underlying model and can allow for improved power. In practice, it is difficult to know which kernel to use a priori because this depends on the unknown underlying trait architecture and selecting the kernel which gives the lowest P‐value can lead to inflated type I error. Therefore, we propose practical strategies for KM testing when multiple candidate kernels are present based on constructing composite kernels and based on efficient perturbation procedures. We demonstrate through simulations and real data applications that the procedures protect the type I error rate and can lead to substantially improved power over poor choices of kernels and only modest differences in power vs. using the best candidate kernel.

Microfluidic chemical cytometry of peptide degradation in single drug-treated acute myeloid leukemia cells.

Microfluidic systems show great promise for single-cell analysis; however, as these technologies mature, their utility must be validated by studies of biologically relevant processes. An important biomedical application of these systems is characterization of tumor cell heterogeneity. In this work, we used a robust microfluidic platform to explore the heterogeneity of enzyme activity in single cells treated with a chemotherapeutic drug. Using chemical cytometry, we measured peptide degradation in the U937 acute myeloid leukemia (AML) cell line in the presence and absence of the aminopeptidase inhibitor Tosedostat (CHR-2797). The analysis of 99 untreated cells revealed rapid and consistent degradation of the peptide reporter within 20 min of loading. Results from drug-treated cells showed inhibited, but ongoing degradation of the reporter. Because the device operates at an average sustained throughput of 37 ± 7 cells/h, we were able to sample cells over the course of this time-dependent degradation. In data from 498 individual drug-treated cells, we found a linear dependence of degradation rate on amount of substrate loaded superimposed upon substantial heterogeneity in peptide processing in response to inhibitor treatment. Importantly, these data demonstrated the potential of microfluidic systems to sample biologically relevant analytes and time-dependent processes in large numbers of single cells.

The role of antigen cross-presentation from leukemia blasts on immunity to the leukemia-associated antigen PR1.

Cross-presentation is an important mechanism by which exogenous tumor antigens are presented to elicit immunity. Because neutrophil elastase (NE) and proteinase-3 (P3) expression is increased in myeloid leukemia, we investigated whether NE and P3 are cross-presented by dendritic cells (DC) and B cells, and whether the NE and P3 source determines immune outcomes. We show that NE and P3 are elevated in leukemia patient serum and that levels correlate with remission status. We demonstrate cellular uptake of NE and P3 into lysosomes, ubiquitination, and proteasome processing for cross-presentation. Using anti-PR1/human leukocyte antigen-A2 monoclonal antibody, we provide direct evidence that B-cells cross-present soluble and leukemia-associated NE and P3, whereas DCs cross-present only leukemia-associated NE and P3. Cross-presentation occurred at early time points but was not associated with DC or B-cell activation, suggesting that NE and P3 cross-presentation may favor tolerance. Furthermore, we show aberrant subcellular localization of NE and P3 in leukemia blasts to compartments that share common elements of the classic major histocompatibility class I antigen-presenting pathway, which may facilitate cross-presentation. Our data demonstrate distinct mechanisms for cross-presentation of soluble and cell-associated NE and P3, which may be valuable in understanding immunity to PR1 in leukemia.

Common minor histocompatibility antigen discovery based upon patient clinical outcomes and genomic data

Background Minor histocompatibility antigens (mHA) mediate much of the graft vs. leukemia (GvL) effect and graft vs. host disease (GvHD) in patients who undergo allogeneic stem cell transplantation (SCT) [1], [2], [3], [4]. Therapeutic decision making and treatments [5] based upon mHAs will require the evaluation of multiple candidate mHAs and the selection of those with the potential to have the greatest impact on clinical outcomes. We hypothesized that common, immunodominant mHAs, which are presented by HLA-A, B, and C molecules, can mediate clinically significant GvL and/or GvHD, and that these mHAs can be identified through association of genomic data with clinical outcomes. Methodology/Principal Findings Because most mHAs result from donor/recipient cSNP disparities, we genotyped 57 myeloid leukemia patients and their donors at 13,917 cSNPs [6]. We correlated the frequency of genetically predicted mHA disparities with clinical evidence of an immune response and then computationally screened all peptides mapping to the highly associated cSNPs for their ability to bind to HLA molecules. As proof-of-concept, we analyzed one predicted antigen, T4A, whose mHA mismatch trended towards improved overall and disease free survival in our cohort. T4A mHA mismatches occurred at the maximum theoretical frequency for any given SCT. T4A-specific CD8+ T lymphocytes (CTLs) were detected in 3 of 4 evaluable post-transplant patients predicted to have a T4A mismatch. Conclusions/Significance Our method is the first to combine clinical outcomes data with genomics and bioinformatics methods to predict and confirm a mHA. Refinement of this method should enable the discovery of clinically relevant mHAs in the majority of transplant patients and possibly lead to novel immunotherapeutics [5].

A Novel HLA-A*0201 Restricted Peptide Derived from Cathepsin G Is an Effective Immunotherapeutic Target in Acute Myeloid Leukemia

Purpose: Immunotherapy targeting aberrantly expressed leukemia-associated antigens has shown promise in the management of acute myeloid leukemia (AML). However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. In this study, we characterize the role of the myeloid azurophil granule protease cathepsin G (CG) as a novel target for AML immunotherapy. Experimental Design: We used Immune Epitope Database and in vitro binding assays to identify immunogenic epitopes derived from CG. Flow cytometry, immunoblotting, and confocal microscopy were used to characterize the expression and processing of CG in AML patient samples, leukemia stem cells, and normal neutrophils. Cytotoxicity assays determined the susceptibility of AML to CG-specific cytotoxic T lymphocytes (CTL). Dextramer staining and cytokine flow cytometry were conducted to characterize the immune response to CG in patients. Results: CG was highly expressed and ubiquitinated in AML blasts, and was localized outside granules in compartments that facilitate antigen presentation. We identified five HLA-A*0201 binding nonameric peptides (CG1-CG5) derived from CG, and showed immunogenicity of the highest HLA-A*0201 binding peptide, CG1. We showed killing of primary AML by CG1-CTL, but not normal bone marrow. Blocking HLA-A*0201 abrogated CG1-CTL–mediated cytotoxicity, further confirming HLA-A*0201-dependent killing. Finally, we showed functional CG1-CTLs in peripheral blood from AML patients following allogeneic stem cell transplantation. Conclusion: CG is aberrantly expressed and processed in AML and is a novel immunotherapeutic target that warrants further development. Clin Cancer Res; 19(1); 247–57. ©2012 AACR.

Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity μSPE device

We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3600 micropillars. Activation of the PMMA micropillars by UV/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (∼20 fmol) of membrane proteins could be isolated and recovered with ∼89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the μSPE device using computational simulations of different micropillar geometries to guide future device designs.