Philippe Szankasi

BCR-ABL1 compound mutations in tyrosine kinase inhibitor–resistant CML: frequency and clonal relationships

BCR-ABL1 compound mutations can confer high-level resistance to imatinib and other ABL1 tyrosine kinase inhibitors (TKIs). The third-generation ABL1 TKI ponatinib is effective against BCR-ABL1 point mutants individually, but remains vulnerable to certain BCR-ABL1 compound mutants. To determine the frequency of compound mutations among chronic myeloid leukemia patients on ABL1 TKI therapy, in the present study, we examined a collection of patient samples (N = 47) with clear evidence of 2 BCR-ABL1 kinase domain mutations by direct sequencing. Using a cloning and sequencing method, we found that 70% (33/47) of double mutations detected by direct sequencing were compound mutations. Sequential, branching, and parallel routes to compound mutations were common. In addition, our approach revealed individual and compound mutations not detectable by direct sequencing. The frequency of clones harboring compound mutations with more than 2 missense mutations was low (10%), whereas the likelihood of silent mutations increased disproportionately with the total number of mutations per clone, suggesting a limited tolerance for BCR-ABL1 kinase domain missense mutations. We conclude that compound mutations are common in patients with sequencing evidence for 2 BCR-ABL1 mutations and frequently reflect a highly complex clonal network, the evolution of which may be limited by the negative impact of missense mutations on kinase function.

Detection of FLT3 Internal Tandem Duplication in Targeted, Short-Read-Length, Next-Generation Sequencing Data

A recurrent somatic mutation frequently found in cytogenetically normal acute myeloid leukemia (AML) is internal tandem duplication (ITD) in the fms-related tyrosine kinase 3 gene (FLT3). This mutation is generally detected in the clinical laboratory by PCR and electrophoresis-based product sizing. As the number of clinically relevant somatic mutations in AML increases, it becomes increasingly attractive to incorporate FLT3 ITD testing into multiplex assays for many somatic mutations simultaneously, using next-generation sequencing (NGS). However, the performance of most NGS analysis tools for identifying medium-size insertions such as FLT3 ITD mutations is largely unknown. We used a multigene, targeted NGS assay to obtain deep sequence coverage (>1000-fold) of FLT3 and 26 other genes from 22 FLT3 ITD-positive and 29 ITD-negative specimens to examine the performance of several commonly used NGS analysis tools for identifying FLT3 ITD mutations. ITD mutations were present in hybridization-capture sequencing data, and Pindel was the only tool out of the seven tested that reliably detected these insertions. Pindel had 100% sensitivity (95% CI = 83% to 100%) and 100% specificity (95% CI = 88% to 100%) in our samples; Pindel provided accurate ITD insertion sizes and was able to detect ITD alleles present at estimated frequencies as low as 1%. These data demonstrate that FLT3 ITDs can be reliably detected in panel-based, next-generation sequencing assays.

Targeted next generation sequencing of clinically significant gene mutations and translocations in leukemia

Leukemias are currently subclassified based on the presence of recurrent cytogenetic abnormalities and gene mutations. These molecular findings are the basis for risk-adapted therapy; however, such data are generally obtained by disparate methods in the clinical laboratory, and often rely on low-resolution techniques such as fluorescent in situ hybridization. Using targeted next generation sequencing, we demonstrate that the full spectrum of prognostically significant gene mutations including translocations, single nucleotide variants (SNVs), and insertions/deletions (indels) can be identified simultaneously in multiplexed sequence data. As proof of concept, we performed hybrid capture using a panel of 20 genes implicated in leukemia prognosis (covering a total of 1 Mbp) from five leukemia cell lines including K562, NB4, OCI-AML3, kasumi-1, and MV4–11. Captured DNA was then sequenced in multiplex on an Illumina HiSeq. Using an analysis pipeline based on freely available software we correctly identified DNA-level translocations in three of the three cell lines where translocations were covered by our capture probes. Furthermore, we found all published gene mutations in commonly tested genes including NPM1, FLT3, and KIT. The same methodology was applied to DNA extracted from the bone marrow of a patient with acute myeloid leukemia, and identified a t(9;11) translocation with single base accuracy as well other gene mutations. These results indicate that targeted next generation sequencing can be successfully applied in the clinical laboratory to identify a full spectrum of DNA mutations ranging from SNVs and indels to translocations. Such methods have the potential to both greatly streamline and improve the accuracy of DNA-based diagnostics.

Ncf1 (p47phox) is essential for direct regulatory T cell mediated suppression of CD4+ effector T cells.

Background Multiple mechanisms have been advanced to account for CD4+FOXP3+ regulatory T cell (Treg)-mediated suppression of CD4+ effector T cells (Teffs) but none appear to completely explain suppression. Previous data indicates that Tregs may affect the microenvironment redox state. Given the inherent redox sensitivity of T cells, we tested the hypothesis that oxidants may mediate the direct suppression of Teffs by Tregs. Methodology/Principal Findings Tregs and Teffs were isolated from the spleens of wild type (WT) C57BL/6 mice or Ncf1(p47phox)-deficient C57BL/6 mice which lack NADPH oxidase function. Teffs were labeled with CFSE and co-cultured with unlabeled Tregs at varying Treg:Teff ratios in the presence of anti-CD3/CD28 coated beads for 3 days in suppression assays. Treg-mediated suppression was quantified by flow cytometric analysis of CFSE dilution in Teffs. The presence of the antioxidants n-acetylcysteine (NAC) or 2-mercaptoethanol or inhibitors of NADPH oxidase (diphenyleneiodonium and VAS-2870) resulted in reduced WT Treg-mediated suppression. The observed suppression was in part dependent upon TGFβ as it was partially blocked with neutralizing antibodies. The suppression of Teff proliferation induced by exogenous TGFβ treatment could be overcome with NAC. Ncf1-deficient Teff were slightly but significantly less sensitive than WT Teff to suppression by exogenous TGFβ. Ncf1-deficient Tregs suppressed Ncf1-deficient Teff very poorly compared to wild type controls. There was partial but incomplete reconstitution of suppression in assays with WT Tregs and Ncf1-deficient Teff. Conclusions/Significance We present evidence that NADPH oxidase derived ROS plays a role in the direct Treg mediated suppression of CD4+ effector T cells in a process that is blocked by thiol-containing antioxidants, NADPH oxidase inhibitors or a lack of Ncf1 expression in Tregs and Teffs. Oxidants may represent a potential new target for therapeutic modulation of Treg function.

The proteomic signature of NPM/ALK reveals deregulation of multiple cellular pathways

Constitutive expression of the chimeric NPM/ALK fusion protein encoded by the t(2;5)(p32;q35) is a key oncogenic event in the pathogenesis of most anaplastic large cell lymphomas (ALCLs). The proteomic network alterations produced by this aberration remain largely uncharacterized. Using a mass spectrometry (MS)-driven approach to identify changes in protein expression caused by the NPM/ALK fusion, we identified diverse NPM/ALK-induced changes affecting cell proliferation, ribosome synthesis, survival, apoptosis evasion, angiogenesis, and cytoarchitectural organization. MS-based findings were confirmed using Western blotting and/or immunostaining of NPM/ALK-transfected cells and ALK-deregulated lymphomas. A subset of the proteins distinguished NPM/ALK-positive ALCLs from NPM/ALK-negative ALCLs and Hodgkin lymphoma. The multiple NPM/ALK-deregulated pathways identified by MS analysis also predicted novel biologic effects of NPM/ALK expression. In this regard, we showed loss of cell adhesion as a consequence of NPM/ALK expression in a kinase-dependent manner, and sensitivity of NPM/ALK-positive ALCLs to inhibition of the RAS, p42/44ERK, and FRAP/mTOR signaling pathways. These findings reveal that the NPM/ALK alteration affects diverse cellular pathways, and provide novel insights into NPM/ALK-positive ALCL pathobiology. Our studies carry important implications for the use of MS-driven approaches for the elucidation of neoplastic pathobiology, the identification of novel diagnostic biomarkers, and pathogenetically relevant therapeutic targets.

A new DNA-based test for detection of nucleophosmin exon 12 mutations by capillary electrophoresis

Mutations in nucleophosmin (NPM1) exon 12 are thought to be the most common genetic event in acute myelogenous leukemia (AML) and to confer favorable clinical prognoses. In this report, we describe a simple molecular test for the detection of NPM1 exon 12 mutations in patients with AML using polymerase chain reaction amplification of genomic DNA followed by the analysis of amplification products by capillary electrophoresis. Mutations were reproducibly detected when present in at least 5% of cells, and all NPM1 exon 12 mutations reported to date in AML could be identified using this method. This method was successfully employed using paraffin-extracted DNA, allowing for the examination of archived clinical specimens, and the assay was validated by the direct sequencing of 33 patient samples. This sensitive test is straightforward to perform and provides important information that can influence both the clinical management and treatment options for many patients with AML.

Use of similar immunoglobulin VH gene segments by MALT lymphomas of the ocular adnexa

Extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue type (MALT lymphomas) develop from acquired reactive infiltrates directed against external or autoantigens. Although some European cases of ocular adnexal MALT lymphoma have been associated with Chlamydia psittaci infections, C. psittaci has not been detected in large studies of US-based cases. To evaluate whether the growth of US-based ocular adnexal MALT lymphomas may be promoted by a similar antigen, we identified and analyzed the expressed immunoglobulin VH genes in 10 cases. Interestingly, the VH genes in two cases used the same VH1 family V1-2 gene segment, and three cases used the same VH4 family V4-34 gene segment. The other five cases all used different gene segments V4-31, V5-51, V3-23, V3-30, and V3-7. All of the VH genes were mutated from germ line, with percent homologies ranging between 96.9 and 89.0%. The distribution of replacement and silent mutations within the VH genes was nonrandom consistent with the maintenance of immunoglobulin function and also strongly suggestive of antigen selection in the six VH genes with highest mutation loads. The CDR3 sequences in two of three VH-34 cases were the same size (15 amino acids) and had similar sizes in the two VH1-2 cases (18 and 16 amino acids). In conclusion, US-based MALT lymphomas of the ocular adnexa preferentially express a limited set of VH gene segments not frequently used by other MALT lymphomas and consistent with some recognizing similar antigens. Analysis of somatic mutations present within the VH genes is also consistent with antigen binding stimulating the growth of these lymphomas.

A Quantitative Allele-Specific PCR Test for the BRAF V600E Mutation Using a Single Heterozygous Control Plasmid for Quantitation: A Model for qPCR Testing without Standard Curves

We describe a novel method for mutant allele quantitation using allele-specific PCR. The method uses a heterozygous plasmid containing one wild-type and one mutant sequence as a calibrator that is run at a single concentration, with each quantitative allele-specific PCR run. PCR data from both calibrator alleles, together with predetermined PCR efficiencies, are used to quantitate the mutant allele burden in unknown specimens. We demonstrate the utility of this method by using it to calculate BRAF V600E allele frequencies in cases of hairy-cell leukemia and show that it generates data that are comparable to those obtained via allele quantitation using conventional standard curves over a wide range of allelic ratios. This method is not subject to errors that may be introduced in traditional standard curves as the result of variations in pippetting or errors in the calculation of the absolute copy numbers of standards. Furthermore, it simplifies the workflow in the clinical laboratory and would provide significant advantages for efforts to standardize clinical quantitative PCR testing.

Combined testing for CCAAT/enhancer-binding protein alpha (CEBPA) mutations and promoter methylation in acute myeloid leukemia demonstrates shared phenotypic features

Loss of function mutations in CCAAT/enhancer binding protein alpha (CEBPA) have been identified in acute myeloid leukemia (AML) and bi-allelic (double) CEBPA mutations are associated with improved prognosis in cases of cytogenetically normal-AML. In a subset of AML patients lacking CEBPA mutations, core promotor methylation of CEBPA has been described and is associated with a gene expression profile similar to the mutated cases including the expression of T cell associated genes such as CD7. However, the overall incidence and pattern of CEBPA mutations and core promoter methylation has not been thoroughly explored in a larger subset of AML with expression of CD7. Here we describe a simple and clinically deployable CEBPA promoter methylation test and the results of combined testing for CEBPA mutations and promoter methylation in 102 cases of AML, including 43 CD7+ cases. Overall, there were 5 methylated cases, 6 cases with double mutations, and 3 cases with single mutations. Significantly, 10 of 43 CD7+ cases (23%) had either methylated or double-mutated CEBPA. The CD7+ subset included all 5 methylated cases and 5 of the 6 cases with double mutations. All 3 cases with single mutations were CD7-. No case exhibited both hypermethylation and mutations. We find that promoter methylation accounts for half of those CD7+ cases with CEBPA dysregulating abnormalities. Furthermore, methylated cases and those with bi-allelic CEBPA mutations have similar phenotypic features including expression of CD7 and lack of co-incident NPM1 mutations. Our study suggests that methylation testing may be as important as mutation testing for identifying AML cases with CEBPA dysregulation and may be indicated in the routine prognostic workup of AML.

A Comparison of Deep Sequencing of TCRG Rearrangements vs Traditional Capillary Electrophoresis for Assessment of Clonality in T-Cell Lymphoproliferative Disorders

OBJECTIVES To design and evaluate a next-generation sequencing (NGS)-based method for T-cell receptor γ (TCRG) gene-based T-cell clonality testing on the Ion Torrent Personal Genome Machine (Life Technologies, Carlsbad, CA) platform. METHODS We analyzed a series of peripheral blood, bone marrow, and formalin-fixed paraffin-embedded tissue specimens with NGS vs traditional capillary electrophoresis methods. RESULTS Using a custom analysis algorithm that we developed, our NGS assay identified between 2,215 and 48,222 unique TCRG rearrangements in a series of 48 samples. We established criteria for assigning clonality based on parameters derived from both the relative and absolute frequencies of reads. In a comparison with standard capillary electrophoresis, 19 of 19 polyclonal samples and 24 of 27 samples that appeared clonal were in agreement. The three discrepant samples demonstrated some of the pitfalls of amplicon length-based testing. Dilution studies with T-lymphoid cell lines demonstrated that a known clonal sequence could be routinely identified when present in as few as 0.1% of total cells demonstrating suitability in residual disease testing. A series of samples was also analyzed on a second NGS platform and yielded very similar results with respect to the frequency and sequence of the clonal rearrangement. CONCLUSIONS In this proof-of-concept study, we describe an NGS-based T-cell clonality assay that is suitable for routine clinical testing either alone or as an adjunct to traditional methods.