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author:("Zhang, mwandi")
1.  Evolution and impact of subclonal mutations in chronic lymphocytic leukemia 
Cell  2013;152(4):714-726.
Summary
Clonal evolution is a key feature of cancer progression and relapse. We studied intratumoral heterogeneity in 149 chronic lymphocytic leukemia (CLL) cases by integrating whole-exome sequence and copy number to measure the fraction of cancer cells harboring each somatic mutation. We identified driver mutations as predominantly clonal (e.g., MYD88, trisomy 12 and del(13q)) or subclonal (e.g., SF3B1, TP53), corresponding to earlier and later events in CLL evolution. We sampled leukemia cells from 18 patients at two timepoints. Ten of 12 CLL cases treated with chemotherapy (but only 1 of 6 without treatment) underwent clonal evolution, predominantly involving subclones with driver mutations (e.g., SF3B1, TP53) that expanded over time. Furthermore, presence of a subclonal driver mutation was an independent risk factor for rapid disease progression. Our study thus uncovers patterns of clonal evolution in CLL, providing insights into its stepwise transformation, and links the presence of subclones with adverse clinical outcome.
doi:10.1016/j.cell.2013.01.019
PMCID: PMC3575604  PMID: 23415222
2.  Mutated BCR-ABL Generates Immunogenic T Cell Epitopes In CML Patients 
Purpose
Characterization of an approach to identify leukemia neoantigens arising in the context of drug resistance.
Experimental Design
We assessed whether leukemia neoantigens could be generated from drug-resistant mutations in BCR-ABL following imatinib relapse in chronic myelogenous leukemia (CML) patients.
Results
We computationally predicted that ~70 peptides derived from 26 BCR-ABL mutations would bind 8 common alleles of MHC class I (IC50<1000 nM). Seven of nine imatinib-resistant CML patients were predicted to generate at least one peptide that binds autologous HLA alleles. We predicted and confirmed that an E255K mutation-derived peptide would bind HLA-A3 with high affinity (IC50=28 nM), and demonstrated that this peptide is endogenously processed and presented. Polyfunctional E255K-specific CD8+ T cells were detected in two imatinib-resistant HLA-A3+ CML patients concurrent with an effective anti-CML response to further therapy.
Conclusions
Our in vitro studies support the hypothesis that leukemia-driven genetic alterations are targeted by the immune system in association with a clinical response, and suggest the possibility of immunizing relapsed CML patients against newly acquired tumor neoantigens.
doi:10.1158/1078-0432.CCR-12-1182
PMCID: PMC3759991  PMID: 22912393
Mutated BCR-ABL; leukemia antigen; chronic myeloid leukemia; T cell
3.  SF3B1 and Other Novel Cancer Genes in Chronic Lymphocytic Leukemia 
The New England journal of medicine  2011;365(26):2497-2506.
Background
The somatic genetic basis of chronic lymphocytic leukemia, a common and clinically heterogeneous leukemia occurring in adults, remains poorly understood.
Methods
We obtained DNA samples from leukemia cells in 91 patients with chronic lymphocytic leukemia and performed massively parallel sequencing of 88 whole exomes and whole genomes, together with sequencing of matched germline DNA, to characterize the spectrum of somatic mutations in this disease.
Results
Nine genes that are mutated at significant frequencies were identified, including four with established roles in chronic lymphocytic leukemia (TP53 in 15% of patients, ATM in 9%, MYD88 in 10%, and NOTCH1 in 4%) and five with unestablished roles (SF3B1, ZMYM3, MAPK1, FBXW7, and DDX3X). SF3B1, which functions at the catalytic core of the spliceosome, was the second most frequently mutated gene (with mutations occurring in 15% of patients). SF3B1 mutations occurred primarily in tumors with deletions in chromosome 11q, which are associated with a poor prognosis in patients with chronic lymphocytic leukemia. We further discovered that tumor samples with mutations in SF3B1 had alterations in pre–messenger RNA (mRNA) splicing.
Conclusions
Our study defines the landscape of somatic mutations in chronic lymphocytic leukemia and highlights pre-mRNA splicing as a critical cellular process contributing to chronic lymphocytic leukemia.
doi:10.1056/NEJMoa1109016
PMCID: PMC3685413  PMID: 22150006
4.  Graft-versus-leukemia antigen CML66 elicits coordinated B and T cell immunity after donor lymphocyte infusion 
Purpose
The target antigens of graft-versus-leukemia that are tumor-associated are incompletely characterized.
Experimental Design
We examined responses developing against CML66, an immunogenic antigen preferentially expressed in myeloid progenitor cells identified from a patient with chronic myelogenous leukemia who attained long-lived remission following CD4+ donor lymphocyte infusion (DLI).
Results
From this patient, CML66-reactive CD8+ T cell clones were detected against an endogenously presented HLA-B*4403-restricted epitope (HDVDALLW). Neither CML66-specific antibody nor T cell responses were detectable in peripheral blood before DLI. However, by one month after DLI, CD8+ T cells were present in peripheral blood, and at 10-fold higher frequency in marrow. Subsequently, plasma antibody to CML66 developed in association with disease remission. Donor-derived CML66-reactive T cells were detected at low levels in vivo in marrow prior to DLI by ELISpot and by a nested polymerase chain reaction-based assay to detect clonotypic T cell receptor sequences, but not in blood of the patient pre-DLI, nor of the graft donor.
Conclusions
CD4+ DLI results in rapid expansion of pre-existing marrow-resident leukemia-specific donor CD8+ T cells, followed by a cascade of antigen-specific immune responses detectable in blood. Our single-antigen analysis thus demonstrates that durable post-transplant tumor immunity is directed in part against nonpolymorphic overexpressed leukemia antigens, that elicit coordinated cellular and humoral immunity.
doi:10.1158/1078-0432.CCR-10-0415
PMCID: PMC2872105  PMID: 20460482
Donor lymphocyte infusion; graft-versus-leukemia; leukemia antigen; chronic myeloid leukemia; T cell
5.  Effective posttransplant antitumor immunity is associated with TLR-stimulating nucleic acid–immunoglobulin complexes in humans 
The Journal of Clinical Investigation  2011;121(4):1574-1584.
Donor lymphocyte infusion (DLI), whereby donor mononuclear cells are infused into patients, is one of the few effective immunotherapeutic strategies that generate long-lasting tumor remissions. We previously demonstrated that chronic myelogenous leukemia (CML) patients treated with DLI develop high-titer plasma antibodies specific for CML-associated antigens, the majority of which have been reported to bind nucleic acids These observations led us to predict that circulating antibody-antigen complexes in DLI-responsive patients carry nucleic acids that can engage innate immune sensors. Consistent with this, we report here that post-DLI plasma from 5 CML patients that responded to DLI treatment induced massive upregulation of MIP-1α, IP-10, and IFN-α in normal blood mononuclear cells. Importantly, this was not observed with plasma obtained before DLI and from DLI nonresponders and imatinib-treated patients. This endogenous immunostimulatory activity required nucleic acid and protein for its adjuvant effect and activated antigen-presenting cells through the RNA and DNA sensors TLR8 and TLR9. Presence of the immunoglobulin Fc receptor CD32 enhanced cellular responses, suggesting that immunoglobulins associate with this activity. Finally, a TLR-induced expression signature was detectable in post-DLI but not pre-DLI blood, consistent with an active circulating TLR8/9-stimulating factor. We have therefore demonstrated that effective tumor immunity correlates with the presence of endogenous nucleic acid–immunoglobulin complexes in patient plasma, thus providing a putative mechanism for the induction of potent antigen-specific immunity against malignant cells.
doi:10.1172/JCI44581
PMCID: PMC3069775  PMID: 21403403
6.  Serologic markers of effective tumor immunity against chronic lymphocytic leukemia include non-mutated B cell antigens 
Cancer research  2010;70(4):1344-1355.
Patients with chronic lymphocytic leukemia (CLL) who relapse after allogeneic transplant may achieve durable remission following donor lymphocytes infusion (DLI), demonstrating the potency of donor-derived immunity in eradicating tumor. We sought to elucidate the antigenic basis of the effective graft-versus-leukemia (GvL) responses associated with DLI for the treatment of CLL by analyzing the specificity of plasma antibody responses developing in two DLI-treated patients who achieved long-term remission without graft-versus-host disease (GvHD). By probing high-density protein microarrays with patient plasma, we discovered 35 predominantly intracellular antigens that elicited high-titer antibody reactivity greater in post-than in pre-DLI plasma. Three antigens, C6orf130, MDS032, and ZFYVE19, were identified by both patients. Along with additional candidate antigens DAPK3, SERBP1 and OGFOD1, these proteins demonstrated higher transcript and protein expression in B cells and CLL cells compared to normal PBMC. DAPK3 and the shared antigens do not represent minor histocompatibility antigens, as their sequences are identical in both donor and tumor. While ZFYVE19, DAPK3 and OGFOD1 elicited minimal antibody reactivity in 12 normal subjects and 12 chemotherapy-treated CLL patients, 5 of 12 CLL patients with clinical GvL responses were serologically reactive to these antigens. Moreover, antibody reactivity against these antigens was temporally correlated with clinical disease regression. These B cell antigens represent promising biomarkers of effective anti-CLL immunity.
doi:10.1158/0008-5472.CAN-09-3143
PMCID: PMC2852266  PMID: 20124481
proteomics; protein microarray; immunology; leukemia; antigen discovery
7.  Antigen targets of remission-inducing immune therapy are expressed on CML progenitor cells 
Cancer research  2010;70(3):906-915.
The curative effect of existing graft-versus-leukemia (GvL)-based therapies such as donor lymphocyte infusion (DLI) for chronic myeloid leukemia (CML) may result from immunologic targeting of self-renewing CML progenitor cells. Patients who achieved durable remission following DLI developed a significant B cell lymphocytosis after treatment, which was not observed in patients unresponsive to DLI. To identify targets of this B cell response, we probed two complementary immunoproteomic platforms, a bacteriophage expression library and a high-density protein microarray, using plasma immunoglobulin from seven CML patients with clinically apparent GvL and without graft-versus-host disease after DLI. In total, 62 antigens elicited greater reactivity from post-DLI versus pre-DLI plasma. More than 70% of the antigens were detectable in CML CD34+ cells in an analysis of HG-FOCUS and HG-U133A Affymetrix microarrays, suggesting that expression in malignant progenitor cells is a feature common to antibody targets of DLI. Higher transcript and protein expression in CML compared to normal CD34+ cells was confirmed for three target antigens (RAB38, TBCE, and DUSP12). Together, they consistently elicited antibody responses in an additional 18 of 21 CML patients with clinical responses to therapy, but not in normal donors and only rarely in patients without CML. Immunologic targets of curative DLI responses thus comprise multiple CML progenitor cell-expressed antigens that may represent potential immunogens for vaccination and/or monitoring of immunotherapeutic strategies designed to eliminate myeloid leukemia stem cells.
doi:10.1158/0008-5472.CAN-09-2303
PMCID: PMC2832197  PMID: 20103624
Donor lymphocyte infusion; chronic myeloid leukemia; hematopoietic stem cell; protein microarray; graft-versus-leukemia
8.  Detection and Toxin Typing of Clostridium perfringens in Formalin-Fixed, Paraffin-Embedded Tissue Samples by PCR▿  
Journal of Clinical Microbiology  2008;47(3):807-810.
Since current microbiology methods are not suitable to detect Clostridium perfringens in formalin-fixed, paraffin-embedded tissue samples, we developed a PCR assay to detect toxin-encoding genes and the 16S rRNA gene of C. perfringens. We successfully detected and genotyped C. perfringens in tissue sections from two autopsy cases.
doi:10.1128/JCM.01324-08
PMCID: PMC2650962  PMID: 19109478
9.  Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification 
Nucleic Acids Research  2006;34(11):e81.
Rolling-circle amplification (RCA) and ramification amplification (RAM, also known as hyperbranched RCA) are isothermal nucleic acid amplification technologies that have gained a great application in in situ signal amplification, DNA and protein microarray assays, single nucleotide polymorphism detection, as well as clinical diagnosis. Real-time detection of RCA or RAM products has been a challenge because of most real-time detection systems, including Taqman and Molecular Beacon, are designed for thermal cycling-based DNA amplification technology. In the present study, we describe a novel fluorescent probe construct, termed molecular zipper, which is specially designed for quantifying target DNA by real-time monitoring RAM reactions. Our results showed that the molecular zipper has very low background fluorescence due to the strong interaction between two strands. Once it is incorporated into the RAM products its double strand region is opened by displacement, therefore, its fluorophore releases a fluorescent signal. Applying the molecular zipper in RAM assay, we were able to detect as few as 10 molecules within 90 min reaction. A linear relationship was observed between initial input of targets and threshold time (R2 = 0.985). These results indicate that molecular zipper can be applied to real-time monitoring and qualification of RAM reaction, implying an amenable method for automatic RAM-based diagnostic assays.
doi:10.1093/nar/gkl261
PMCID: PMC1488881  PMID: 16822854
10.  Use of Ramification Amplification Assay for Detection of Escherichia coli O157:H7 and Other E. coli Shiga Toxin-Producing Strains 
Journal of Clinical Microbiology  2005;43(12):6086-6090.
Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) strains are important human pathogens that are mainly transmitted through the food chain. These pathogens have a low infectious dose and may cause life-threatening illnesses. However, detection of this microorganism in contaminated food or a patient's stool specimens presents a diagnostic challenge because of the low copy number in the sample. Often, a more sensitive nucleic acid amplification method, such as PCR, is required for rapid detection of this microorganism. Ramification amplification (RAM) is a recently introduced isothermal DNA amplification technique that utilizes a circular probe for target detection and achieves exponential amplification through the mechanism of primer extension, strand displacement, and ramification. In this study, we synthesized a circular probe specific for the Shiga toxin 2 gene (stx2). Our results showed that as few as 10 copies of stx2 could be detected, indicating that the RAM assay was as sensitive as conventional PCR. We further tested 33 isolates of E coli O157:H7, STEC, Shigella dysenteriae, and nonpathogenic E. coli by RAM assay. Results showed that all 27 STEC isolates containing the stx2 gene were identified by RAM assay, while S. dysenteriae and nonpathogenic E. coli isolates were undetected. The RAM results were 100% in concordance with those of PCR. Because of its simplicity and isothermal amplification, the RAM assay could be a useful method for detecting STEC in food and human specimens.
doi:10.1128/JCM.43.12.6086-6090.2005
PMCID: PMC1317159  PMID: 16333102
11.  Detection of Chlamydia trachomatis by Isothermal Ramification Amplification Method: a Feasibility Study 
Journal of Clinical Microbiology  2002;40(1):128-132.
Chlamydia trachomatis is the leading cause of sexually transmitted disease in the United States. Effective screening for this agent can facilitate prompt treatment and prevent its sequelae. The recent introduction of liquid-based cytology has made possible the simultaneous screening of cervical intraepithelial lesions and detection of C. trachomatis in a single collection vial. In this study we determined whether cytological fluid could support DNA-based amplification for the detection of C. trachomatis. Three methods were compared, including ramification amplification (RAM), real-time PCR with molecular beacon, and Abbott’s ligase chain reaction (LCx). RAM is a novel, recently introduced, isothermal DNA amplification technique that utilizes a circular probe for target detection and achieves exponential amplification through the mechanism of primer extension, strand displacement, and ramification. Our results show that RAM can detect as few as 10 C. trachomatis elementary bodies in less than 2 h, comparable to results with real-time PCR. Thirty clinical specimens collected in PreservCyt solution were tested by LCx, real-time PCR, and RAM. Among 30 specimens, 15 were positive by PCR and LCx and 14 were positive by RAM. One specimen missed by RAM had an inadequate amount of residual cellular material. Our results show that nucleic acid amplification methods can serve to detect C. trachomatis and presumably other sexually transmitted agents in cytological fluid and that the RAM assay can be an alternative to PCR and LCx because of its simplicity and isothermal amplification.
doi:10.1128/JCM.40.1.128-132.2002
PMCID: PMC120135  PMID: 11773105

Results 1-11 (11)