A Comparative Analysis of Molecular Genetic and Conventional Cytogenetic Detection of Diagnostically Important Translocations in More than 400 Cases of Acute Leukemia, Highlighting the Frequency of False-negative Conventional Cytogenetics

Rebecca L. King, MD; Mojdeh Naghashpour, MD, PhD; Christopher D. Watt, MD, PhD; Jennifer J.D. Morrissette, PhD; Adam Bagg, MD

Disclosures

Am J Clin Pathol. 2011;135(6):921-928. 

In This Article

Materials and Methods

Diagnostic Samples and Study Population

Data were collected on adult patients who had diagnostic specimens submitted for MRP at the Hospital of the University of Pennsylvania, Philadelphia, during a 5.5-year period (July 2004 through December 2009). Patients were excluded if they were ultimately given a diagnosis other than AL. The specimens consisted of peripheral blood or bone marrow aspirate collected into EDTA for MRP and sodium heparin for CC. Diagnoses were established by conventional morphologic, histologic, cytochemical, immunophenotypic, and genetic criteria. The institutional review board of the University of Pennsylvania approved the study.

RNA Isolation

RNA was extracted with a silica gel–based membrane system, the RNAeasy Mini Kit (Qiagen, Valencia, CA) or the QIAmp RNA Blood Mini kit (Qiagen) according to the manufacturer's specifications and following the recommended protocol adjustments for fresh samples.

Multiplex RT-PCR

MRP was performed by 1 of 2 commercially available assays used at our institution during the study period, Hemavision-7 (HV-7; DNA Technology A/S, Aarhus, Denmark) and Signature LTx (LTx; Asuragen, Austin, TX). Both assays detect 7 frequent translocations/inversions in AL, including the most commonly occurring variants thereof. These are the t(1;19), t(12;21), inv(16), t(8;21), t(4;11), t(15;17), and t(9;22) Table 1.

Hemavision-7 The HV-7 system was used from July 2004 through April 2006. The system was used with the reagents, concentrations, and volumes indicated in the manufacturer's specifications, as previously published.[32] In contrast with the LTx method, the HV-7 uses an agarose gel–based readout. In this system, a specimen was scored positive if a distinct, sharp, and appropriately sized band was detected. Owing to difficulty obtaining reagents, this method was discontinued in our laboratory as of May 2006.

Signature LTx The LTx assay was used between May 2006 and December 2009. The system was used with the reagents, concentrations, and volumes indicated in the manufacturer's specifications. In brief, complementary DNA was synthesized using approximately 400 ng of RNA (3 μL), 4 μL of Signature LTx RT Primer Mix (Asuragen), and 3 μL of Signature LTx Diluent (Asuragen). This mixture was then incubated in a thermocycler at 70°C for 5 minutes and then cooled to 4°C. A master mix consisting of 4 μL of Signature LTx Buffer I (Asuragen), 4 μL of Signature LTx Buffer II (Asuragen), 1 μL of MMLV reverse transcriptase (Asuragen), and 1 μL of Signature LTx RNAse inhibitor (Asuragen) was added to each sample. Samples were then incubated in a thermocycler as follows: 42°C for 45 minutes and 93°C for 10 minutes and then cooled to 4°C.

PCR was performed using 5 μL of Signature LTx DNA Primer Mix (Asuragen), 5 μL of Signature LTx DNA Amp Buffer (Asuragen), 0.5 μL of AmpliTAQ Gold (5U/μL; Applied Biosystems, Foster City, CA), 10 μL of Signature LTx Diluent, and 5 μL of complementary DNA product for a total volume of 25 μL per reaction. PCR conditions were as follows: 1 cycle of 95°C for 10 minutes; 45 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds; and hold at 4°C.

Hybridization was performed using 45 μL of Signature LTx Bead Mix (Asuragen) with 5 μL of PCR product. Hybridization conditions were as follows: 95°C for 5 minutes and 52°C for 25 minutes.

Following hybridization, the samples were transferred to the Luminex instrument (Luminex, Austin, TX) where 25 μL of 1× Signature LTx Conjugate (Asuragen) was added to the mixture. Detection on the Luminex instrument was then performed according to the manufacturer's protocol. In this system, a specimen was scored positive for a specific translocation if the mean fluorescence intensity (MFI) was 400 or more. Specimen RNA quality was assessed through the amplification and detection of endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Samples with a GAPDH MFI of less than 400 and a translocation MFI of less than 400 were reported as inconclusive. In a preliminary study, the validation of the LTx assay was accomplished by comparing its performance characteristics with the HV-7 in our laboratory, which revealed a sensitivity and specificity of 100% for diagnostic specimens.[33] The LTx MRP assay can be completed in 6 hours, allowing for a same-day turnaround time for urgent cases.

Cytogenetic Analysis

CC was performed on unstimulated 24-hour cultures of bone marrow or peripheral blood cells. Trypsin/Giemsabanded metaphases were prepared and analyzed using standard techniques of colchicine arrest, hypotonic treatment, and 3:1 of methanol/acetic acid fixation. In most cases, a karyotype was reported based on the analysis of at least 20 metaphases.

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