NPM1 for MRD? Droplet Like It's Hot Wertheim, Gerald B.W; Bagg, Adam
The Journal of molecular diagnostics : JMD,
07/2017, Letnik:
19, Številka:
4
Journal Article
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This commentary highlights the article by Mencia-Trinchant et al that describes a novel digital PCR assay for sensitive detection of minimal residual disease in NPM1 mutated acute myeloid leukemia.
Epigenetic dysregulation involving alterations in DNA methylation is a hallmark of various types of cancer, including acute myeloid leukemia (AML). Although specific cancer types and clinical ...aggressiveness of tumors can be determined by DNA methylation status, the assessment of DNA methylation at multiple loci is not routinely performed in the clinical laboratory.
We recently described a novel microsphere-based assay for multiplex evaluation of DNA methylation. In the current study, we validated and used an improved assay termed expedited microsphere HpaII small fragment Enrichment by Ligation-mediated PCR (xMELP) that can be performed with appropriate clinical turnaround time.
Using the xMELP assay in conjunction with a new 17-locus random forest classifier that has been trained using 344 AML samples, we were able to segregate an independent cohort of 70 primary AML patients into methylation-determined subgroups with significantly distinct mortality risk (P = 0.009). We also evaluated precision, QC parameters, and preanalytic variables of the xMELP assay and determined the sensitivity of the random forest classifier score to failure at 1 or more loci.
Our results demonstrate that xMELP performance is suitable for implementation in the clinical laboratory and predicts AML outcome in an independent patient cohort.
Cytochemical Staining Paessler, Michele E; Helfrich, Marybeth; Wertheim, Gerald B W
Methods in molecular biology (Clifton, N.J.),
2017, Letnik:
1633
Journal Article
Historically, the diagnosis and classification of acute leukemia involved morphologic review of blasts in the peripheral blood and bone marrow smears and cytochemical staining. Cytochemical stains, ...which are enzymatic colorimetric reactions that occur in the cells of interest, were necessary to assign and confirm myeloid and lymphoid lineage. In the current WHO 2008 Classification of leukemia, immunophenotyping and cytogenetic analysis have largely replaced cytochemical staining in the characterization of acute leukemias. Nonetheless, cytochemical testing remains a useful adjunct assay for the proper classification of acute leukemia in a number of diagnostic settings. This chapter reviews the principles of the most common cytochemical stains, their procedures and guides to interpretation, and results in acute myeloid leukemia.
Epigenetic dysregulation, including aberrant methylation of cytosine residues in DNA, is a hallmark of cancer and clearly results in oncogenic cellular alterations such as transcriptional attenuation ...of tumor suppressors and genomic instability. A number of studies have examined DNA methylation alterations in patients with acute myeloid leukemia (AML) and have shown that analysis of multilocus methylation patterns can identify biologically distinct AML subclasses and can predict patient prognosis. In order to utilize the prognostic capability of methylation analysis in a clinical setting, we have developed a microsphere-based HpaII tiny fragment enrichment by ligation-mediated PCR (xMELP) assay to interrogate the methylation state of genomic multiple loci along with a random forest-based classification algorithm that correlates DNA methylation status with patient prognosis. These tools can be easily implemented in a clinical molecular pathology laboratory and can be utilized for more accurate risk stratification of AML patients.
We previously identified a SNF1/AMPK-related protein kinase, Hunk, from a mammary tumor arising in an MMTV-neu transgenic mouse. The function of this kinase is unknown. Using targeted deletion in ...mice, we now demonstrate that Hunk is required for the metastasis of c-myc-induced mammary tumors, but is dispensable for normal development. Reconstitution experiments revealed that Hunk is sufficient to restore the metastatic potential of Hunk-deficient tumor cells, as well as defects in migration and invasion, and does so in a manner that requires its kinase activity. Consistent with a role for this kinase in the progression of human cancers, the human homologue of Hunk is overexpressed in aggressive subsets of carcinomas of the ovary, colon, and breast. In addition, a murine gene expression signature that distinguishes Hunk-wild type from Hunk-deficient mammary tumors predicts clinical outcome in women with breast cancer in a manner consistent with the pro-metastatic function of Hunk in mice. These findings identify a direct role for Hunk kinase activity in metastasis and establish an in vivo function for this kinase.
Background: Pallister-Killian syndrome (PKS) is typically recognized by its features that include developmental delay, seizures, sparse temporal hair, and facial dysmorphisms. PKS is most frequently ...caused by mosaic supernumerary isochromosome 12p. Case Presentation: Here, we report a patient with PKS who was subsequently diagnosed with Burkitt lymphoma. Following the successful treatment of lymphoma, this patient demonstrated very mild intellectual disability despite the diagnosis of PKS, which is usually associated with severe developmental delay. Discussion: This is the first reported patient with PKS and a hematologic malignancy. Although there is no significant reported association of tetrasomy 12p with cancer, the co-occurrence of two rare findings in this patient suggests a potential relationship. The localization of AICDA, a gene for which overexpression has been implicated in promoting t(8;14) noted in our patient’s lymphoma, raises a potential mechanism of pathogenesis. In addition, this case indicates that children with PKS can demonstrate near-normal cognitive development.
Aberrant regulation of DNA methylation is characteristic of cancer cells and clearly influences phenotypes of various malignancies. Despite clear correlations between DNA methylation and patient ...outcome, tests that directly measure multiple-locus DNA methylation are typically expensive and technically challenging. Previous studies have demonstrated that the prognosis of patients with acute myeloid leukemia can be predicted by the DNA methylation pattern of 18 loci. We have developed a novel strategy, termed microsphere HpaII tiny fragment enrichment by ligation-mediated PCR (MELP), to simultaneously analyze the DNA methylation pattern at these loci using methylation-specific DNA digestion, fluorescently labeled microspheres, and branched DNA hybridization. The method uses techniques that are inexpensive and easily performed in a molecular laboratory. MELP accurately reflects the methylation levels at each locus analyzed and segregates patients with acute myeloid leukemia into prognostic subgroups. Our results demonstrate the usefulness of MELP as a platform for simultaneous evaluation of DNA methylation of multiple loci.
ABSTRACT
Normal developmental events such as puberty, pregnancy, and parity influence the susceptibility of the mammary gland to tumorigenesis in both humans and rodent model systems. Unfortunately, ...constitutive transgenic mouse models that rely on mammary‐specific promoters to control transgene expression have limited utility for studying the effect of developmental events on breast cancer risk since the hormonal signals governing these events also markedly influence transgene expression levels. A novel transgenic mouse system is described that uses the MMTV‐LTR to drive expression of the reverse tetracycline‐dependent transactivator rtTA. Transgenic mice expressing rtTA in the mammary epithelium were crossed with reporter lines bearing tet operator‐controlled transgenes. We tested the ability to spatially, temporally, and quantitatively control reporter gene expression after administration of doxycycline to bitransgenic mice. Transgene expression using this system can be rapidly induced and deinduced, is highly mammary specific, can be reproducibly titrated over a wide range of expression levels, and is essentially undetectable in the uninduced state. Homogeneous transgene expression throughout the mammary epithelium can be achieved. This system permits transgene expression to be restricted to any desired stage of postnatal mammary gland development. We have developed a mammary‐specific, doxycycline‐inducible transgenic mouse model for studying the effect of mammary gland development on transgene‐mediated phenotypes. Unlike other mammary‐specific, transgenic systems that have been described, this system combines spatially homogeneous transgene expression in the mammary epithelium during puberty, pregnancy, lactation, and involution with the use of an orally administered, inexpensive, and widely available inducing agent. This system offers new opportunities for the transgenic analysis of mammary gland biology in vivo.—Gunther, E. J., Belka, G. K., Wertheim, G. B. W., Wang, J., Hartman, J. L., Boxer, R. B., Chodosh, L. A. A novel doxycycline‐inducible system for the transgenic analysis of mammary gland biology. FASEB J. 16, 283–292 (2002)
This commentary highlights the article by Spencer et al that outlines a novel next-generation sequencing-based method for the detection of FLT3 mutations.
Acute myeloid leukemia (AML) is not a single pathologic entity but represents a heterogeneous group of malignancies. This heterogeneity is exemplified by the variable clinical outcomes that are ...observed in patients with AML, and it is largely the result of diverse mutations within the leukemic cells. These mutations range from relatively large genetic alterations, such as gains, losses, and translocations of chromosomes, to single nucleotide changes. Detection of many of these mutations is required for accurate diagnosis, prognosis, and treatment of patients with AML. As such, many testing modalities have been developed and are currently employed in clinical laboratories to ascertain mutational status at prognostically and therapeutically critical loci. The assays include those that specifically identify large chromosomal alterations, such as conventional metaphase analysis and fluorescence in situ hybridization, and methods that are geared more toward analysis of small mutations, such as PCR with allele-specific oligonucleotide primers. Furthermore, newer tests, including array analysis and next-generation sequencing, which can simultaneously probe numerous molecular aberrancies within tumor cells, are likely to become commonplace in AML diagnostics. Each testing method clearly has advantages and disadvantages, an understanding of which should influence the choice of test in various clinical circumstances. To aid such understanding, this review discusses both genetic mutations in AML and the clinical tests-including their pros and cons-that may be used to probe these abnormalities. Additionally, we highlight the significance of genetic testing by describing cases in which results of genetic testing significantly influence clinical management of patients with AML.
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