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Find video protocols related to scientific articles indexed in Pubmed.
Cytosine deamination is a major cause of baseline noise in next-generation sequencing.
Mol Diagn Ther
PUBLISHED: 08-06-2014
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As next-generation sequencing (NGS) becomes a major sequencing platform in clinical diagnostic laboratories, it is critical to identify artifacts that constitute baseline noise and may interfere with detection of low-level gene mutations. This is especially critical for applications requiring ultrasensitive detection, such as molecular relapse of solid tumors and early detection of cancer. We recently observed a ~10-fold higher frequency of C:G > T:A mutations than the background noise level in both wild-type peripheral blood and formalin-fixed paraffin-embedded samples. We hypothesized that these might represent cytosine deamination events, which have been seen using other platforms.
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Improved FLT3/ITD PCR assay predicts outcome following allogeneic transplant for AML.
Biol. Blood Marrow Transplant.
PUBLISHED: 06-10-2014
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Acute myeloid leukemia (AML) patients harboring internal tandem duplication (ITD) mutations of the FMS-like tyrosine kinase 3 (FLT3) gene carry a poor prognosis. While allogeneic transplantation may improve outcomes, relapse occurs frequently. The FLT3/ITD mutation has been deemed an unsuitable minimal residual disease (MRD) marker because it is unstable and because the standard assay for the mutation is relatively insensitive. The FLT3 mutation is undetectable by polymerase chain reaction (PCR) at pre- or post-transplant time points in many FLT3/ITD AML patients who subsequently relapse following transplant. We report the application of a new technique, tandem duplication PCR (TD-PCR), for detecting MRD in FLT3/ITD AML patients. Between October 2004 and January 2012, 54 FLT3/ITD AML patients in remission underwent transplantation at our institution. Of 37 patients with available Day 60 marrow samples, 28 (76%) were evaluable for MRD detection. In seven (25%) of the 28 patients, the FLT3/ITD mutation was detectable by TD-PCR, but not by standard PCR, on day 60. Six out of the seven patients (86%) with MRD by TD-PCR have relapsed to date compared with only 2 of 21 (10%) patients who were negative for MRD (p = 0.0003). The ability to detect MRD by this sensitive technique may provide an opportunity for early clinical intervention.
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Clinical validation of KRAS, BRAF, and EGFR mutation detection using next-generation sequencing.
Am. J. Clin. Pathol.
PUBLISHED: 05-20-2014
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To validate next-generation sequencing (NGS) technology for clinical diagnosis and to determine appropriate read depth.
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The evolution of treatment strategies for patients with chronic myeloid leukemia relapsing after allogeneic bone marrow transplant: can tyrosine kinase inhibitors replace donor lymphocyte infusions?
Leuk. Lymphoma
PUBLISHED: 04-10-2014
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The optimal treatment for chronic myeloid leukemia (CML) relapsing following allogeneic bone marrow transplant (alloBMT) is unknown. We performed a single-center retrospective analysis of 71 consecutive patients undergoing alloBMT for CML from 1995 to 2008. A multi-state model was used to quantify cumulative incidences of complete molecular response (CMR) and death following alloBMT. The primary analysis was comparison of three treatment interventions (tyrosine kinase inhibitor: TKI, donor lymphocyte infusion: DLI, and TKI + DLI) for relapsed disease post-alloBMT. Forty-five (63%) patients relapsed post-alloBMT (molecular relapse: n = 16, cytogenetic relapse: n = 20, hematologic relapse: n = 2, advanced phase relapse: n = 7) and 40 patients underwent one of three treatments: TKI-only (n = 13), DLI-only (n = 11) or TKI + DLI (n = 16). Although not statistically significant, the TKI-only group had the highest cumulative incidence of CMR and lowest cumulative incidence of death compared to DLI and TKI + DLI. These data support the finding that TKI therapy is active in the post-alloBMT setting.
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Granulocyte-macrophage colony stimulating factor (GM-CSF) enhances the clinical responses to interferon-? (IFN) in newly diagnosed chronic myeloid leukemia (CML).
Leuk. Res.
PUBLISHED: 04-08-2014
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The majority of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors (TKIs) remain with residual disease. In contrast to TKIs, interferon (IFN) is directly toxic to CML progenitor cells, and myeloid growth factors such as GM-CSF may enhance IFN's cytotoxicity. We performed a phase 2 study of IFN+GM-CSF in 58 newly diagnosed CML patients before imatinib approval. Short-term clinical responses included: 60% major cytogenetic response, 28% complete cytogenetic response and 19% complete molecular response. Six patients remain off all therapy for CML (range: 15 months-12 years) after IFN+GM-CSF treatment. IFN+GM-CSF shows promise as an adjunctive therapy for CML.
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Genetic Profiling by Single-Nucleotide Polymorphism-Based Array Analysis Defines Three Distinct Subtypes of Orbital Meningioma.
Brain Pathol.
PUBLISHED: 04-01-2014
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Orbital meningiomas can be classified as primary optic nerve sheath (ON) meningiomas, primary intraorbital ectopic (Ob) meningiomas and spheno-orbital (Sph-Ob) meningiomas based on anatomic site. Single-nucleotide polymorphism (SNP)-based array analysis with the Illumina 300K platform was performed on formalin-fixed, paraffin-embedded tissue from 19 orbital meningiomas (5 ON, 4 Ob and 10 Sph-Ob meningiomas). Tumors were World Health Organization (WHO) grade I except for two grade II meningiomas, and one was NF2-associated. We found genomic alterations in 68% (13 of 19) of orbital meningiomas. Sph-Ob tumors frequently exhibited monosomy 22/22q loss (70%; 7/10) and deletion of chromosome 1p, 6q and 19p (50% each; 5/10). Among genetic alterations, loss of chromosome 1p and 6q were more frequent in clinically progressive tumors. Chromosome 22q loss also was detected in the majority of Ob meningiomas (75%; 3/4) but was infrequent in ON meningiomas (20%; 1/5). In general, Ob tumors had fewer chromosome alterations than Sph-Ob and ON tumors. Unlike Sph-Ob meningiomas, most of the Ob and ON meningiomas did not progress even after incomplete excision, although follow-up was limited in some cases. Our study suggests that ON, Ob and Sph-Ob meningiomas are three molecularly distinct entities. Our results also suggest that molecular subclassification may have prognostic implications.
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Haplotype counting by next-generation sequencing for ultrasensitive human DNA detection.
J Mol Diagn
PUBLISHED: 03-28-2014
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Human identity testing is critical to the fields of forensics, paternity, and hematopoietic stem cell transplantation. Most bone marrow (BM) engraftment testing currently uses microsatellites or short tandem repeats that are resolved by capillary electrophoresis. Single-nucleotide polymorphisms (SNPs) are theoretically a better choice among polymorphic DNA; however, ultrasensitive detection of SNPs using next-generation sequencing is currently not possible because of its inherently high error rate. We circumvent this problem by analyzing blocks of closely spaced SNPs, or haplotypes. As proof-of-principle, we chose the HLA-A locus because it is highly polymorphic and is already genotyped to select proper donors for BM transplant recipients. We aligned common HLA-A alleles and identified a region containing 18 closely spaced SNPs, flanked by nonpolymorphic DNA for primer placement. Analysis of cell line mixtures shows that the assay is accurate and precise, and has a lower limit of detection of approximately 0.01%. The BM from a series of hematopoietic stem cell transplantation patients who tested as all donor by short tandem repeat analysis demonstrated 0% to 1.5% patient DNA. Comprehensive analysis of the human genome using the 1000 Genomes database identified many additional loci that could be used for this purpose. This assay may prove useful to identify hematopoietic stem cell transplantation patients destined to relapse, microchimerism associated with solid organ transplantation, forensic applications, and possibly patient identification.
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Utility of BRAF mutation detection in fine-needle aspiration biopsy samples read as "suspicious for papillary thyroid carcinoma"
Head Neck
PUBLISHED: 03-19-2014
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The purpose of this study was to evaluate the diagnostic utility of BRAF mutation testing on thyroid nodules "suspicious for papillary thyroid carcinoma" (PTC) cytology.
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Tumor cellularity as a quality assurance measure for accurate clinical detection of BRAF mutations in melanoma.
Mol Diagn Ther
PUBLISHED: 03-08-2014
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Detection of BRAF mutations is an established standard of care to predict small-molecule inhibitor (vemurafenib) response in metastatic melanoma. Molecular assays should be designed to detect not only the most common p.V600E mutation, but also p.V600K and other non-p.V600E mutations.
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False positives in multiplex PCR-based next-generation sequencing have unique signatures.
J Mol Diagn
PUBLISHED: 03-02-2014
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Next-generation sequencing shows great promise by allowing rapid mutational analysis of multiple genes in human cancers. Recently, we implemented the multiplex PCR-based Ion AmpliSeq Cancer Hotspot Panel (>200 amplicons in 50 genes) to evaluate EGFR, KRAS, and BRAF in lung and colorectal adenocarcinomas. In 10% of samples, automated analysis identified a novel G873R substitution mutation in EGFR. By examining reads individually, we found this mutation in >5% of reads in 50 of 291 samples and also found similar events in 18 additional amplicons. These apparent mutations are present only in short reads and within 10 bases of either end of the read. We therefore hypothesized that these were from panel primers promiscuously binding to nearly complementary sequences of nontargeted amplicons. Sequences around the mutations matched primer binding sites in the panel in 18 of 19 cases, thus likely corresponding to panel primers. Furthermore, because most primers did not show this effect, we demonstrated that next-generation sequencing may be used to better design multiplex PCR primers through iterative elimination of offending primers to minimize mispriming. Our results indicate the need for careful sequence analysis to avoid false-positive mutations that can arise in multiplex PCR panels. The AmpliSeq Cancer panel is a valuable tool for clinical diagnostics, provided awareness of potential artifacts.
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Microsatellite instability confounds engraftment analysis of hematopoietic stem-cell transplantation.
Appl. Immunohistochem. Mol. Morphol.
PUBLISHED: 02-27-2014
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Polymorphic short tandem-repeat, or microsatellite, loci have been widely used to analyze chimerism status after allogeneic hematopoietic stem-cell transplantation. In molecular diagnostic laboratories, it is recommended to calculate mixed chimerism for at least 2 informative loci and to avoid microsatellite loci on chromosomes with copy number changes. In this report, we show that microsatellite instability observed in 2 patients with acute leukemia may confound chimerism analysis. Interpretation errors may occur even if 2 to 3 loci are analyzed because of length variation in multiple microsatellite loci. Although microsatellite loci with length variation should not be selected for chimerism analysis, the presence of microsatellite instability, like copy number alteration because of aberrant chromosomes, provides evidence of recurrent or residual cancer cells after hematopoietic stem-cell transplantation.
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Rb Loss is Characteristic of Prostatic Small Cell Neuroendocrine Carcinoma.
Clin. Cancer Res.
PUBLISHED: 12-09-2013
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Small cell neuroendocrine carcinoma of the prostate is likely to become increasingly common with recent advances in pharmacologic androgen suppression. Thus, developing molecular markers of small cell differentiation in prostate cancer will be important to guide diagnosis and therapy of this aggressive tumor.
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Software-automated counting of Ki-67 proliferation index correlates with pathologic grade and disease progression of follicular lymphomas.
Am. J. Clin. Pathol.
PUBLISHED: 09-19-2013
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To examine the accuracy of software-assisted measurement of the Ki-67 proliferation index (PI) and its correlation with the grade and clinical progression of follicular lymphoma (FL).
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Tandem duplication PCR: an ultrasensitive assay for the detection of internal tandem duplications of the FLT3 gene.
Diagn. Mol. Pathol.
PUBLISHED: 07-13-2013
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Internal tandem duplication (ITD) mutations of the FLT3 gene have been associated with a poor prognosis in acute myeloid leukemia. Detection of ITD-positive minor clones at the initial diagnosis and during the minimal residual disease stage may be essential. We previously designed a delta-PCR strategy to improve the sensitivity to 0.1% ITD-positive leukemia cells and showed that minor mutants with an allele burden of <1% can be clinically significant. In this study, we report on tandem duplication PCR (TD-PCR), a modified inverse PCR assay, and demonstrate a limit of detection of a few molecules of ITD mutants. The TD-PCR was initially designed to confirm ITD mutation of an amplicon, which was undetectable by capillary electrophoresis and was incidentally isolated by a molecular fraction collecting tool. Subsequently, TD-PCR detected ITD mutation in 2 of 77 patients previously reported as negative for ITD mutation by a standard PCR assay. TD-PCR can also potentially be applied to monitor minimal residual disease with high analytic sensitivity in a portion of ITD-positive acute myeloid leukemia patients. Further studies using TD-PCR to detect ITD mutants at diagnosis may clarify the clinical significance of those ITD mutants with extremely low allele burden.
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Pleural Fluid Cytology of the Polymorphous Variant of EBV-Positive Diffuse Large B-Cell Lymphoma: First Report and Distinction from a Reactive Process.
Case Rep Pathol
PUBLISHED: 06-21-2013
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EBV-positive diffuse large B-cell lymphoma of the elderly is a newly described aggressive lymphoma predominantly affecting patients >50 years of age. Patients may present with nodal and/or extranodal involvement. The lung is one of the more common extranodal sites. The incidence of pleural fluid involvement is less well described. In one study by Oyama et al., pleural effusions were noted in nine percent of cases. Identification of pleural fluid involvement could be important as it may carry prognostic importance in staging (it typically occurs more often in cases with widespread disease), and it could be a relatively easy means of establishing a diagnosis in newly presenting cases. We report the first description of the pleural fluid cytology in a case of EBV-positive diffuse large B-cell lymphoma of the elderly.
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Absence of post-transplantation lymphoproliferative disorder after allogeneic blood or marrow transplantation using post-transplantation cyclophosphamide as graft-versus-host disease prophylaxis.
Biol. Blood Marrow Transplant.
PUBLISHED: 06-18-2013
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Immunosuppressive regimens that effectively prevent graft-versus-host disease (GVHD) after allogeneic blood or marrow transplantation (allo-BMT) have been associated with an increased incidence of post-transplantation lymphoproliferative disorder (PTLD) in the first year after transplantation. We evaluated the incidence of PTLD associated with the use of high-dose post-transplantation cyclophosphamide (PTCy) as GVHD prophylaxis. Between 2000 and 2011, a total of 785 adult allo-BMT recipients were given PTCy as GVHD prophylaxis at the Johns Hopkins Hospital, including 313 patients who received PTCy as sole GVHD prophylaxis. HLA-haploidentical or unrelated donor graft transplantation was performed in 526 patients (67%). No cases of PTLD occurred during the first year after allo-BMT in this series. PTLD is a rare occurrence after allo-BMT using PTCy, even in high-risk alternative donor transplantations.
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Liquid bead array technology in the detection of common translocations in acute and chronic leukemias.
Methods Mol. Biol.
PUBLISHED: 05-14-2013
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Hematologic malignancies often have specific chromosomal translocations that promote cancer initiation and progression. Translocation identification is often vital in the diagnosis, prognosis, and treatment of malignancies. A variety of methods including metaphase cytogenetics, in situ hybridization, microarray techniques, Southern blotting, and many variations of PCR are used to identify translocations. While all these techniques have utility, many have drawbacks limiting their clinical usefulness: high cost, slow turnaround time, low density, large sample requirements, high complexity, and difficult validation and standardization. Multiplexed RT-PCR combined with liquid bead array detection overcomes many of these limitations, allowing simultaneous amplification and detection of multiple translocations within one patient sample. This system has high reliability, reproducibility, and flexibility; low cost and low complexity; rapid turnaround time; and appropriate analyte density. Recently, Asuragen Inc. has developed a multiplexed RT-PCR liquid bead array panel that simultaneously analyzes 12 fusion transcripts found in four major types of hematologic malignancies, allowing rapid and efficient diagnosis. In this chapter, we review liquid bead array technology in relation to the specific hematologic translocations analyzed in the Signature LTx panel.
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Multi-institutional retrospective cohort study of spontaneous pneumothorax.
Pathol. Res. Pract.
PUBLISHED: 03-19-2013
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Spontaneous pneumothorax (SP) affects only a subset of patients in a variety of disorders. This multi-institutional study attempted to identify unique clinicopathologic features of spontaneous pneumothorax (SP) in a large cohort. A total of 111 cases from 109 patients were retrieved from 3 institutions over an 11-year period: 27 women, 82 men. 66 were smokers, 16 were non-smokers, 27 unknown. Sixty-six cases (61%) had identifiable disorders (secondary SP). Seven patients had a family history of lung disease: 2 of pneumothorax, 2 of lung cancer, and 1 each of emphysema, COPD, and not further classified. Forty-three cases (32 men, 11 women) were primary. The average age was 28 (men) and 37 (women). 17 smoked, 14 were non-smokers, and 12 unknown. In 26%, a unique recently described entity, pneumothorax-associated fibroblastic lesion (PAFL), was noted. 16% of this subset also had cellular alveolar septae. Abundant intraalveolar macrophages were noted in 12 cases. Five cases of unknown etiology had extensive bullous lung disease not otherwise classified. One patient had unusual cellular areas suggestive of LAM but negative for HMB-45. In conclusion, several distinct morphologic lesions were identified: PAFL, a subset with cellular septae, and a third with numerous intraalveolar macrophages.
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Detection of minor clones with internal tandem duplication mutations of FLT3 gene in acute myeloid leukemia using delta-PCR.
Diagn. Mol. Pathol.
PUBLISHED: 02-02-2013
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Internal tandem duplication (ITD) mutations of the FLT3 gene have been associated with inferior prognosis of acute myeloid leukemia. Detection of minor clones or minimal residual clones with ITD mutations is desirable, but is challenging when the mutant signal determined by polymerase chain reaction (PCR) and capillary electrophoresis is weak. In this study, we applied delta-PCR, which is a triple-primer strategy, to ensure PCR specificity and improve the sensitivity to 0.1% leukemic cells with ITD mutation. We also applied a reference peak to calculate ITD allelic burdens of <2% threshold of technical limitation for evaluating the relative ratio of 2 signals by capillary electrophoresis. Delta-PCR was able to detect single or multiple ITD mutations with an allelic burden (peak height ratio of mutant allele and wild-type allele) ranging from 0.4% to >100% among all 31 cases with previous documented ITD mutations. In one of the 3 cases with previously reported negative ITD mutation in the initial diagnostic specimen and ITD-positive results in the follow-up specimens, an ITD of 0.04% allele burden was retrospectively detected in the initial diagnosis specimen using delta-PCR. We also demonstrated that minor ITD mutant clones with an allelic burden of <1% present at diagnosis may become a dominant clone at the later refractory status, suggesting that detection of leukemic clones with allelic burdens of <1% may be clinically significant. Delta-PCR can detect ITD mutations with improved sensitivity and specificity and may be useful for the detection of minimal residual leukemia.
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Targeted pathologic evaluation of bone marrow donors identifies previously undiagnosed marrow abnormalities.
Biol. Blood Marrow Transplant.
PUBLISHED: 01-31-2013
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Potential bone marrow donors are screened to ensure the safety of both the donor and recipient. At our institution, potential donors with abnormal peripheral blood cell counts, a personal history of malignancy, or age >60 years are evaluated to ensure that they are viable candidates for donation. Evaluation of the marrow includes morphologic, flow cytometric, and cytogenetic studies. A total of 122 potential donors were screened between the years of 2001 and 2011, encompassing approximately 10% of all donors. Of the screened potential donors, the mean age was 59 years and there were 59 men and 63 women. The donors were screened because of age >60 years (n = 33), anemia (n = 22), cytopenias other than anemia (n = 27), elevated peripheral blood counts without a concurrent cytopenia (n = 20), elevated peripheral blood counts with a concurrent cytopenia (n = 10), history of malignancy (n = 4), abnormal peripheral blood differential (n = 3), prior graft failure (n = 1), history of treatment with chemotherapy (n = 1), and body habitus (n = 1). Marrow abnormalities were detected in 9% (11 of 122) of donors. These donors were screened because of anemia (5 of 22, 23%), age >60 years (2 of 33, 6%), history of malignancy (2 of 4, 50%), elevated peripheral blood counts (1 of 20, 5%), and body habitus (1 of 1, 100%). Abnormalities included plasma cell dyscrasia (n = 3), abnormal marrow cellularity (n = 3), clonal cytogenetic abnormalities (n = 2), low-grade myelodysplastic syndrome (1), a mutated JAK2 V617F allele (n = 1), and monoclonal B cell lymphocytosis (n = 1). Our experience indicates that extended screening of potential donors identifies a significant number of donors with previously undiagnosed marrow abnormalities.
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Outcomes of related donor HLA-identical or HLA-haploidentical allogeneic blood or marrow transplantation for peripheral T cell lymphoma.
Biol. Blood Marrow Transplant.
PUBLISHED: 01-11-2013
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The role of allogeneic blood or marrow transplantation (alloBMT) for peripheral T cell lymphoma (PTCL) remains to be defined. There is growing interest in reduced-intensity conditioning (RIC) regimens and/or utilization of human leukocyte antigen haploidentical (haplo) grafts given concerns about treatment-associated toxicities and donor availability. We reviewed the outcomes of 44 consecutive, related donor alloBMTs for PTCL performed at Johns Hopkins Hospital from 1994 to 2011, including 18 RIC/haplo alloBMTs. Patients receiving RIC (n = 24) were older, with median age of 59 years (range, 24 to 70), than patients receiving myeloablative conditioning (MAC, n = 20), with median age of 46 years (range, 18 to 64), P = .01. The median age at RIC/haplo alloBMT was 60 years. The estimated 2-year progression-free survival (PFS) was 40% (95% confidence interval [CI], 26% to 55%) and overall survival (OS) was 43% (95% CI, 28% to 59%). In older patients (?60, n = 14), the estimated 2-year PFS and OS were 38% (95% CI, 18% to 79%) and 45% (95% CI, 24% to 86%), respectively. On unadjusted analysis, there was a tendency toward superior outcomes for alloBMT in first remission versus beyond first remission, with an estimated 2-year PFS of 53% (95% CI, 33% to 77%) versus 29% (95% CI, 9% to 45%), P = .08. On competing risk analysis, the 1-year cumulative incidence of relapse was 38% for MAC/HLA-identical alloBMTs and 34% for RIC/haplo alloBMTs. Estimated 1-year nonrelapse mortality was 10% for MAC and 8% for RIC (11% for RIC/haplo alloBMT). On unadjusted landmark analysis, patients with acute grade II-IV or chronic graft-versus-host disease (GVHD) had a 17% probability of relapse (95% CI, 0% to 39%), compared with 66% (95% CI, 48% to 84%) in patients without GVHD, P = .04. Utilization of RIC and alternative donors expands treatment options in PTCL to those who are older and unable to tolerate high-dose conditioning, with outcomes comparable with approaches using myeloablative regimens and HLA-matched donors. AlloBMT may be appropriate in first remission in select high-risk cases.
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Analysis of hematopoietic stem cell transplant engraftment: use of loss or gain of microsatellite alleles to identify residual hematopoietic malignancy.
Diagn. Mol. Pathol.
PUBLISHED: 11-18-2011
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Polymorphic short tandem repeat (STR), or microsatellite, loci have been widely used to analyze chimerism status after allogeneic hematopoietic stem cell transplantation. The presence of a patients DNA, as identified by STR analysis, may indicate residual or recurrent malignant disease or may represent normal hematopoiesis of patient origin. The ratio of patient-derived to donor-derived alleles is used to calculate the relative amount of patient cells (both benign and malignant) to donor cells. STRs on chromosomes known to be gained or lost in a patients tumor are generally ignored because it is difficult to perform meaningful calculations of mixed chimerism. However, in this study, we present evidence that STR loci on gained or lost chromosomes are useful in distinguishing the benign or malignant nature of chimeric DNA. In the peripheral blood or bone marrow of 4 hematopoietic stem cell transplantation patients with leukemia or lymphoma, we identified tumor DNA on the basis of STR loci showing copy number alteration. We propose that a targeted evaluation of STR loci showing altered copy number in posttransplant chimerism analysis can provide evidence of residual cancer cells.
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Lymphomatoid papulosis followed by pityriasis lichenoides: a common pathogenesis?
Am J Dermatopathol
PUBLISHED: 11-02-2011
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Pityriasis lichenoides (PL) and lymphomatoid papulosis (LyP) are uncommon idiopathic eruptions with overlapping clinical and histological features. Although current opinion indicates that PL and LyP are distinct and separate entities, molecular genetic evidence of T-cell clonality in both conditions suggests that an etiopathogenic relationship may exist. We report a patient who was diagnosed with LyP type B in 1985 followed by PL after 11 years. We hypothesize that LyP followed by PL in the same patient reflects differences in the host immune response to a common antigenic stimulus.
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Multi-institutional phase 2 clinical and pharmacogenomic trial of tipifarnib plus etoposide for elderly adults with newly diagnosed acute myelogenous leukemia.
Blood
PUBLISHED: 10-14-2011
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Tipifarnib (T) exhibits modest activity in elderly adults with newly diagnosed acute myelogenous leukemia (AML). Based on preclinical synergy, a phase 1 trial of T plus etoposide (E) yielded 25% complete remission (CR). We selected 2 comparable dose levels for a randomized phase 2 trial in 84 adults (age range, 70-90 years; median, 76 years) who were not candidates for conventional chemotherapy. Arm A (T 600 mg twice a day × 14 days, E 100 mg days 1-3 and 8-10) and arm B (T 400 mg twice a day × 14 days, E 200 mg days 1-3 and 8-10) yielded similar CR, but arm B had greater toxicity. Total CR was 25%, day 30 death rate 7%. A 2-gene signature of high RASGRP1 and low aprataxin (APTX) expression previously predicted for T response. Assays using blasts from a subset of 40 patients treated with T plus E on this study showed that AMLs with a RASGRP1/APTX ratio of more than 5.2 had a 78% CR rate and negative predictive value 87%. This ratio did not correlate with outcome in 41 patients treated with conventional chemotherapies. The next T-based clinical trials will test the ability of the 2-gene signature to enrich for T responders prospectively. This study is registered at www.clinicaltrials.gov as #NCT00602771.
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Importance of immunoglobulin heavy chain variable region mutational status in del(13q) chronic lymphocytic leukemia.
Leuk. Lymphoma
PUBLISHED: 08-18-2011
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Among prognostic factors for chronic lymphocytic leukemia (CLL), immunoglobulin heavy chain variable region (IGHV) mutation status and DNA analysis appear to be the most important. However, there is limited clinical outcome information for patients with the favorable-risk del(13q) and poor-risk unmutated IGHV. We retrospectively screened all patients with CLL at our institution between 2004 and June 2010 for del(13q) who also had an IGHV analysis. Unmutated IGHV was found in 38/79 patients; age, Rai stage, prior therapy, and time to evaluation were similar to those for patients with mutated IGHV. Unmutated patients were nearly four times more likely to harbor additional chromosomal aberrations compared to mutated patients (p?
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Ex vivo graft purging and expansion of autologous blood progenitor cell products from patients with multiple myeloma.
Cancer Res.
PUBLISHED: 06-06-2011
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Autologous peripheral blood progenitor cell (PBPC) transplantation is the treatment of choice for selected myeloma patients. However, tumor cells contaminating the apheresis product are a potential source of relapse. Here we report a sequential purging strategy targeting mature and immature clonogenic myeloma cell populations in the autograft. Thawed PBPC products of myeloma patients were treated with rituximab to kill CD138(-)20(+) B cells (highly clonogenic immature cells), and bortezomib to target CD138(+) cells (normal and differentiated myeloma plasma cells), followed by coculture with allogeneic mesenchymal stem cells (MSC) from normal donors. After 7 days of coculture, nonadherent cells were removed and cultured in the absence of MSC for an additional 7 days. Then, efficacy of purging (removal of CD138(-)20(+) and CD138(+) cells) was assessed by flow cytometry and PCR. We used our ex vivo purging strategy to treat frozen aphereses from 16 patients. CD138(+) and CD138(-)20(+)(19(+)) cells present in the initial products were depleted more than 3 and 4 logs, respectively based on 10(6) flow-acquisition events, and to levels below the limit of detection by PCR. In contrast, total nucleated cell (TNC), CD34(+) cell, and colony-forming cell numbers were increased by approximately 12 to 20, 8-, and 23-fold, respectively. Overall, ex vivo treatment of apheresis products with rituximab, bortezomib, and coculture with normal donor MSC depleted mature and immature myeloma cells from clinical aphereses while expanding the normal hematopoietic progenitor cell compartment.
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Clonal immunoglobulin DNA in the plasma of patients with AIDS lymphoma.
Blood
PUBLISHED: 03-09-2011
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Immunoglobulin (Ig) gene rearrangements are used to define clonality of suspected B-lineage malignancy in tissue samples. To determine whether such rearrangements could be identified in plasma, we screened plasma from 14 consecutive patients with AIDS-related lymphoma with multiplex Ig primers. Clonally rearranged Ig DNA was detected in plasma from 7 of 14 patients. Patients in whom clonal Ig DNA remained detectable after combination chemotherapy died with lymphoma. Tumor was available from 1 patient, and the IgH amplification products from plasma and tumor were sequenced and confirmed to be identical. Ig DNA rearrangements in plasma may be useful as a lymphoma-specific tumor marker, and failure to clear clonal Ig DNA may identify patients at high risk for failure of standard therapy.
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Genomic changes in gliomas detected using single nucleotide polymorphism array in formalin-fixed, paraffin-embedded tissue: superior results compared with microsatellite analysis.
J Mol Diagn
PUBLISHED: 02-22-2011
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Deletion or loss of heterozygosity (LOH) in chromosomes 1p and 19q in oligodendrogliomas (ODGs) have diagnostic, prognostic, and therapeutic implications. Current clinical assays are limited because the probes or primers interrogate only limited genomic segments. We investigated the use of single nucleotide polymorphism (SNP) arrays for identifying genomic changes in gliomas from FFPE tissues. DNA was extracted from FFPE tissues of 30 brain tumor cases (15 ODGs and 15 non-ODGs) and assayed on the Illumina array with 300,000 markers. SNP results were compared with standard short tandem repeat (STR) assays of chromosomes 1p and 19q. Fifteen ODGs had LOH by STR and deletion by array on both 1p and 19q. Ten non-ODGs had no evidence of LOH on 1p and 19q by STR, seven of which had no abnormalities for these chromosomes; three had partial deletions by SNP array. Five non-ODG cases had partial LOH or deletion by both assays. No major discordance was found between SNP array and STR results. Advantages of SNP arrays include no need for an accompanying normal sample, the ability to find small segmental deletions, the potential to distinguish between deletions and copy neutral LOH, and whole-genome screening to allow discovery of new, significant loci. Assessment of genomic changes in routine glioma specimens using SNP arrays is feasible and has great potential as an accurate clinical diagnostic test.
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Characterization of chronic myeloid leukemia stem cells.
Am. J. Hematol.
PUBLISHED: 02-12-2011
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Although tyrosine kinase inhibitors have redefined the care of chronic myeloid leukemia (CML), these agents have not proved curative, likely due to resistance of the leukemia stem cells (LSC). While a number of potential therapeutic targets have emerged in CML, their expression in the LSC remains largely unknown. We therefore isolated subsets of CD34(+) stem/progenitor cells from normal donors and from patients with chronic phase or blast crisis CML. These cell subsets were then characterized based on ability to engraft immunodeficient mice and expression of candidate therapeutic targets. The CD34(+)CD38(-) CML cell population with high aldehyde dehydrogenase (ALDH) activity was the most enriched for immunodeficient mouse engrafting capacity. The putative targets: PROTEINASE 3, SURVIVIN, and hTERT were expressed only at relatively low levels by the CD34(+)CD38(-)ALDH(high) CML cells, similar to the normal CD34(+)CD38(-)ALDH(high) cells and less than in the total CML CD34(+) cells. In fact, the highest expression of these antigens was in normal, unfractionated CD34(+) cells. In contrast, PRAME and WT1 were more highly expressed by all CML CD34(+) subsets than their normal counterparts. Thus, ALDH activity appears to enrich for CML stem cells, which display an expression profile that is distinct from normal stem/progenitor cells and even the CML progenitors. Indeed, expression of a putative target by the total CD34(+) population in CML does not guarantee expression by the LSC. These expression patterns suggest that PROTEINASE 3, SURVIVIN, and hTERT are not optimal therapeutic targets in CML stem cells; whereas PRAME and WT1 seem promising.
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Early lymphocyte recovery after intensive timed sequential chemotherapy for acute myelogenous leukemia: peripheral oligoclonal expansion of regulatory T cells.
Blood
PUBLISHED: 10-08-2010
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Few published studies characterize early lymphocyte recovery after intensive chemotherapy for acute myelogenous leukemia (AML). To test the hypothesis that lymphocyte recovery mirrors ontogeny, we characterized early lymphocyte recovery in 20 consecutive patients undergoing induction timed sequential chemotherapy for newly diagnosed AML. Recovering T lymphocytes were predominantly CD4(+) and included a greatly expanded population of CD3(+)CD4(+)CD25(+)Foxp3(+) T cells. Recovering CD3(+)CD4(+)CD25(+)Foxp3(+) T cells were phenotypically activated regulatory T cells and showed suppressive activity on cytokine production in a mixed lymphocyte reaction. Despite an initial burst of thymopoiesis, most recovering regulatory T cells were peripherally derived. Furthermore, regulatory T cells showed marked oligoclonal skewing, suggesting that their peripheral expansion was antigen-driven. Overall, lymphocyte recovery after chemotherapy differs from ontogeny, specifically identifying a peripherally expanded oligoclonal population of activated regulatory T lymphocytes. These differences suggest a stereotyped immunologic recovery shared by patients with newly diagnosed AML after induction timed sequential chemotherapy. Further insight into this oligoclonal regulatory T-cell population will be fundamental toward developing effective immunomodulatory techniques to improve survival for patients with AML.
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Performance and clinical evaluation of a sensitive multiplex assay for the rapid detection of common NPM1 mutations.
J Mol Diagn
PUBLISHED: 07-08-2010
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Determination of NPM1 mutation status has become essential for the molecular classification of acute myeloid leukemias (AML). Methods with high clinical sensitivity and specificity adapted to the molecular laboratory workflow are required for the diagnosis, prognosis, and monitoring of AML with normal karyotype. We report here the development and evaluation of a novel, streamlined, RNA-based assay for the rapid multiplex detection of common NPM1 mutations in a 96-well assay format. Using synthetic transcripts and total RNA from leukemic cell lines, we show that the assay can specifically detect NPM1 wild-type and mutants A, B, D, or J transcripts in the same reaction. Dilution experiments indicate an assay dynamic range >4 log units with an analytical sensitivity of approximately 0.01%. Evaluation of 69 clinical specimens at initial diagnosis resulted in 100% agreement with reference methods. Of patients with AML with normal karyotype, 53% carried one of four different mutations. The assay was also combined with other laboratory-developed tests to simultaneously detect NPM1 mutant transcripts and fusion transcripts resulting from t(8;21) or inv(16) in a single reaction well. Overall, these results show that the assay is a versatile and specific tool for the screening of NPM1 mutations in patients with AML. Its high analytical sensitivity further suggests potential utility for the monitoring of residual disease in AML with normal karyotype.
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Extended follow-up of autologous bone marrow transplantation with 4-hydroperoxycyclophosphamide (4-HC) purging for indolent or transformed non-Hodgkin lymphomas.
Biol. Blood Marrow Transplant.
PUBLISHED: 06-22-2010
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Autologous blood or marrow transplantation (ABMT) for low-grade lymphomas can prolong event-free survival (EFS) but requires long-term follow-up. We report one of the longest follow-ups to a prospective transplantation study in such diseases. On a phase II study, 80 patients with low-grade, transformed, or mantle cell lymphoma received ABMT with 4-hydroperoxycyclophosphamide (4-HC) purging as part of initial or salvage therapy. Diagnoses included nontransformed follicular lymphoma in 63% and transformed lymphoma in 15%. With 16.6-year median follow-up for survival, actuarial 10-year EFS and overall survival (OS) were 34% (95% confidence interval [CI], 25%-46%) and 45% (35%-57%). Median EFS and OS were 3.0 and 8.0 years. Early nonrelapse mortality incidence was 8%; myelodysplastic syndrome or leukemia incidence was 4%. Most relapses occurred within 3 years, with a median time to diagnosis of relapse of 1.8 years (range: 0.1-15.6 years). On multivariate analysis, age >50 years, ?3 prior chemotherapy regimens, and ABMT after relapse were associated with significantly inferior survival. Fifteen patients (19%) were event-free >15 years after transplantation, raising the possibility of a plateau in the progression-free survival curve. Thus, 4-HC-purged ABMT can produce extended remissions in a subgroup of patients with indolent lymphomas.
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?-PCR, A Simple Method to Detect Translocations and Insertion/Deletion Mutations.
J Mol Diagn
PUBLISHED: 06-06-2010
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PCR detection of chromosomal translocations and small insertion/deletion mutations is challenging when potential amplicon size varies greatly. Molecular diagnostic laboratories face such difficulties with the BCL2-IGH translocation in follicular lymphoma and with internal tandem duplication mutation of the FLT3 gene in leukemia, where breakpoints are widely distributed, mutations may be multiple, signal strength is low, and background noise is elevated. We developed a strategy, called ?-PCR, that ensures PCR specificity and identifies individual breakpoints. ?-PCR uses two forward primers (external and internal) and a reverse primer simultaneously. The internal primer functions as a probe with a defined distance ? from the external primer. For follicular lymphoma, we prepared upstream, BCL2-specific primers for potential breakpoints to pair with a common, downstream VLJH primer. Multiplexed PCR amplicons are sized by capillary electrophoresis. Each of the upstream pairs has a defined interval separating them that uniquely identifies the breakpoint. The presence of two amplicons with a defined size difference confirms validity of the rearrangement and identity of the specific breakpoint, even if signal strength is low. By testing 40 follicular lymphoma and 12 control specimens from formalin-fixed, paraffin-embedded (FFPE) blocks, we showed that multiplex ?-PCR is a simple, sensitive strategy to identify translocations with multiple breakpoints or partners. The strategy was also applied to detect minor leukemic clones with internal tandem duplication mutations and could have broader applications for other insertion/deletion and duplication mutations.
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Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications.
J Mol Diagn
PUBLISHED: 04-29-2010
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Mutations in codons 12 and 13 of the KRAS oncogene are relatively common in colorectal and lung adenocarcinomas. Recent data indicate that these mutations result in resistance to anti-epidermal growth factor receptor therapy. Therefore, we assessed Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS codon 12/13 mutations in formalin-fixed paraffin-embedded samples, including 58 primary and 42 metastatic colorectal adenocarcinomas, 63 primary and 17 metastatic lung adenocarcinomas, and 20 normal colon samples. Of 180 tumor samples, 62.2% were KRAS mutant positive, and 37.8% were negative. Melting curve analysis yielded no false positive or false negative results, but had 10% equivocal calls. Melting curve analysis also resulted in 4 cases with melting curves inconsistent with either wild-type or codon 12/13 mutations. These patterns were generated from samples with double mutants in codons 12/13 and with mutations outside of codons 12/13. Pyrosequencing yielded no false positive or false negative results as well. However, two samples from one patient yielded a pyrogram that was flagged as abnormal, but the mutation subtype could not be determined. Finally, using an electronic cutoff of 10%, Sanger sequencing showed 11.1% false positives and 6.1% false negatives. In our hands, the limit of detection for Sanger sequencing, pyrosequencing, and melting curve analysis was approximately 15 to 20%, 5%, and 10% mutant alleles, respectively.
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Auer rod-like inclusions in a low-grade B-cell leukemia.
Ann Diagn Pathol
PUBLISHED: 04-02-2010
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Auer rods define neoplastic blasts of myeloid origin. However, azurophilic, needle-shaped, Auer rod-like inclusions have rarely been reported in the neoplastic cells of patients with B-cell malignancies, including chronic lymphocytic leukemia/small lymphocytic lymphoma. Here, we report a case of low-grade B-cell leukemia most consistent with chronic lymphocytic leukemia/small lymphocytic lymphoma that displayed numerous intracytoplasmic, needle-shaped, azurophilic rods resembling Auer rods. Furthermore, we review the literature on Auer rod-like structures in B-cell neoplasms.
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K562/GM-CSF immunotherapy reduces tumor burden in chronic myeloid leukemia patients with residual disease on imatinib mesylate.
Clin. Cancer Res.
PUBLISHED: 01-06-2010
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Chronic myeloid leukemia (CML) can be responsive to T-cell-mediated immunity. K562/granulocyte macrophage-colony stimulating factor (GM-CSF) is a GM-CSF producing vaccine derived from a CML cell line that expresses several CML-associated antigens. A pilot study was developed to determine if K562/GM-CSF immunotherapy could improve clinical responses to imatinib mesylate (IM) in patients with chronic myeloid leukemia.
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Resolution of pansclerotic morphea after treatment with antithymocyte globulin.
Nat Rev Rheumatol
PUBLISHED: 08-28-2009
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A previously healthy 50-year-old man presented with thickening and hardening of the skin on his trunk, neck and upper extremities that had started after the appearance of a 5 cm web-like patch of blood vessels on his upper chest and progressed over 4 months. He also reported difficulties with swallowing and a 20 kg weight loss.
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A rare e14a3 (b3a3) BCR-ABL fusion transcript in chronic myeloid leukemia: diagnostic challenges in clinical laboratory practice.
J Mol Diagn
PUBLISHED: 06-04-2009
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Patients with chronic myelogenous leukemia have a t(9;22)(q34;q11.2) or variant translocation that results in a BCR-ABL fusion gene. BCR-ABL detection by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) is the standard practice for monitoring residual disease in patients with chronic myelogenous leukemia who receive tyrosine kinase inhibitor therapies. In this study, we describe a patient who tested positive for the BCR-ABL translocation by fluorescence in situ hybridization and cytogenetic analysis but tested negative by qRT-PCR molecular analysis at the time of diagnosis. Further PCR analysis and DNA sequencing with alternative primer sets demonstrated the presence of an e14a3 (also known as b3a3) BCR-ABL fusion. The e14a3 fusion is rare, but may be underreported as a result of many commercially available and laboratory-developed primer sets that fail to detect breakpoints in the ABL gene that are downstream of intron 1. For this patient, if the qRT-PCR assay had been used to monitor disease response/progression after treatment and not in conjunction with fluorescence in situ hybridization or cytogenetics at the time of diagnosis, the negative result would have been misinterpreted as molecular remission.
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Circulating clonotypic B cells in classic Hodgkin lymphoma.
Blood
PUBLISHED: 02-02-2009
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Although Hodgkin and Reed-Sternberg (HRS) cells are B lymphoid cells, they are unlike any normal cells of that lineage. Moreover, the limited proliferative potential of HRS cells belies the clinical aggressiveness of Hodgkin lymphoma (HL). More than 20 years ago, the L428 HL cell line was reported to contain a small population of phenotypic B cells that appeared responsible for the continued generation of HRS cells. This observation, however, has never been corroborated, and such clonotypic B cells have never been documented in HL patients. We found that both the L428 and KM-H2 HL cell lines contained rare B-cell subpopulations responsible for the generation and maintenance of the predominant HRS cell population. The B cells within the HL cell lines expressed immunoglobulin light chain, the memory B-cell antigen CD27, and the stem cell marker aldehyde dehydrogenase (ALDH). Clonal CD27(+)ALDH(high) B cells, sharing immunoglobulin gene rearrangements with lymph node HRS cells, were also detected in the blood of most newly diagnosed HL patients regardless of stage. Although the clinical significance of circulating clonotypic B cells in HL remains unclear, these data suggest they may be the initiating cells for HL.
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A unique, histopathologic lesion in a subset of patients with spontaneous pneumothorax.
Arch. Pathol. Lab. Med.
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Spontaneous pneumothorax can be idiopathic (primary), or it can occur in association with an underlying predisposing condition (secondary). Spontaneous pneumothorax may be a harbinger of an undiagnosed clinical condition, which may be associated with serious systemic abnormalities, making early recognition and diagnosis important. The pulmonary pathology of some of these disorders has not been fully elucidated.
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Terminal myeloid differentiation in vivo is induced by FLT3 inhibition in FLT3/ITD AML.
Blood
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A hallmark of cancer is the disruption of differentiation within tumor cells. Internal tandem duplication mutations of the FLT3 kinase (FLT3/ITD) occur commonly in acute myeloid leukemia (AML) and are associated with poor survival, leading to efforts to develop FLT3 kinase inhibitors. However, FLT3 inhibitors have thus far met with limited success, inducing only a clearance of peripheral blasts with minimal BM responses. Quizartinib is a novel potent and selective FLT3 inhibitor currently being studied in clinical trials. In 13 of 14 FLT3/ITD AML patients with normal karyotype treated with quizartinib, we observed terminal myeloid differentiation of BM blasts in association with a clinical differentiation syndrome. The single patient whose blasts failed to differentiate had a preexisting C/EBP? mutation and another developed a C/EBP? mutation at disease progression, suggesting a mechanism of resistance to FLT3 inhibition. In vitro, in primary blasts cocultured with human BM stroma, FLT3 inhibition with quizartinib induced cell-cycle arrest and differentiation rather than apoptosis. The present study is the first description of terminal differentiation of cancer cells in patients treated with a tyrosine kinase inhibitor. These data highlight the importance of the differentiation block in the patho-genesis of AML.
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Flow cytometry of lesional skin enhances the evaluation of cutaneous B-cell lymphomas.
J. Cutan. Pathol.
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Cutaneous B-cell lymphoproliferative lesions can pose diagnostic challenges. This study investigates the utlility of flow cytometry in 42 cases of suspected cutaneous B-cell lymphoma.
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Flow cytometric findings in hemophagocytic lymphohistiocytosis.
Am. J. Clin. Pathol.
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Hemophagocytic lymphohistiocytosis (HLH) is an often fatal hyperinflammatory syndrome. HLH may be inherited, but it more commonly arises secondary to Epstein-Barr virus (EBV) or other infections, hematologic malignancies, or rheumatologic diseases. We identified 17 patients diagnosed with HLH who had flow cytometric analysis of peripheral blood or bone marrow performed at the time of diagnosis. Two patients had primary HLH, and the others had HLH secondary to EBV infection, hematologic malignancies, rheumatologic conditions, or tuberculosis. The marrow typically showed a reactive lymphocytosis and a marked left shift in myelopoiesis regardless of the etiology. Qualitative abnormalities were also found in several cases, including T-cell abnormalities in the majority of the EBV-associated HLH cases. While not specific, flow cytometric findings in HLH are different from the findings in uninvolved marrow samples, and care should be taken not to overinterpret immunophenotypic findings in these cases as indicative of a primary marrow disorder or lymphoma.
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Fine-needle aspiration of metastatic papillary thyroid carcinoma found in the liver.
Diagn. Cytopathol.
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Papillary thyroid carcinoma (PTC) metastasis to the liver is a rare event and may cause diagnostic dilemmas. In this study, we have reviewed the cytological features of metastatic PTC to the liver, tested the BRAF V600E status of these lesions, and discussed potential diagnostic pitfalls. The pathological archives at the Johns Hopkins Hospital were searched for metastatic PTC. A total of 247 cases were identified; four cases were found to have metastases to the liver. Three of these cases were available for molecular testing to determine BRAF V600E status. All patients were female with ages ranging from 39- to 66-years old. Local lymph node involvement was found in all patients at the time of thyroidectomy. The average time of liver metastasis discovery following thyroidectomy was 16 years. The cytomorphology revealed predominantly microacini or two-dimensional clusters. Tumor cells were small-to-intermediate in size with oval nuclei, fine chromatin, nuclear grooves, rare intranuclear pseudoinclusions, and mitoses. In all cases, immunohistochemical stains for thyroglobulin and thyroid transcription factor 1 (TTF-1) were positive. All these tested cases were negative for the BRAF V600E mutation. The differential diagnosis includes adenocarcinoma and neuroendocrine neoplasms. The most important morphologic features for diagnosing PTC are the presence of pale finely granular chromatin, nuclear grooves, and intranuclear pseudoinclusions. A thorough review of the patients clinical history and the use of appropriate IHC stains are essential in reaching an accurate diagnosis. Further molecular characterization is necessary to identify the changes most strongly associated with distant metastasis.
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Phase 2 study of rituximab-ABVD in classical Hodgkin lymphoma.
Blood
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In classical Hodgkin lymphoma, circulating clonotypic malignant cells express CD20, which potentially explains the observed activity of rituximab. This multicenter phase 2 study investigated the combination of rituximab-ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) for stage II-IV untreated classical Hodgkin lymphoma. A goal was to assess the behavior of circulating clonotypic B cells clinically. Of 49 evaluable patients, 69% had stage IIB-IV disease; 8% had CD20(+) Hodgkin and Reed-Sternberg cells. Rituximab-ABVD was generally well tolerated. Delivered relative dose intensity was 94% for AVD and 79% for bleomycin. After 6 cycles, 81% of patients were in complete remission. Only 8% received radiation therapy. The actuarial 3-year event-free and overall survival rates were 83% and 98%, respectively. EBV copy number in plasma fell dramatically during cycle 1 in patients with EBV(+) tumors. Persistence of detectable circulating clonotypic B cells was associated with a greater relapse frequency (P < .05). Rituximab-ABVD and clonotypic B cells warrant additional study in classical Hodgkin lymphoma.
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Clinical and pathologic features of secondary acute promyelocytic leukemia.
Am. J. Clin. Pathol.
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Acute promyelocytic leukemia (APL) is a relatively common form of acute myeloid leukemia (AML) that has an excellent prognosis. In contrast, secondary acute myeloid leukemias, including therapy-related AML and AML with myelodysplasia-related changes, have a relatively poor prognosis. We identified 9 cases of APL at our institution in which there was a history of chemotherapy, radiotherapy, chronic immunosuppression, or antecedent myelodysplastic syndrome. The clinical and pathologic findings in these cases of secondary APL were compared with the clinical and pathologic findings in cases of de novo APL. We found that secondary and de novo APL had abnormal promyelocytes with similar morphologic and immunophenotypic features, comparable cytogenetic findings, comparable rates of FMS-like tyrosine kinase mutations, and similar rates of recurrent disease and death. These data suggest that secondary APL is similar to de novo APL and, thus, should be considered distinct from other secondary acute myeloid neoplasms.
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A virtual pyrogram generator to resolve complex pyrosequencing results.
J Mol Diagn
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We report a freely available software program, Pyromaker, which generates simulated traces for pyrosequencing results based on user inputs. Simulated pyrograms can aid in the analysis of complex pyrosequencing results in which various hypothesized mutations can be tested, and the resultant pyrograms can be matched with the actual pyrogram. We validated the software using the actual pyrograms for common KRAS gene mutations as well as several mutations in the BRAF, GNAS, and p53 genes. We demonstrate that all 18 possible single-base mutations within codons 12 and 13 of KRAS generate unique pyrosequencing traces and highlight the distinctions between them. We further show that all reported codon 12 and 13 complex mutations produce unique pyrograms. However, some complex mutations are indistinguishable from single-base mutations. For complicated pyrograms, Pyromaker was used in two modes, one in which hypothesis-based simulated pyrograms were pattern-matched with the actual pyrograms. In a second strategy with only the pyrogram, Pyromaker was used to identify the underlying mutation by iteratively reconstructing the mutant pyrogram. Either strategy was able to successfully identify the complex mutations, which were confirmed by cloning and sequencing. Using two examples of KRAS codon 12 mutations (specifically GGT?TTT, G12F and GGT?GAG, G12E), we report which combinations of five approaches permit unambiguous mutation identification. The most efficient approach was found to be pyrosequencing with Pyromaker.
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The importance of IGHV mutational status in del(11q) and del(17p) chronic lymphocytic leukemia.
Clin Lymphoma Myeloma Leuk
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Most patients with CLL with a poor-risk cytogenetic profile have an unmutated IGHV sequence. Limited clinical information exists for patients with CLL who have a poor-risk cytogenetic profile and a mutated or good-risk IGHV status. We retrospectively screened all patients with CLL seen at our institution from 2006 onward who harbored a del(11q) or del(17p) CLL detected by fluorescence in situ hybridization (FISH) analysis for whom an IGHV analysis was requested. In 66 evaluable patients, 50 (76%) had an unmutated IGHV sequence. Thirty-nine patients (59%) had del(11q) and 27 patients (41%) had del(17p); no patient in this series had both del(11q) and del(17p). The patients initial clinical presentations were similar in both mutational groups. Patients with an unmutated IGHV sequence were more likely to receive treatment and to have a shorter survival, with an estimated 3-year overall survival (OS) of 81% compared with 100% in the group with a mutated IGHV sequence (log rank, P = .06). These data suggest that IGHV mutational status has prognostic relevance even in patients with CLL who are defined as poor risk by genomic FISH analysis.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.