Jonathan A. Schumacher

Validation of cDNA microarray gene expression data obtained from linearly amplified RNA.

Background: DNA microarray technology has permitted the analysis of global gene expression profiles for several diseases, including cancer. However, standard hybridisation and detection protocols require micrograms of mRNA for microarray analysis, limiting broader application of this technology to small excisional biopsies, needle biopsies, and/or microdissected tissue samples. Therefore, linear amplification protocols to increase the amount of RNA have been developed. The correlation between the results of microarray experiments derived from non-amplified RNA and amplified samples needs to be evaluated in detail. Methods: Total RNA was amplified and replicate hybridisation experiments were performed with linearly amplified (aRNA) and non-amplified mRNA from tonsillar B cells and the SUDHL-6 cell line using cDNA microarrays containing approximately 4500 genes. The results of microarray differential expression using either source of RNA (mRNA or aRNA) were also compared with those found using real time quantitative reverse transcription polymerase chain reaction (QRT-PCR). Results: Microarray experiments using aRNA generated reproducible data displaying only small differences to data obtained from non-amplified mRNA. The quality of the starting total RNA template and the concentration of the promoter primer used to synthesise cDNA were crucial components of the linear amplification reaction. Approximately 80% of selected upregulated and downregulated genes identified by microarray analysis using linearly amplified RNA were confirmed by QRT-PCR using non-amplified mRNA as the starting template. Conclusions: Linear RNA amplification methods can be used to generate high fidelity microarray expression data of comparable quality to data generated by microarray methods that use non-amplified mRNA samples.

Detection of the c-kit D816V mutation in systemic mastocytosis by allele-specific PCR

Aims: The c-kit D816V activating mutation is found in >80% of cases of systemic mastocytosis (SM) and represents a potential drug target. Furthermore, because D816V is one of the diagnostic criteria for SM, it is clinically relevant to determine whether the mutation is present. Traditional techniques such as DNA sequencing are often not sensitive enough to detect mutations in low-abundance tumour cells, including SM. Here, an allele-specific assay to detect the D816V mutation in DNA from archived formalin-fixed paraffin-embedded tissues is described. Methods: A two-tube PCR format was employed to amplify c-kit exon 17 as a control and an allele-specific reaction to selectively amplify the D816V mutant allele using standard oligonucleotides. A D816V-mutant plasmid control was generated by site-directed mutagenesis of wild-type cells. 14 cases of SM, one D816V-positive seminoma sample, and 35 cases without SM were analysed using the assay. Results: The assay successfully amplified D816V in the mutant plasmid control, 13/14 cases of SM, and confirmed D816V in a seminoma sample. In addition, D816V was not amplified in 35/35 cases without SM. Serial dilution experiments demonstrated sensitivity down to <1%. Conclusion: A sensitive, specific and cost-effective assay to detect the D816V mutation in archived formalin-fixed paraffin-embedded tissues from cases of SM has been developed.

T-cell clonality assessment by next-generation sequencing improves detection sensitivity in mycosis fungoides

BACKGROUND T-cell receptor (TCR) clonality assessment is a principal diagnostic test in the management of mycosis fungoides (MF). However, current polymerase chain reaction-based methods may produce ambiguous results, often because of low abundance of clonal T lymphocytes, resulting in weak clonal peaks that cannot be size-resolved by contemporary capillary electrophoresis (CE). OBJECTIVE We sought to determine if next-generation sequencing (NGS)-based detection has increased sensitivity for T-cell clonality over CE-based detection in MF. METHODS Clonality was determined by an NGS-based method in which the TCR-γ variable region was polymerase chain reaction amplified and the products sequenced to establish the identity of rearranged variable and joining regions. RESULTS Of the 35 MF cases tested, 29 (85%) showed a clonal T-cell rearrangement by NGS, compared with 15 (44%) by standard CE detection. Three patients with MF had follow-up testing that showed identical, clonal TCR sequences in subsequent skin biopsy specimens. LIMITATIONS Clonal T-cell populations have been described in benign conditions; evidence of clonality alone, by any method, is not sufficient for diagnosis. CONCLUSION TCR clonality assessment by NGS has superior sensitivity compared with CE-based detection. Further, NGS enables tracking of specific clones across multiple time points for more accurate identification of recurrent MF.

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.

Coexisting and cooperating mutations in NPM1-mutated acute myeloid leukemia

NPM1 insertion mutations represent a common recurrent genetic abnormality in acute myeloid leukemia (AML) patients. The frequency of these mutations varies from approximately 30% overall up to 50% in patients with a normal karyotype. Several recent studies have exploited advances in massively parallel sequencing technology to shed light on the complex genomic landscape of AML. We hypothesize that variant allele fraction (VAF) data derived from massively parallel sequencing studies may provide further insights into the clonal architecture and pathogenesis of NPM1-driven leukemogenesis. Diagnostic peripheral blood or bone marrow samples from NPM1-mutated AML patients (n=120) were subjected to targeted sequencing using a panel of fifty-seven genes known to be commonly mutated in myeloid malignancies. NPM1 mutations were always accompanied by additional mutations and NPM1 had the highest VAF in only one case. Nearly all NPM1-mutated AML patients showed concurrent mutations in genes involved in regulation of DNA methylation (DNMT3A, TET2, IDH1, IDH2), RNA splicing (SRSF2, SF3B1), or in the cohesin complex (RAD21, SMC1A, SMC3, STAG2). Mutations in these genes had higher median VAFs that were higher (40% or greater) than the co-existing NPM1 mutations (median VAF 16.8%). Mutations associated with cell signaling pathways (FLT3, NRAS, and PTPN11) are also frequently encountered in NPM1-mutated AML cases, but had relatively low VAFs (7.0-11.9%). No cases of NPM1-mutated AML with a concurrent IDH2R172 mutation were observed, suggesting that these variants are mutually exclusive. Overall, these data suggest that NPM1 mutations are a secondary or late event in the pathogenesis of AML and are preceded by founder mutations in genes that may be associated with recently described preclinical states such as clonal hematopoiesis of indeterminate potential or clonal cytopenias of undetermined significance.

Concurrent detection of targeted copy number variants and mutations using a myeloid malignancy next generation sequencing panel allows comprehensive genetic analysis using a single testing strategy

Currently, comprehensive genetic testing of myeloid malignancies requires multiple testing strategies with high costs. Somatic mutations can be detected by next generation sequencing (NGS) but copy number variants (CNVs) require cytogenetic methods including karyotyping, fluorescence in situ hybidization and microarray. Here, we evaluated a new method for CNV detection using read depth data derived from a targeted NGS mutation panel. In a cohort of 270 samples, we detected pathogenic mutations in 208 samples and targeted CNVs in 68 cases. The most frequent CNVs were 7q deletion including LUC7L2 and EZH2, TP53 deletion, ETV6 deletion, gain of RAD21 on 8q, and 5q deletion, including NSD1 and NPM1. We were also able to detect exon‐level duplications, including so‐called KMT2A (MLL) partial tandem duplication, in 9 cases. In the 63 cases that were negative for mutations, targeted CNVs were observed in 4 cases. Targeted CNV detection by NGS had very high concordance with single nucleotide polymorphism microarray, the current gold standard. We found that ETV6 deletion was strongly associated with TP53 alterations and 7q deletion was associated with mutations in TP53, KRAS and IDH1. This proof‐of‐concept study demonstrates the feasibility of using the same NGS data to simultaneously detect both somatic mutations and targeted CNVs.

Utility of Linearly Amplified RNA for RT-PCR Detection of Chromosomal Translocations: Validation Using the t(2;5)(p23;q35) NPM-ALK Chromosomal Translocation

The requirement for sufficient quantities of starting RNA has limited the ability to evaluate multiple transcripts using reverse transcriptase-polymerase chain reaction (RT-PCR). In this study, we demonstrate the utility of linear RNA amplification for RT-PCR analysis of multiple gene transcripts including a chromosomal translocation, using the t(2;5)(p23;q35) as a model. RNA from the t(2;5)-positive cell line, SU-DHL-1, and the t(2;5)-negative cell line, HUT-78, was extracted and exposed to two rounds of linear amplification. RT-PCR using cDNA from the resultant amplified (a) RNA and total RNA resulted in the 177 bp NPM-ALK fusion gene product from the SU-DHL-1 cell line, but not from aRNA or total RNA from the HUT-78 cell line. DNA sequencing of the RT-PCR products from total and aRNA of SU-DHL-1 cells demonstrated identical sequences corresponding to the NPM-ALK fusion gene. Evaluation of 25 snap-frozen tissue samples, including eight NPM-ALK-positive ALCLs demonstrated 100% concordance of t(2;5) detection between cDNA from total RNA and that from aRNA. Our results show that linear amplification of RNA can enhance starting RNA greater than 200-fold and can be used for rapid and specific detection of multiplex gene expression from a variety of sources. This method can generate a renewable archive of representative cDNA, which can be used for retrospective screening of stored samples as well as positive controls for the clinical molecular diagnostic laboratory.

A novel approach to quantitating leukemia fusion transcripts by qRT-PCR without the need for standard curves.

Acute myeloid leukemia patients with recurrent cytogenetic abnormalities including inv(16);CBFB-MYH11 and t(15;17);PML-RARA may be assessed by monitoring the levels of the corresponding abnormal fusion transcripts by quantitative reverse transcription-PCR (qRT-PCR). Such testing is important for evaluating the response to therapy and for the detection of early relapse. Existing qRT-PCR methods are well established and in widespread use in clinical laboratories but they are laborious and require the generation of standard curves. Here, we describe a new method to quantitate fusion transcripts in acute myeloid leukemia by qRT-PCR without the need for standard curves. Our approach uses a plasmid calibrator containing both a fusion transcript sequence and a reference gene sequence, representing a perfect normalized copy number (fusion transcript copy number/reference gene transcript copy number; NCN) of 1.0. The NCN of patient specimens can be calculated relative to that of the single plasmid calibrator using experimentally derived PCR efficiency values. We compared the data obtained using the plasmid calibrator method to commercially available assays using standard curves and found that the results obtained by both methods are comparable over a broad range of values with similar sensitivities. Our method has the advantage of simplicity and is therefore lower in cost and may be less subject to errors that may be introduced during the generation of standard curves.

Detection and Quantification of Acute Myeloid Leukemia-Associated Fusion Transcripts

Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based detection of abnormal fusion transcripts is an important strategy for the diagnosis and monitoring of patients with acute myeloid leukemia (AML) with t(8;21)(q22;q22); RUNX1-RUNX1T1, inv(16)(p13.1;q22); CBFB-MYH11 or t(15;17)(q22;q12); PML-RARA. In RT-qPCR assays, patient-derived cDNA is subjected to amplification using PCR primers directed against the fusion transcript of interest as well as a reference gene for normalization. Quantification is typically performed by constructing standard curves for each PCR run using a series of plasmid standards of known concentration that harbor the same fusion transcript or the same reference gene of interest. Fusion transcripts and reference gene copy numbers are then calculated in patient samples using these standard curves. The process of constructing standard curves is laborious and consumes additional reagents. In this chapter, we give the method details for a multiplex RT-qPCR strategy to detect and quantify the acute myeloid leukemia (AML)-associated fusion transcripts PML-RARA in patients with t(15;17) without the need for standard curves. This general method can also be applied to other AML-associated fusion transcripts such as CBFB-MYH11 and RUNX1-RUNX1T1.

Systemic mastocytosis in a patient with Cowden syndrome

cases have been reported who received infusions of fX containing products, with poor recovery and short survival of fX [4–6]. We report a 52-year-old woman with AL amyloidosis and acquired fX deficiency who received a three-factor prothrombin complex concentrate (PCC, BebulinVR ) perioperatively for thyroidectomy, resulting in adequate hemostasis. She initially presented with nephrotic syndrome and IgG/lambda monoclonal gammopathy. She underwent autologous stem cell transplant (ASCT) in 1999. She required renal transplantation in 2004 despite the absence of clear hematologic relapse. She underwent a second ASCT in 2010, after hematologic relapse but remained with measurable, but low disease activity. In 2013, a thyroid nodule was found and biopsy revealed papillary carcinoma. Evaluation of elevated preoperative activated partial thromboplastin time (37.100) and prothrombin time (14.700) was significant for fX deficiency (13%). Factors II, V, VII, VIII, and IX were normal. Mixing studies showed no evidence of an inhibitor. She had no history of spontaneous bleeding. Because of the reported poor response to infused fX, [4–6], and the critical site of the surgery, we wanted to determine response to the PCC before thyroidectomy. She received a test dose of 50 IU/kg (5,000 IU). Her fX level increased from 19 to 37%, and persisted above 30% for 22 hr. This was felt to be a sufficient response for her upcoming thyroidectomy. For surgery, PCC 50 IU/kg was administered at 0 hr (preop), 8 hr, and 14 hr, and 25 IU/kg at 42 hr. Surgery was from 4.5 to 7.5 hr after the initial dose of PCC. Adequate hemostasis was achieved during surgery and no major complications occurred. For each PCC infusion (Fig. 1), fX increased by approximately half of the fIX increase, although the fX:fIX ratio in BebulinVR is >1:1. Half-life determination is not possible from our data, but there was no evidence of rapid clearance, as judged by levels persistently above baseline from 14 through 42 hr. This indicates reduced recovery, but preserved survival of infused fX. Our patient illustrates that PCC may be successfully used to increase fX levels in amyloidosis, differing from prior reports. [4–6] Her adequate response may be related to her low-level of disease at the time. If one has the opportunity to perform a test dose prior to planned invasive procedure, PCC may be a safe and effective hemostatic therapy for some patients with fX deficiency associated with amyloidosis. ANYA LITVAK, ANITA KUMAR, RICHARD J. WONG, LARRY SMITH, HANI HASSOUN, AND GERALD A. SOFF* Department of Medicine, Memorial Sloan-Kettering Cancer Center, Hematology Service, New York, New York Department of Surgery, Memorial Sloan-Kettering Cancer Center, Hematology Service, New York, New York Department of Laboratory Medicine, Memorial Sloan-Kettering Cancer Center, Hematology Service, New York, New York Conflict of interest: Nothing to report *Correspondence to: Gerald Soff; Department of Medicine, Memorial Sloan-Kettering Cancer Center, Hematology Service, Box 324 1275 York Avenue, New York, NY 10065. E-mail: soffg@mskcc.org Received for publication: 8 July 2014; Revised: 20 July 2014; Accepted: 21 July 2014 Published online: 24 July 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ajh.23813