Gene/Protein Disease Symptom Drug Enzyme Compound
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Metastatic neuroblastoma is a poor-prognosis malignancy arising during childhood that overexpresses the L1-cell adhesion molecule (CD171). We have previously described a tumor L1-cell adhesion molecule-specific, single chain antibody-derived, chimeric antigen receptor designated CE7R for re-directing the antigen-specific effector functioning of cytolytic T lymphocytes. Here, we report on the feasibility of isolating, and the safety of infusing, autologous CD8(+) cytolytic T lymphocyte clones co-expressing CE7R and the selection-suicide expression enzyme HyTK in children with recurrent/refractory neuroblastoma. The cytolytic T lymphocyte products were derived from peripheral blood mononuclear cells that were subjected to polyclonal activation, plasmid vector electrotransfer, limiting dilution hygromycin selection, and expansion to numbers sufficient for adoptive transfer. In total, 12 infusions (nine at 10(8) cells/m(2), three at 10(9) cells/m(2)) were administered to six patients. No overt toxicities to tissues known to express L1-cell adhesion molecule (e.g., central nervous system, adrenal medulla, and sympathetic ganglia) were observed. The persistence of cytolytic T lymphocyte clones in the circulation, measured by vector-specific quantitative polymerase chain reaction, was short (1-7 days) in patients with bulky disease, but significantly longer (42 days) in a patient with a limited disease burden. This first-in-humans pilot study sets the stage for clinical trials employing adoptive transfer in the context of minimal residual disease.
Mol Ther 2007 Apr
PMID:Adoptive transfer of chimeric antigen receptor re-directed cytolytic T lymphocyte clones in patients with neuroblastoma. 1729 5

Patients with chronic myeloid leukemia harbor the chromosomal translocation t(9;22), which corresponds to fusion of the BCR and ABL genes at the DNA level. The translated fusion product is an oncogenic protein with increased ABL tyrosine kinase activity causing cell transformation. To date, reverse transcriptase-polymerase chain reaction is considered the most sensitive method available for detecting low copy numbers of the BCR-ABL gene fusion. Recently, Cepheid introduced its GeneXpert-based assay for the identification of the BCR-ABL gene fusion in cells from blood samples. This system comprises a walk-away self-contained instrument that combines cartridge-based microfluidic sample preparation with reverse transcriptase-polymerase chain reaction-based fluorescent signal detection and BCR-ABL and ABL Ct (threshold cycle) determination. The difference between the BCR-ABL Ct and ABL Ct (DeltaCt) is expected to represent the ratio of the two populations of mRNAs and ultimately the percentage of neoplastic cells present. We tested whether this BCR-ABL fusion detection system could be used as a clinical diagnostic tool for monitoring patients with minimal residual disease of chronic myelogenous leukemia. We report similar performance characteristics, including limit of detection, specificity, sensitivity, and precision, of this automated BCR-ABL fusion detection system to those of a manual TaqMan reverse transcriptase-polymerase chain reaction-based test.
J Mol Diagn 2007 Apr
PMID:Evaluation of the Cepheid GeneXpert BCR-ABL assay. 1738 14

Accurate monitoring of minimal residual disease (MRD) is critical for the management of metastatic neuroblastoma (NB). We evaluated cyclin D1 (CCND1), a cell-cycle control gene, as a novel MRD marker of NB. Using quantitative reverse transcriptase-polymerase chain reaction, we studied CCND1 expression in 133 solid tumors of different histological types, including 39 NB tumors, and examined its potential clinical utility as an early response marker in the bone marrows before and after treatment of 118 stage 4 patients enrolled after induction chemotherapy in an immunotherapy protocol. Based on 40 normal marrow and peripheral blood samples, a CCND1 transcript value greater than the mean + 2 SD was defined as positive. Sensitivity of this assay was one NB cell in 10(6) normal mononuclear cells. CCND1 transcript levels were high in NB, breast cancer, and Ewing family tumors. Among the NB patients evaluated, early (2.5 months from protocol entry) marrow response was strongly associated with both progression-free (P=0.0001) and overall survival (P=0.0006). CCND1 response remained predictive of survival among a subset of 66 patients who had no histological evidence of marrow disease before immunotherapy. We conclude that CCND1 has potential clinical utility as a novel molecular marker of MRD in the bone marrow of patients with metastatic NB.
J Mol Diagn 2007 Apr
PMID:Cyclin D1, a novel molecular marker of minimal residual disease, in metastatic neuroblastoma. 1738 16

In addition to its loss playing a pivotal role in the development of a childhood kidney malignancy, the Wilms tumour 1 gene (WT1) has emerged as an important factor in normal and malignant haematopoiesis. Preferentially expressed in CD34+ haematopoietic progenitors and down-regulated in more-differentiated cells, the WT1 transcription factor has been implicated in regulation of apoptosis, proliferation and differentiation. Putative target genes, such as BCL2, MYC, A1 and cyclin E, may cooperate with WT1 to modulate cell growth. However, the effects of WT1 on target gene expression appear to be isoform-specific. Certain WT1 isoforms are over-represented in leukaemia, but the exact mechanisms underlying the role of WT1 in transformation remain unclear. The ubiquity of WT1 in haematological malignancies has led to efforts to exploit it as a marker for minimal residual disease and as a prognostic factor, with conflicting results. In vitro killing of tumour cells by WT1-specific CD8+ cytotoxic T lymphocytes facilitated design of Phase I vaccine trials that showed clinical regression of WT1-positive tumours. Alternative methods employing WT1-specific immunotherapy are being investigated and might ultimately be used to optimise multimodal therapy of haematological malignancies.
Expert Rev Mol Med 2007 May 24
PMID:The role of the Wilms tumour gene (WT1) in normal and malignant haematopoiesis. 1752 67

Monitoring of minimal residual disease (MRD) in patients with acute or chronic myeloid disorders is routinely performed after allogeneic or autologous transplantation. The detection of MRD helps to identify patients who are at high risk for leukemic relapse after transplantation. The most commonly used techniques for MRD detection are qualitative and quantitative PCR methods, fluorescence in situ hybridization (FISH), fluorescence-activated cell sorting (FACS), and cytogenetic analysis, which are often performed complementary in order to assess more precisely MRD. Here, we describe the most used sensitive real-time reverse-transcription (RT)-PCR methods for chronic and acute myeloid disorders. Besides protocols for real-time RT-PCR and multiplex RT-PCR procedures for the most common fusion-gene transcripts in acute and chronic myeloid disorders, methods for detection of disease-specific genetic mutated alterations as FLT3 gene-length mutations, and aberrantly expressed genes as WT1 gene transcripts, are described in detail for daily use.
Methods Mol Med 2007
PMID:Molecular methods used for detection of minimal residual disease following hematopoietic stem cell transplantation in myeloid disorders. 1766 50

Intensified treatments aimed at maximal tumor reduction are an important therapeutic option for patients affected by B-cell malignancies. The possibility of obtaining a relevant number of clinical complete remissions after these treatments prompted the application of molecular techniques for the detection of extremely low numbers of residual malignant cells. These cells can be present either in the stem cell graft or, during the follow-up, in the bone marrow of patients attaining a clinical complete remission. The most sensitive and widely used techniques for minimal residual disease (MRD) assessment are those based on the PCR method. These methods allow the detection of autologous graft contamination and the identification of patients at high risk of disease recurrence by means of post-transplant MRD monitoring. In this setting, quantitative PCR assays can evaluate the kinetics of tumor clone growth in complete remission (CR) patients showing a persistence of PCR detectable tumor cells with standard qualitative methods.
Methods Mol Med 2007
PMID:Molecular methods used for the detection of autologous graft contamination in lymphoid disorders. 1766 51

In patients with chronic myeloid leukemia, the use of real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for measuring BCR-ABL1 transcripts has become standard methodology for the diagnosis and monitoring of minimal residual disease. In 2004 and 2005, 38 different laboratories from North America participated in three separate sample exchanges using real-time qRT-PCR to measure RNA transcript levels in unknown diluents of a BCR-ABL1-positive cell line, K562. In this study we compared results of quantitative testing for BCR-ABL1 from laboratories using different platforms, internal controls, reagents, and calculation methods. Our data showed that there can be considerable variability of results from laboratory to laboratory, with log reduction calculations varying from 1.6 to 3 log between laboratories at the same dilution. We found that none of the variables tested had a significant impact on the results reported, except for the use of ABL1 as the internal control (P < 0.001). Laboratories that used ABL1 consistently underreported their log reduction values. Regardless of the specific methodology and platform used for real-time qRT-PCR testing, it is important for laboratories to participate in proficiency testing to ensure consistent and acceptable test accuracy and sensitivity. Our study emphasizes the need for optimization of real-time qRT-PCR before offering clinical testing and the need for widely available universal standards that can be used for test calibration.
J Mol Diagn 2007 Sep
PMID:Inter-laboratory comparison of chronic myeloid leukemia minimal residual disease monitoring: summary and recommendations. 1769 Feb 11

We have performed combined organ and hematopoietic cell transplantation using a similar conditioning regimen in mice and humans. In the mouse model of MHC-mismatched combined heart and marrow transplantation, we compared conditioning of BALB/c hosts with total lymphoid irradiation (TLI: 10 doses of 240 cGy each) targeted to the spleen, lymph nodes and thymus to conditioning with a single dose of sublethal total body irradiation (TBI; 450 cGy). Conditioning also included three injections of anti-thymocyte serum (ATS), in both groups. C57BL/6 heart grafts, marrow cells and blood mononuclear cells were transplanted 24 h after the completion of irradiation. Blood mononuclear cells were added to the marrow cells to engender severe graft versus host disease (GVHD) that is present after combined organ and hematopoietic cell transplantation in humans given non-myeloablative conditioning. Both TLI and TBI conditioned groups accepted the organ grafts and became stable chimeras. However, the TBI group all died of GVHD during the 100-day observation period. The TLI group survived during the same period without clinical signs of GVHD. These hosts were tolerized to the donor organ grafts, since third party grafts were rejected rapidly when transplanted after 100 days. When NK T-cell-deficient CD1d(-/-) BALB/c hosts were used instead of wild-type hosts in the TLI/ATS conditioned group, then all hosts survived but all rejected the organ grafts and almost all failed to develop stable chimerism. None developed GVHD. Since host NK T cells were required for graft acceptance and NK T cells are activated after recognition of CD1d on antigen presenting cells, we compared heart and marrow graft survival from wild-type versus CD1d(-/-) donors after transplantation to TLI and ATS conditioned wild-type hosts. Whereas marrow and heart grafts from wild-type donors were accepted, almost all grafts from CD1d donors were rejected. Grafts from control Jalpha18(-/-) donors that were NK T cell deficient but expressed CD1d were all accepted. The results indicate that host NK T cells facilitate graft acceptance by recognizing CD1d on donor cells. We applied the TLI conditioning regimen using 10 doses of 80 cGy each and 5 doses of rabbit ATG to human recipients of HLA-matched G-CSF "mobilized" blood mononuclear cell transplants for the treatment of leukemia and lymphoma [R. Lowsky, T. Takahashi, Y.P. Liu, et al., Protective conditioning for acute graft-versus-host disease. N. Engl. J. Med. 353 (2005) 1321-1331.]. Currently more than 100 transplants have been performed, and the incidence of acute GVHD has been about 4% when both MRD and MUD transplants are combined. Almost all recipients became complete chimeras after receiving grafts that contained 2-3x10(8) CD3(+) T cells/kg. In further studies, we applied the same TLI and ATG conditioning regimen to combined kidney and G-CSF "mobilized" blood stem cell transplantation from HLA-matched sibling donors. The hematopoietic grafts in the latter protocol were selected CD34(+) cells with 1x10(6) CD3(+) T cells/kg added back to the hematopoietic cells. Preliminary results indicate that stable mixed chimerism can be achieved using this protocol allowing for complete immunosuppressive drug withdrawal without GVHD or subsequent rejection episodes. Thus, conditioning with TLI based regimens can simultaneously protect against organ graft rejection and GVHD. Levels of chimerism are dependent upon the content of donor T cells in the hematopoietic graft.
Blood Cells Mol Dis
PMID:Protective conditioning against GVHD and graft rejection after combined organ and hematopoietic cell transplantation. 1782 36

Identification of genetic abnormalities in pathological samples is critical for accurate diagnosis, risk stratification, detection of minimal residual disease, and assessment of response to therapy. Interphase fluorescence in situ hybridization analysis is the standard cytogenetic assay used by many laboratories to detect specific clonal karyotypic aberrations in formalin-fixed, paraffin-embedded tissue. However, direct correlation with immunophenotype or morphology in individual cells is rarely performed because the procedural steps are labor intensive and usually require extensive troubleshooting. In this study, we present a sequential fluorescence in situ hybridization-based technique that uses the identical archived bone marrow smears or paraffin-embedded tissue sections previously evaluated by a pathologist for morphological or immunohistochemical characteristics. This approach is relatively straightforward, using uncomplicated pretreatment and hybridization conditions and basic equipment attached to an automated image analyzer with image capture software to record the location of targeted cells for genotypic/phenotype correlation. Furthermore, the method has proved reliable and reproducible on test samples regardless of specimen age, tissue type, or referring institution.
J Mol Diagn 2007 Nov
PMID:Successful application of a direct detection slide-based sequential phenotype/genotype assay using archived bone marrow smears and paraffin embedded tissue sections. 1797 26

Nucleophosmin-1 (NPM1) mutations represent the most frequent gene alteration in acute myeloid leukemia (AML). The most common NPM1 mutation type, accounting for 75 to 80% of cases, is referred to as mutation A (NPM1-mutA). These NPM1 alterations have been shown to possess prognostic significance because they appear to identify patients who will benefit from chemotherapy. Given the high prevalence and stability of these mutations over the course of disease, NPM1 mutations may serve as ideal targets for minimal residual disease (MRD) assessment in AML. Current detection methods are costly, require sophisticated equipment, and are often not sufficiently sensitive. We report here an allele-specific (ASO)-RT-PCR assay that enables rapid and sensitive detection of NPM1-mutA. A semi-nested ASO-PCR method was also designed to increase the sensitivity of our assay for the monitoring of MRD. We analyzed bone marrow cells collected from 52 patients with AML at presentation. NPM1-mutA was detected in leukemic cells from 21 patients. Assay specificity was confirmed by capillary electrophoresis and DNA sequencing. ASO-RT-PCR and semi-nested ASO-PCR assays could detect NPM1-mutA with sensitivities of 10(-2) and 10(-5), respectively. Results obtained here verify that our ASO-RT-PCR assay is a specific and sensitive method for the routine screening of NPM1-mutA, as well as for MRD monitoring of AML patients with this alteration. This method is convenient and easily applicable in countries with limited resources and no access to real-time quantitative PCR-based technologies.
J Mol Diagn 2008 May
PMID:An allele-specific rt-PCR assay to detect type A mutation of the nucleophosmin-1 gene in acute myeloid leukemia. 1840 13


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